Comparative Genetic Variability in HIV-1 Subtype C vpu Gene in Early Age Groups of Infants.

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Sharma, Uma; Gupta, Poonam; Gupta, Sunil; Venkatesh, S; Husain, Mohammad

2018-01-01

Identifying the genetic variability in vertically transmitted viruses in early infancy is important to understand the disease progression. Being important in HIV-1 disease pathogenesis, vpu gene, isolated from young infants was investigated to understand the viral characteristics. Blood samples were obtained from 80 HIV-1 positive infants, categorized in two age groups; acute (6-18 months). A total of 77 PCR positive samples, amplified for vpu gene, were sequenced and analyzed. 73 isolates belonged to subtype C. Analysis of heterogeneity of amino acid sequences in infant groups showed that in the sequences of acute age group both insertions and deletions were present while in the early age group only deletions were present. In the acute age group, a deletion of 3 residues (RAE) in the first alfa helix in one sequence and insertions of 1-2 residues (DM, GH, G and H) in the second alfa helix in 4 sequences were observed. In the early age group, deletion of 2 residues (VN) in the cytoplasmic tail region in 2 sequences was observed. Length of the amino terminal was observed to be gradually increasing with the increasing age of the infants. Protein Variation Effect Analyzer software showed that deleterious mutations were more in the acute than the early age group. Entropy analysis revealed that heterogeneity of the residues was comparatively higher in the sequences of acute than the early age group. Mutations observed in the helixes may affect the conformation and lose the ability to degrade CD4 receptors. Heterogeneity was decreasing with the increasing ages of the infants, indicating positive selection for robust virion survival. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

RNA-binding proteins involved in post-transcriptional regulation in bacteria

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Elke eVan Assche

2015-03-01

Full Text Available Post-transcriptional regulation is a very important mechanism to control gene expression in changing environments. In the past decade, a lot of interest has been directed towards the role of small RNAs in bacterial post-transcriptional regulation. However, small RNAs are not the only molecules controlling gene expression at this level, RNA-binding proteins play an important role as well. CsrA and Hfq are the two best studied bacterial proteins of this type, but recently, additional proteins involved in post-transcriptional control have been identified. This review focuses on the general working mechanisms of post-transcriptionally active RNA-binding proteins, which include (i adaptation of the susceptibility of mRNAs and sRNAs to RNases, (ii modulating the accessibility of the ribosome binding site of mRNAs, (iii recruiting and assisting in the interaction of mRNAs with other molecules and (iv regulating transcription terminator / antiterminator formation, and gives an overview of both the well-studied and the newly identified proteins that are involved in post-transcriptional regulatory processes. Additionally, the post-transcriptional mechanisms by which the expression or the activity of these proteins is regulated, are described. For many of the newly identified proteins, however, mechanistic questions remain. Most likely, more post-transcriptionally active proteins will be identified in the future.

The Q Motif Is Involved in DNA Binding but Not ATP Binding in ChlR1 Helicase.

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Hao Ding

Full Text Available Helicases are molecular motors that couple the energy of ATP hydrolysis to the unwinding of structured DNA or RNA and chromatin remodeling. The conversion of energy derived from ATP hydrolysis into unwinding and remodeling is coordinated by seven sequence motifs (I, Ia, II, III, IV, V, and VI. The Q motif, consisting of nine amino acids (GFXXPXPIQ with an invariant glutamine (Q residue, has been identified in some, but not all helicases. Compared to the seven well-recognized conserved helicase motifs, the role of the Q motif is less acknowledged. Mutations in the human ChlR1 (DDX11 gene are associated with a unique genetic disorder known as Warsaw Breakage Syndrome, which is characterized by cellular defects in genome maintenance. To examine the roles of the Q motif in ChlR1 helicase, we performed site directed mutagenesis of glutamine to alanine at residue 23 in the Q motif of ChlR1. ChlR1 recombinant protein was overexpressed and purified from HEK293T cells. ChlR1-Q23A mutant abolished the helicase activity of ChlR1 and displayed reduced DNA binding ability. The mutant showed impaired ATPase activity but normal ATP binding. A thermal shift assay revealed that ChlR1-Q23A has a melting point value similar to ChlR1-WT. Partial proteolysis mapping demonstrated that ChlR1-WT and Q23A have a similar globular structure, although some subtle conformational differences in these two proteins are evident. Finally, we found ChlR1 exists and functions as a monomer in solution, which is different from FANCJ, in which the Q motif is involved in protein dimerization. Taken together, our results suggest that the Q motif is involved in DNA binding but not ATP binding in ChlR1 helicase.

Involvement of two classes of binding sites in the interactions of cyclophilin B with peripheral blood T-lymphocytes.

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Denys, A; Allain, F; Carpentier, M; Spik, G

1998-12-15

Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein, mainly associated with the secretory pathway, and is released in biological fluids. We recently reported that CyPB specifically binds to T-lymphocytes and promotes enhanced incorporation of CsA. The interactions with cellular binding sites involved, at least in part, the specific N-terminal extension of the protein. In this study, we intended to specify further the nature of the CyPB-binding sites on peripheral blood T-lymphocytes. We first provide evidence that the CyPB binding to heparin-Sepharose is prevented by soluble sulphated glycosaminoglycans (GAG), raising the interesting possibility that such interactions may occur on the T-cell surface. We then characterized CyPB binding to T-cell surface GAG and found that these interactions involved the N-terminal extension of CyPB, but not its conserved CsA-binding domain. In addition, we determined the presence of a second CyPB binding site, which we termed a type I site, in contrast with type II for GAG interactions. The two binding sites exhibit a similar affinity but the expression of the type I site was 3-fold lower. The conclusion that CyPB binding to the type I site is distinct from the interactions with GAG was based on the findings that it was (1) resistant to NaCl wash and GAG-degrading enzyme treatments, (2) reduced in the presence of CsA or cyclophilin C, and (3) unmodified in the presence of either the N-terminal peptide of CyPB or protamine. Finally, we showed that the type I binding sites were involved in an endocytosis process, supporting the hypothesis that they may correspond to a functional receptor for CyPB.

Involvement of the N-terminal part of cyclophilin B in the interaction with specific Jurkat T-cell binding sites.

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Mariller, C; Haendler, B; Allain, F; Denys, A; Spik, G

1996-07-15

Cyclophilin B (CyPB) is secreted in biological fluids such as blood or milk and binds to a specific receptor present on the human lymphoblastic cell line Jurkat and on human peripheral blood lymphocytes. This study was intended to specify the areas of CyPB that are involved in the interaction with the receptor. A synthetic peptide corresponding to the first 24 N-terminal amino acid residues of CyPB was shown to specifically recognize the receptor. Moreover, modification of Arg18 of CyPB by p-hydroxyphenlglyoxal led to a dramatic loss of affinity for the receptor. However, when this residue was replaced by an alanine residue using site-directed mutagenesis, no modification of the binding properties was found, suggesting that Arg18 is not directly involved but is sufficiently close to the interaction site to interfere with the binding when modified. Competitive binding experiments using a chimaeric protein made up of the 24 N-terminal amino acid residues of CyPB fused to the cyclophilin A core sequence confirmed the involvement of this region of CyPB in receptor binding.

The involvement of coordinative interactions in the binding of dihydrolipoamide dehydrogenase to titanium dioxide-Localization of a putative binding site.

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Dayan, Avraham; Babin, Gilad; Ganoth, Assaf; Kayouf, Nivin Samir; Nitoker Eliaz, Neta; Mukkala, Srijana; Tsfadia, Yossi; Fleminger, Gideon

2017-08-01

Titanium (Ti) and its alloys are widely used in orthodontic and orthopedic implants by virtue to their high biocompatibility, mechanical strength, and high resistance to corrosion. Biointegration of the implants with the tissue requires strong interactions, which involve biological molecules, proteins in particular, with metal oxide surfaces. An exocellular high-affinity titanium dioxide (TiO 2 )-binding protein (TiBP), purified from Rhodococcus ruber, has been previously studied in our lab. This protein was shown to be homologous with the orthologous cytoplasmic rhodococcal dihydrolipoamide dehydrogenase (rhDLDH). We have found that rhDLDH and its human homolog (hDLDH) share the TiO 2 -binding capabilities with TiBP. Intrigued by the unique TiO 2 -binding properties of hDLDH, we anticipated that it may serve as a molecular bridge between Ti-based medical structures and human tissues. The objective of the current study was to locate the region and the amino acids of the protein that mediate the protein-TiO 2 surface interaction. We demonstrated the role of acidic amino acids in the nonelectrostatic enzyme/dioxide interactions at neutral pH. The observation that the interaction of DLDH with various metal oxides is independent of their isoelectric values strengthens this notion. DLDH does not lose its enzymatic activity upon binding to TiO 2 , indicating that neither the enzyme undergoes major conformational changes nor the TiO 2 binding site is blocked. Docking predictions suggest that both rhDLDH and hDLDH bind TiO 2 through similar regions located far from the active site and the dimerization sites. The putative TiO 2 -binding regions of both the bacterial and human enzymes were found to contain a CHED (Cys, His, Glu, Asp) motif, which has been shown to participate in metal-binding sites in proteins. Copyright © 2017 John Wiley & Sons, Ltd.

Involvement of two classes of binding sites in the interactions of cyclophilin B with peripheral blood T-lymphocytes.

OpenAIRE

Denys, A; Allain, F; Carpentier, M; Spik, G

1998-01-01

Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein, mainly associated with the secretory pathway, and is released in biological fluids. We recently reported that CyPB specifically binds to T-lymphocytes and promotes enhanced incorporation of CsA. The interactions with cellular binding sites involved, at least in part, the specific N-terminal extension of the protein. In this study, we intended to specify further the nature of the CyPB-binding sites on peripheral blood T-lymphocytes...

Gemin5: A Multitasking RNA-Binding Protein Involved in Translation Control

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David Piñeiro

2015-04-01

Full Text Available Gemin5 is a RNA-binding protein (RBP that was first identified as a peripheral component of the survival of motor neurons (SMN complex. This predominantly cytoplasmic protein recognises the small nuclear RNAs (snRNAs through its WD repeat domains, allowing assembly of the SMN complex into small nuclear ribonucleoproteins (snRNPs. Additionally, the amino-terminal end of the protein has been reported to possess cap-binding capacity and to interact with the eukaryotic initiation factor 4E (eIF4E. Gemin5 was also shown to downregulate translation, to be a substrate of the picornavirus L protease and to interact with viral internal ribosome entry site (IRES elements via a bipartite non-canonical RNA-binding site located at its carboxy-terminal end. These features link Gemin5 with translation control events. Thus, beyond its role in snRNPs biogenesis, Gemin5 appears to be a multitasking protein cooperating in various RNA-guided processes. In this review, we will summarise current knowledge of Gemin5 functions. We will discuss the involvement of the protein on translation control and propose a model to explain how the proteolysis fragments of this RBP in picornavirus-infected cells could modulate protein synthesis.

DNA-binding protects p53 from interactions with cofactors involved in transcription-independent functions.

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Lambrughi, Matteo; De Gioia, Luca; Gervasio, Francesco Luigi; Lindorff-Larsen, Kresten; Nussinov, Ruth; Urani, Chiara; Bruschi, Maurizio; Papaleo, Elena

2016-11-02

Binding-induced conformational changes of a protein at regions distant from the binding site may play crucial roles in protein function and regulation. The p53 tumour suppressor is an example of such an allosterically regulated protein. Little is known, however, about how DNA binding can affect distal sites for transcription factors. Furthermore, the molecular details of how a local perturbation is transmitted through a protein structure are generally elusive and occur on timescales hard to explore by simulations. Thus, we employed state-of-the-art enhanced sampling atomistic simulations to unveil DNA-induced effects on p53 structure and dynamics that modulate the recruitment of cofactors and the impact of phosphorylation at Ser215. We show that DNA interaction promotes a conformational change in a region 3 nm away from the DNA binding site. Specifically, binding to DNA increases the population of an occluded minor state at this distal site by more than 4-fold, whereas phosphorylation traps the protein in its major state. In the minor conformation, the interface of p53 that binds biological partners related to p53 transcription-independent functions is not accessible. Significantly, our study reveals a mechanism of DNA-mediated protection of p53 from interactions with partners involved in the p53 transcription-independent signalling. This also suggests that conformational dynamics is tightly related to p53 signalling. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Characterization of Microfibrillar-associated Protein 4 (MFAP4) as a Tropoelastin- and Fibrillin-binding Protein Involved in Elastic Fiber Formation

DEFF Research Database (Denmark)

Pilecki, Bartosz; Holm, Anne T; Schlosser, Anders

2016-01-01

primarily assembles into trimeric and hexameric structures of homodimers. Binding analysis revealed that MFAP4 specifically binds tropoelastin and fibrillin-1 and -2, as well as the elastin cross-linking amino acid desmosine, and that it co-localizes with fibrillin-1-positive fibers in vivo. Site......-directed mutagenesis disclosed residues Phe(241) and Ser(203) in MFAP4 as being crucial for type I collagen, elastin, and tropoelastin binding. Furthermore, we found that MFAP4 actively promotes tropoelastin self-assembly. In conclusion, our data identify MFAP4 as a new ligand of microfibrils and tropoelastin involved...

Lack of adaptation to human tetherin in HIV-1 Group O and P

Science.gov (United States)

2011-01-01

Background HIV-1 viruses are categorized into four distinct groups: M, N, O and P. Despite the same genomic organization, only the group M viruses are responsible for the world-wide pandemic of AIDS, suggesting better adaptation to human hosts. Previously, it has been reported that the group M Vpu protein is capable of both down-modulating CD4 and counteracting BST-2/tetherin restriction, while the group O Vpu cannot antagonize tetherin. This led us to investigate if group O, and the related group P viruses, possess functional anti-tetherin activities in Vpu or another viral protein, and to further map the residues required for group M Vpu to counteract human tetherin. Results We found a lack of activity against human tetherin for both the Vpu and Nef proteins from group O and P viruses. Furthermore, we found no evidence of anti-human tetherin activity in a fully infectious group O proviral clone, ruling out the possibility of an alternative anti-tetherin factor in this virus. Interestingly, an activity against primate tetherins was retained in the Nef proteins from both a group O and a group P virus. By making chimeras between a functional group M and non-functional group O Vpu protein, we were able to map the first 18 amino acids of group M Vpu as playing an essential role in the ability of the protein to antagonize human tetherin. We further demonstrated the importance of residue alanine-18 for the group M Vpu activity. This residue lies on a diagonal face of conserved alanines in the TM domain of the protein, and is necessary for specific Vpu-tetherin interactions. Conclusions The absence of human specific anti-tetherin activities in HIV-1 group O and P suggests a failure of these viruses to adapt to human hosts, which may have limited their spread. PMID:21955466

Lack of adaptation to human tetherin in HIV-1 Group O and P

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Haworth Kevin G

2011-09-01

Full Text Available Abstract Background HIV-1 viruses are categorized into four distinct groups: M, N, O and P. Despite the same genomic organization, only the group M viruses are responsible for the world-wide pandemic of AIDS, suggesting better adaptation to human hosts. Previously, it has been reported that the group M Vpu protein is capable of both down-modulating CD4 and counteracting BST-2/tetherin restriction, while the group O Vpu cannot antagonize tetherin. This led us to investigate if group O, and the related group P viruses, possess functional anti-tetherin activities in Vpu or another viral protein, and to further map the residues required for group M Vpu to counteract human tetherin. Results We found a lack of activity against human tetherin for both the Vpu and Nef proteins from group O and P viruses. Furthermore, we found no evidence of anti-human tetherin activity in a fully infectious group O proviral clone, ruling out the possibility of an alternative anti-tetherin factor in this virus. Interestingly, an activity against primate tetherins was retained in the Nef proteins from both a group O and a group P virus. By making chimeras between a functional group M and non-functional group O Vpu protein, we were able to map the first 18 amino acids of group M Vpu as playing an essential role in the ability of the protein to antagonize human tetherin. We further demonstrated the importance of residue alanine-18 for the group M Vpu activity. This residue lies on a diagonal face of conserved alanines in the TM domain of the protein, and is necessary for specific Vpu-tetherin interactions. Conclusions The absence of human specific anti-tetherin activities in HIV-1 group O and P suggests a failure of these viruses to adapt to human hosts, which may have limited their spread.

Binding of the human "electron transferring flavoprotein" (ETF) to the medium chain acyl-CoA dehydrogenase (MCAD) involves an arginine and histidine residue.

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Parker, Antony R

2003-10-01

The interaction between the "electron transferring flavoprotein" (ETF) and medium chain acyl-CoA dehydrogenase (MCAD) enables successful flavin to flavin electron transfer, crucial for the beta-oxidation of fatty acids. The exact biochemical determinants for ETF binding to MCAD are unknown. Here we show that binding of human ETF, to MCAD, was inhibited by 2,3-butanedione and diethylpyrocarbonate (DEPC) and reversed by incubation with free arginine and hydroxylamine respectively. Spectral analyses of native ETF vs modified ETF suggested that flavin binding was not affected and that the loss of ETF activity with MCAD involved modification of one ETF arginine residue and one ETF histidine residue respectively. MCAD and octanoyl-CoA protected ETF against inactivation by both 2,3-butanedione and DEPC indicating that the arginine and histidine residues are present in or around the MCAD binding site. Comparison of exposed arginine and histidine residues among different ETF species, however, indicates that arginine residues are highly conserved but that histidine residues are not. These results lead us to conclude that this single arginine residue is essential for the binding of ETF to MCAD, but that the single histidine residue, although involved, is not.

Development of complex simulation suite 'VEB' and application area expansion - 15326

International Nuclear Information System (INIS)

Obraztsov, E.; Kapista, D.; Kremnev, I.; Korokhov, T.; Kukhtevich, V.; Bezlepkin, V.

2015-01-01

Complex simulation suite 'Virtual Unit of NPP' (CSS 'VEB') is a set of connected software tools and computer codes that allow the simulation of various physical processes occurring in a power nuclear reactor (initially VVER - pressurized water reactor). A coupled computational model developed by means of CSS 'VEB' is based on a specific design and called an NPP Virtual Power Unit (VPU). And in November 2012 a pilot version of the Leningrad NPP-2 VPU (NPP-2006 design with water-cooled power reactor) successfully passed acceptance tests. The paper presents further development of CSS 'VEB' and application area expansion both on new tasks such as VPU calculations for the Probability Safety Analysis (PSA) and all modeling of other NPP types, like sodium-cooled reactor (that means a possibility to create a VPU not only for NPP with PWR). In the first part of this paper we will recall the basic idea and concept of CSS 'VEB' and VPU. Then we will describe the main components of CSS 'VEB' and their improvements. In the last part we will briefly describe the broadening of VPU uses

RPA-Binding Protein ETAA1 Is an ATR Activator Involved in DNA Replication Stress Response.

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Lee, Yuan-Cho; Zhou, Qing; Chen, Junjie; Yuan, Jingsong

2016-12-19

ETAA1 (Ewing tumor-associated antigen 1), also known as ETAA16, was identified as a tumor-specific antigen in the Ewing family of tumors. However, the biological function of this protein remains unknown. Here, we report the identification of ETAA1 as a DNA replication stress response protein. ETAA1 specifically interacts with RPA (Replication protein A) via two conserved RPA-binding domains and is therefore recruited to stalled replication forks. Interestingly, further analysis of ETAA1 function revealed that ETAA1 participates in the activation of ATR signaling pathway via a conserved ATR-activating domain (AAD) located near its N terminus. Importantly, we demonstrate that both RPA binding and ATR activation are required for ETAA1 function at stalled replication forks to maintain genome stability. Therefore, our data suggest that ETAA1 is a new ATR activator involved in replication checkpoint control. Copyright © 2016 Elsevier Ltd. All rights reserved.

Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

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Gangi Setty, Thanuja [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Cho, Christine [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Govindappa, Sowmya [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Apicella, Michael A. [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Ramaswamy, S., E-mail: ramas@instem.res.in [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India)

2014-07-01

Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

International Nuclear Information System (INIS)

Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

2014-01-01

Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states

Minor Coat and Heat Shock Proteins Are Involved in the Binding of Citrus Tristeza Virus to the Foregut of Its Aphid Vector, Toxoptera citricida.

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Killiny, N; Harper, S J; Alfaress, S; El Mohtar, C; Dawson, W O

2016-11-01

Vector transmission is a critical stage in the viral life cycle, yet for most plant viruses how they interact with their vector is unknown or is explained by analogy with previously described relatives. Here we examined the mechanism underlying the transmission of citrus tristeza virus (CTV) by its aphid vector, Toxoptera citricida, with the objective of identifying what virus-encoded proteins it uses to interact with the vector. Using fluorescently labeled virions, we demonstrated that CTV binds specifically to the lining of the cibarium of the aphid. Through in vitro competitive binding assays between fluorescent virions and free viral proteins, we determined that the minor coat protein is involved in vector interaction. We also found that the presence of two heat shock-like proteins, p61 and p65, reduces virion binding in vitro Additionally, treating the dissected mouthparts with proteases did not affect the binding of CTV virions. In contrast, chitinase treatment reduced CTV binding to the foregut. Finally, competition with glucose, N-acetyl-β-d-glucosamine, chitobiose, and chitotriose reduced the binding. These findings together suggest that CTV binds to the sugar moieties of the cuticular surface of the aphid cibarium, and the binding involves the concerted activity of three virus-encoded proteins. Limited information is known about the specific interactions between citrus tristeza virus and its aphid vectors. These interactions are important for the process of successful transmission. In this study, we localized the CTV retention site as the cibarium of the aphid foregut. Moreover, we demonstrated that the nature of these interactions is protein-carbohydrate binding. The viral proteins, including the minor coat protein and two heat shock proteins, bind to sugar moieties on the surface of the foregut. These findings will help in understanding the transmission mechanism of CTV by the aphid vector and may help in developing control strategies which interfere

Retinoid-binding proteins: similar protein architectures bind similar ligands via completely different ways.

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Yu-Ru Zhang

Full Text Available BACKGROUND: Retinoids are a class of compounds that are chemically related to vitamin A, which is an essential nutrient that plays a key role in vision, cell growth and differentiation. In vivo, retinoids must bind with specific proteins to perform their necessary functions. Plasma retinol-binding protein (RBP and epididymal retinoic acid binding protein (ERABP carry retinoids in bodily fluids, while cellular retinol-binding proteins (CRBPs and cellular retinoic acid-binding proteins (CRABPs carry retinoids within cells. Interestingly, although all of these transport proteins possess similar structures, the modes of binding for the different retinoid ligands with their carrier proteins are different. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we analyzed the various retinoid transport mechanisms using structure and sequence comparisons, binding site analyses and molecular dynamics simulations. Our results show that in the same family of proteins and subcellular location, the orientation of a retinoid molecule within a binding protein is same, whereas when different families of proteins are considered, the orientation of the bound retinoid is completely different. In addition, none of the amino acid residues involved in ligand binding is conserved between the transport proteins. However, for each specific binding protein, the amino acids involved in the ligand binding are conserved. The results of this study allow us to propose a possible transport model for retinoids. CONCLUSIONS/SIGNIFICANCE: Our results reveal the differences in the binding modes between the different retinoid-binding proteins.

Extended hormone binding site of the human thyroid stimulating hormone receptor: distinctive acidic residues in the hinge region are involved in bovine thyroid stimulating hormone binding and receptor activation.

Science.gov (United States)

Mueller, Sandra; Kleinau, Gunnar; Jaeschke, Holger; Paschke, Ralf; Krause, Gerd

2008-06-27

The human thyroid stimulating hormone receptor (hTSHR) belongs to the glycoprotein hormone receptors that bind the hormones at their large extracellular domain. The extracellular hinge region of the TSHR connects the N-terminal leucine-rich repeat domain with the membrane-spanning serpentine domain. From previous studies we reasoned that apart from hormone binding at the leucine-rich repeat domain, additional multiple hormone contacts might exist at the hinge region of the TSHR by complementary charge-charge recognition. Here we investigated highly conserved charged residues in the hinge region of the TSHR by site-directed mutagenesis to identify amino acids interacting with bovine TSH (bTSH). Indeed, the residues Glu-297, Glu-303, and Asp-382 in the TSHR hinge region are essential for bTSH binding and partially for signal transduction. Side chain substitutions showed that the negative charge of Glu-297 and Asp-382 is necessary for recognition of bTSH by the hTSHR. Multiple combinations of alanine mutants of the identified positions revealed an increased negative effect on hormone binding. An assembled model suggests that the deciphered acidic residues form negatively charged patches at the hinge region resulting in an extended binding mode for bTSH on the hTSHR. Our data indicate that certain positively charged residues of bTSH might be involved in interaction with the identified negatively charged amino acids of the hTSHR hinge region. We demonstrate that the hinge region represents an extracellular intermediate connector for both hormone binding and signal transduction of the hTSHR.

The asymmetric binding of PGC-1α to the ERRα and ERRγ nuclear receptor homodimers involves a similar recognition mechanism.

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Maria Takacs

Full Text Available PGC-1α is a crucial regulator of cellular metabolism and energy homeostasis that functionally acts together with the estrogen-related receptors (ERRα and ERRγ in the regulation of mitochondrial and metabolic gene networks. Dimerization of the ERRs is a pre-requisite for interactions with PGC-1α and other coactivators, eventually leading to transactivation. It was suggested recently (Devarakonda et al that PGC-1α binds in a strikingly different manner to ERRγ ligand-binding domains (LBDs compared to its mode of binding to ERRα and other nuclear receptors (NRs, where it interacts directly with the two ERRγ homodimer subunits.Here, we show that PGC-1α receptor interacting domain (RID binds in an almost identical manner to ERRα and ERRγ homodimers. Microscale thermophoresis demonstrated that the interactions between PGC-1α RID and ERR LBDs involve a single receptor subunit through high-affinity, ERR-specific L3 and low-affinity L2 interactions. NMR studies further defined the limits of PGC-1α RID that interacts with ERRs. Consistent with these findings, the solution structures of PGC-1α/ERRα LBDs and PGC-1α/ERRγ LBDs complexes share an identical architecture with an asymmetric binding of PGC-1α to homodimeric ERR.These studies provide the molecular determinants for the specificity of interactions between PGC-1α and the ERRs, whereby negative cooperativity prevails in the binding of the coactivators to these receptors. Our work indicates that allosteric regulation may be a general mechanism controlling the binding of the coactivators to homodimers.

A Tenebrio molitor GPI-anchored alkaline phosphatase is involved in binding of Bacillus thuringiensis Cry3Aa to brush border membrane vesicles.

Science.gov (United States)

Zúñiga-Navarrete, Fernando; Gómez, Isabel; Peña, Guadalupe; Bravo, Alejandra; Soberón, Mario

2013-03-01

Bacillus thuringiensis Cry toxins recognizes their target cells in part by the binding to glycosyl-phosphatidyl-inositol (GPI) anchored proteins such as aminopeptidase-N (APN) or alkaline phosphatases (ALP). Treatment of Tenebrio molitor brush border membrane vesicles (BBMV) with phospholipase C that cleaves out GPI-anchored proteins from the membranes, showed that GPI-anchored proteins are involved in binding of Cry3Aa toxin to BBMV. A 68 kDa GPI-anchored ALP was shown to bind Cry3Aa by toxin overlay assays. The 68 kDa GPI-anchored ALP was preferentially expressed in early instar larvae in comparison to late instar larvae. Our work shows for the first time that GPI-anchored ALP is important for Cry3Aa binding to T. molitor BBMV suggesting that the mode of action of Cry toxins is conserved in different insect orders. Copyright © 2012 Elsevier Inc. All rights reserved.

Human Blue Cone Opsin Regeneration Involves Secondary Retinal Binding with Analog Specificity.

Science.gov (United States)

Srinivasan, Sundaramoorthy; Fernández-Sampedro, Miguel A; Morillo, Margarita; Ramon, Eva; Jiménez-Rosés, Mireia; Cordomí, Arnau; Garriga, Pere

2018-03-27

Human color vision is mediated by the red, green, and blue cone visual pigments. Cone opsins are G-protein-coupled receptors consisting of an opsin apoprotein covalently linked to the 11-cis-retinal chromophore. All visual pigments share a common evolutionary origin, and red and green cone opsins exhibit a higher homology, whereas blue cone opsin shows more resemblance to the dim light receptor rhodopsin. Here we show that chromophore regeneration in photoactivated blue cone opsin exhibits intermediate transient conformations and a secondary retinoid binding event with slower binding kinetics. We also detected a fine-tuning of the conformational change in the photoactivated blue cone opsin binding site that alters the retinal isomer binding specificity. Furthermore, the molecular models of active and inactive blue cone opsins show specific molecular interactions in the retinal binding site that are not present in other opsins. These findings highlight the differential conformational versatility of human cone opsin pigments in the chromophore regeneration process, particularly compared to rhodopsin, and point to relevant functional, unexpected roles other than spectral tuning for the cone visual pigments. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Mapping Escherichia coli elongation factor Tu residues involved in binding of aminoacyl-tRNA

DEFF Research Database (Denmark)

Wiborg, Ove; Andersen, C; Knudsen, Charlotte Rohde

1996-01-01

Two residues of Escherichia coli elongation factor Tu involved in binding of aminoacyl-tRNA were identified and subjected to mutational analysis. Lys-89 and Asn-90 were each replaced by either Ala or Glu. The four single mutants were denoted K89A, K89E, N90A, and N90E, respectively. The mutants...... were characterized with respect to thermal and chemical stability, GTPase activity, tRNA affinity, and activity in an in vitro translation assay. Most conspicuously tRNA affinities were reduced for all mutants. The results verify our structural analysis of elongation factor Tu in complex with aminoacyl....... Their functional roles are discussed in relation to the structure of elongation factor Tu in complex with aminoacyl-tRNA. Udgivelsesdato: 1996-Aug-23...

Copper tolerance in Frankia sp. strain EuI1c involves surface binding and copper transport.

Science.gov (United States)

Rehan, Medhat; Furnholm, Teal; Finethy, Ryan H; Chu, Feixia; El-Fadly, Gomaah; Tisa, Louis S

2014-09-01

Several Frankia strains have been shown to be copper-tolerant. The mechanism of their copper tolerance was investigated for Frankia sp. strain EuI1c. Copper binding was shown by binding studies. Unusual globular structures were observed on the surface of the bacterium. These globular structures were composed of aggregates containing many relatively smaller "leaf-like" structures. Scanning electron microscopy with energy-dispersive X-ray (SEM-EDAX) analysis of these structures indicated elevated copper and phosphate levels compared to the control cells. Fourier transform infrared spectroscopy (FTIR) analysis indicated an increase in extracellular phosphate on the cell surface of copper-stressed cells. Bioinformatics' analysis of the Frankia sp. strain EuI1c genome revealed five potential cop genes: copA, copZ, copC, copCD, and copD. Experiments with Frankia sp. strain EuI1c using qRT-PCR indicated an increase in messenger RNA (mRNA) levels of the five cop genes upon Cu(2+) stress. After 5 days of Cu(2+) stress, the copA, copZ, copC, copCD, and copD mRNA levels increased 25-, 8-, 18-, 18-, and 25-fold, respectively. The protein profile of Cu(2+)-stressed Frankia sp. strain EuI1c cells revealed the upregulation of a 36.7 kDa protein that was identified as FraEuI1c_1092 (sulfate-binding periplasmic transport protein). Homologues of this gene were only present in the genomes of the Cu(2+)-resistant Frankia strains (EuI1c, DC12, and CN3). These data indicate that copper tolerance by Frankia sp. strain EuI1c involved the binding of copper to the cell surface and transport proteins.

Modeling and designing of variable-period and variable-pole-number undulator

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I. Davidyuk

2016-02-01

Full Text Available The concept of permanent-magnet variable-period undulator (VPU was proposed several years ago and has found few implementations so far. The VPUs have some advantages as compared with conventional undulators, e.g., a wider range of radiation wavelength tuning and the option to increase the number of poles for shorter periods. Both these advantages will be realized in the VPU under development now at Budker INP. In this paper, we present the results of 2D and 3D magnetic field simulations and discuss some design features of this VPU.

Tannic acid and chromic chloride-induced binding of protein to red cells: a preliminary study of possible binding sites and reaction mechanisms.

Science.gov (United States)

Hunt, A F; Reed, M I

1990-07-01

The binding mechanisms and binding sites involved in the tannic acid and chromic chloride-induced binding of protein to red cells were investigated using the binding of IgA paraprotein to red cells as model systems. Inhibition studies of these model systems using amino acid homopolymers and compounds (common as red cell membrane constituents) suggest that the mechanisms involved are similar to those proposed for the conversion of hide or skin collagen to leather, as in commercial tanning. These studies also suggest that tannic acid-induced binding of IgA paraprotein to red cells involves the amino acid residues of L-arginine, L-lysine, L-histidine, and L-proline analogous to tanning with phenolic plant extracts. The amino acid residues of L-aspartate, L-glutamate and L-asparagine are involved in a similar manner in chronic chloride-induced binding of protein to red cells.

Megalin binds and mediates cellular internalization of folate binding protein

DEFF Research Database (Denmark)

Birn, Henrik; Zhai, Xiaoyue; Holm, Jan

2005-01-01

Folate is an essential vitamin involved in a number of biological processes. High affinity folate binding proteins (FBPs) exist both as glycosylphosphatidylinositol-linked, membrane associated folate binding proteins and as soluble FBPs in plasma and some secretory fluids such as milk, saliva...... to express high levels of megalin, is inhibitable by excess unlabeled FBP and by receptor associated protein, a known inhibitor of binding to megalin. Immortalized rat yolk sac cells, representing an established model for studying megalin-mediated uptake, reveal (125)I-labeled FBP uptake which is inhibited...

Combining modelling and mutagenesis studies of synaptic vesicle protein 2A to identify a series of residues involved in racetam binding.

Science.gov (United States)

Shi, Jiye; Anderson, Dina; Lynch, Berkley A; Castaigne, Jean-Gabriel; Foerch, Patrik; Lebon, Florence

2011-10-01

LEV (levetiracetam), an antiepileptic drug which possesses a unique profile in animal models of seizure and epilepsy, has as its unique binding site in brain, SV2A (synaptic vesicle protein 2A). Previous studies have used a chimaeric and site-specific mutagenesis approach to identify three residues in the putative tenth transmembrane helix of SV2A that, when mutated, alter binding of LEV and related racetam derivatives to SV2A. In the present paper, we report a combined modelling and mutagenesis study that successfully identifies another 11 residues in SV2A that appear to be involved in ligand binding. Sequence analysis and modelling of SV2A suggested residues equivalent to critical functional residues of other MFS (major facilitator superfamily) transporters. Alanine scanning of these and other SV2A residues resulted in the identification of residues affecting racetam binding, including Ile273 which differentiated between racetam analogues, when mutated to alanine. Integrating mutagenesis results with docking analysis led to the construction of a mutant in which six SV2A residues were replaced with corresponding SV2B residues. This mutant showed racetam ligand-binding affinity intermediate to the affinities observed for SV2A and SV2B.

Two distinctive β subunits are separately involved in two binding sites of imidacloprid with different affinities in Locusta migratoria manilensis.

Science.gov (United States)

Bao, Haibo; Liu, Yang; Zhang, Yixi; Liu, Zewen

2017-08-01

Due to great diversity of nicotinic acetylcholine receptor (nAChR) subtypes in insects, one β subunit may be contained in numerous nAChR subtypes. In the locust Locusta migratoria, a model insect species with agricultural importance, the third β subunits (Locβ3) was identified in this study, which reveals at least three β subunits in this insect species. Imidacloprid was found to bind nAChRs in L. migratoria central nervous system at two sites with different affinities, with K d values of 0.16 and 10.31nM. The specific antisera (L1-1, L2-1 and L3-1) were raised against fusion proteins at the large cytoplasmic loop of Locβ1, Locβ2 and Locβ3 respectively. Specific immunodepletion of Locβ1 with antiserum L1-1 resulted in the selective loss of the low affinity binding site for imidacloprid, whereas the immunodepletion of Locβ3 with L3-1 caused the selective loss of the high affinity site. Dual immunodepletion with L1-1 and L3-1 could completely abolish imidacloprid binding. In contrast, the immunodepletion of Locβ2 had no significant effect on the specific [ 3 H]imidacloprid binding. Taken together, these data indicated that Locβ1 and Locβ3 were respectively contained in the low- and high-affinity binding sites for imidacloprid in L. migratoria, which is different to the previous finding in Nilaparvata lugens that Nlβ1 was in two binding sites for imidacloprid. The involvement of two β subunits separately in two binding sites may decrease the risk of imidacloprid resistance due to putative point mutations in β subunits in L. migratoria. Copyright © 2017 Elsevier B.V. All rights reserved.

An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase

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Wong, Jaslyn E. M. M. [Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus (Denmark); Midtgaard, Søren Roi [University of Copenhagen, Universitetsparken 5, 2100 Copenhagen (Denmark); Gysel, Kira [Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus (Denmark); Thygesen, Mikkel B.; Sørensen, Kasper K.; Jensen, Knud J. [University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C (Denmark); Stougaard, Jens; Thirup, Søren; Blaise, Mickaël, E-mail: mickael.blaise@cpbs.cnrs.fr [Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus (Denmark)

2015-03-01

The crystal and solution structures of the T. thermophilus NlpC/P60 d, l-endopeptidase as well as the co-crystal structure of its N-terminal LysM domains bound to chitohexaose allow a proposal to be made regarding how the enzyme recognizes peptidoglycan. LysM domains, which are frequently present as repetitive entities in both bacterial and plant proteins, are known to interact with carbohydrates containing N-acetylglucosamine (GlcNAc) moieties, such as chitin and peptidoglycan. In bacteria, the functional significance of the involvement of multiple LysM domains in substrate binding has so far lacked support from high-resolution structures of ligand-bound complexes. Here, a structural study of the Thermus thermophilus NlpC/P60 endopeptidase containing two LysM domains is presented. The crystal structure and small-angle X-ray scattering solution studies of this endopeptidase revealed the presence of a homodimer. The structure of the two LysM domains co-crystallized with N-acetyl-chitohexaose revealed a new intermolecular binding mode that may explain the differential interaction between LysM domains and short or long chitin oligomers. By combining the structural information with the three-dimensional model of peptidoglycan, a model suggesting how protein dimerization enhances the recognition of peptidoglycan is proposed.

Mannose-Binding Lectin Binds to Amyloid Protein and Modulates Inflammation

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Mykol Larvie

2012-01-01

Full Text Available Mannose-binding lectin (MBL, a soluble factor of the innate immune system, is a pattern recognition molecule with a number of known ligands, including viruses, bacteria, and molecules from abnormal self tissues. In addition to its role in immunity, MBL also functions in the maintenance of tissue homeostasis. We present evidence here that MBL binds to amyloid β peptides. MBL binding to other known carbohydrate ligands is calcium-dependent and has been attributed to the carbohydrate-recognition domain, a common feature of other C-type lectins. In contrast, we find that the features of MBL binding to Aβ are more similar to the reported binding characteristics of the cysteine-rich domain of the unrelated mannose receptor and therefore may involve the MBL cysteine-rich domain. Differences in MBL ligand binding may contribute to modulation of inflammatory response and may correlate with the function of MBL in processes such as coagulation and tissue homeostasis.

Aspartic acid 397 in subunit B of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae forms part of a sodium-binding site, is involved in cation selectivity, and affects cation-binding site cooperativity.

Science.gov (United States)

Shea, Michael E; Juárez, Oscar; Cho, Jonathan; Barquera, Blanca

2013-10-25

The Na(+)-pumping NADH:quinone complex is found in Vibrio cholerae and other marine and pathogenic bacteria. NADH:ubiquinone oxidoreductase oxidizes NADH and reduces ubiquinone, using the free energy released by this reaction to pump sodium ions across the cell membrane. In a previous report, a conserved aspartic acid residue in the NqrB subunit at position 397, located in the cytosolic face of this protein, was proposed to be involved in the capture of sodium. Here, we studied the role of this residue through the characterization of mutant enzymes in which this aspartic acid was substituted by other residues that change charge and size, such as arginine, serine, lysine, glutamic acid, and cysteine. Our results indicate that NqrB-Asp-397 forms part of one of the at least two sodium-binding sites and that both size and charge at this position are critical for the function of the enzyme. Moreover, we demonstrate that this residue is involved in cation selectivity, has a critical role in the communication between sodium-binding sites, by promoting cooperativity, and controls the electron transfer step involved in sodium uptake (2Fe-2S → FMNC).

Stereochemistry of charged nitrogen-aromatic interactions and its involvement in ligand-receptor binding

Science.gov (United States)

Verdonk, Marcel L.; Boks, Gertjan J.; Kooijman, Huub; Kanters, Jan A.; Kroon, Jan

1993-04-01

Recently, new evidence was found for the involvement of charged nitrogen-aromatic interactions in ligand-receptor binding. In this study we report two favourable orientations of a phenyl ring with respect to a R-N+(CH3)3 group, based on crystal structure statistics from the Cambridge Structural Database. In the first orientation, the phenyl ring is situated in between the substituents at about 4.5 Å from the nitrogen atom, and the ring is approximately oriented on the sphere around the nitrogen atom. In the second orientation, the phenyl ring is situated in the same direction as one of the N-C bonds at about 6.0 Å from the nitrogen atom, and the ring is tilted with respect to the sphere around the nitrogen atom. The same two orientations were also found in the crystal structures of three ligand-receptor complexes, which implies that these orientations probably play a major role in molecular recognition mechanisms.

Predicting binding within disordered protein regions to structurally characterised peptide-binding domains.

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Waqasuddin Khan

Full Text Available Disordered regions of proteins often bind to structured domains, mediating interactions within and between proteins. However, it is difficult to identify a priori the short disordered regions involved in binding. We set out to determine if docking such peptide regions to peptide binding domains would assist in these predictions.We assembled a redundancy reduced dataset of SLiM (Short Linear Motif containing proteins from the ELM database. We selected 84 sequences which had an associated PDB structures showing the SLiM bound to a protein receptor, where the SLiM was found within a 50 residue region of the protein sequence which was predicted to be disordered. First, we investigated the Vina docking scores of overlapping tripeptides from the 50 residue SLiM containing disordered regions of the protein sequence to the corresponding PDB domain. We found only weak discrimination of docking scores between peptides involved in binding and adjacent non-binding peptides in this context (AUC 0.58.Next, we trained a bidirectional recurrent neural network (BRNN using as input the protein sequence, predicted secondary structure, Vina docking score and predicted disorder score. The results were very promising (AUC 0.72 showing that multiple sources of information can be combined to produce results which are clearly superior to any single source.We conclude that the Vina docking score alone has only modest power to define the location of a peptide within a larger protein region known to contain it. However, combining this information with other knowledge (using machine learning methods clearly improves the identification of peptide binding regions within a protein sequence. This approach combining docking with machine learning is primarily a predictor of binding to peptide-binding sites, and is not intended as a predictor of specificity of binding to particular receptors.

Mapping EBNA-1 Domains Involved in Binding to Metaphase Chromosomes

Science.gov (United States)

Marechal, Vincent; Dehee, Axelle; Chikhi-Brachet, Roxane; Piolot, Tristan; Coppey-Moisan, Maité; Nicolas, Jean-Claude

1999-01-01

The Epstein-Barr virus (EBV) genome can persist in dividing human B cells as multicopy circular episomes. Viral episomes replicate in synchrony with host cell DNA and are maintained at a relatively constant copy number for a long time. Only two viral elements, the replication origin OriP and the EBNA-1 protein, are required for the persistence of viral genomes during latency. EBNA-1 activates OriP during the S phase and may also contribute to the partition and/or retention of viral genomes during mitosis. Indeed, EBNA-1 has been shown to interact with mitotic chromatin. Moreover, viral genomes are noncovalently associated with metaphase chromosomes. This suggests that EBNA-1 may facilitate the anchorage of viral genomes on cellular chromosomes, thus ensuring proper partition and retention. In the present paper, we have investigated the chromosome-binding activity of EBV EBNA-1, herpesvirus papio (HVP) EBNA-1, and various derivatives of EBV EBNA-1, fused to a variant of the green fluorescent protein. The results show that binding to metaphase chromosomes is a common property of EBV and HVP EBNA-1. Further studies indicated that at least three independent domains (CBS-1, -2, and -3) mediate EBNA-1 binding to metaphase chromosomes. In agreement with the anchorage model, two of these domains mapped to a region that has been previously demonstrated to be required for the long-term persistence of OriP-containing plasmids. PMID:10196336

Identification and genetic characterization of unique HIV-1 A1/C recombinant strain in South Africa.

Science.gov (United States)

Musyoki, Andrew M; Rakgole, Johnny N; Selabe, Gloria; Mphahlele, Jeffrey

2015-03-01

HIV isolates from South Africa are predominantly subtype C. Sporadic isolation of non-C strains has been reported mainly in cosmopolitan cities. HIV isolate j51 was recovered from a rural South African heterosexual female aged 51 years. Near full length amplification of the genome was attempted using PCR with primers targeting overlapping segments of the HIV genome. Analysis of 5593 bp (gag to vpu) at a bootstrap value greater than 70% found that all but the vpu gene was HIV-1 subtype A1. The vpu gene was assigned HIV-1 subtype C. The recombination breaking point was estimated at position 6035+/- 15 bp with reference to the beginning of the HXB2 reference strain. Isolate j51 revealed a unique genome constellation to previously reported recombinant strains with parental A/C backbones from South Africa though a common recombination with subtype C within the vpu gene. Identification of recombinant strains supports continued surveillance of HIV genetic diversity.

The heparin-binding site in tetranectin is located in the N-terminal region and binding does not involve the carbohydrate recognition domain.

Science.gov (United States)

Lorentsen, R H; Graversen, J H; Caterer, N R; Thogersen, H C; Etzerodt, M

2000-04-01

Tetranectin is a homotrimeric plasma and extracellular-matrix protein that binds plasminogen and complex sulphated polysaccharides including heparin. In terms of primary and tertiary structure, tetranectin is related to the collectin family of Ca(2+)-binding C-type lectins. Tetranectin is encoded in three exons. Exon 3 encodes the carbohydrate recognition domain, which binds to kringle 4 in plasminogen at low levels of Ca(2+). Exon 2 encodes an alpha-helix, which is necessary and sufficient to govern the trimerization of tetranectin by assembling into a triple-helical coiled-coil structural element. Here we show that the heparin-binding site in tetranectin resides not in the carbohydrate recognition domain but within the N-terminal region, comprising the 16 amino acid residues encoded by exon 1. In particular, the lysine residues in the decapeptide segment KPKKIVNAKK (tetranectin residues 6-15) are shown to be of primary importance in heparin binding.

Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals a functional heparin binding motif involved in binding to the Duffy antigen receptor for chemokines

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Bolton Michael J

2011-11-01

Full Text Available Abstract Background The HIV surface glycoprotein gp120 (SU, gp120 and the Plasmodium vivax Duffy binding protein (PvDBP bind to chemokine receptors during infection and have a site of amino acid sequence similarity in their binding domains that often includes a heparin binding motif (HBM. Infection by either pathogen has been found to be inhibited by polyanions. Results Specific polyanions that inhibit HIV infection and bind to the V3 loop of X4 strains also inhibited DBP-mediated infection of erythrocytes and DBP binding to the Duffy Antigen Receptor for Chemokines (DARC. A peptide including the HBM of PvDBP had similar affinity for heparin as RANTES and V3 loop peptides, and could be specifically inhibited from heparin binding by the same polyanions that inhibit DBP binding to DARC. However, some V3 peptides can competitively inhibit RANTES binding to heparin, but not the PvDBP HBM peptide. Three other members of the DBP family have an HBM sequence that is necessary for erythrocyte binding, however only the protein which binds to DARC, the P. knowlesi alpha protein, is inhibited by heparin from binding to erythrocytes. Heparitinase digestion does not affect the binding of DBP to erythrocytes. Conclusion The HBMs of DBPs that bind to DARC have similar heparin binding affinities as some V3 loop peptides and chemokines, are responsible for specific sulfated polysaccharide inhibition of parasite binding and invasion of red blood cells, and are more likely to bind to negative charges on the receptor than cell surface glycosaminoglycans.

A tandem sequence motif acts as a distance-dependent enhancer in a set of genes involved in translation by binding the proteins NonO and SFPQ

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Roepcke Stefan

2011-12-01

Full Text Available Abstract Background Bioinformatic analyses of expression control sequences in promoters of co-expressed or functionally related genes enable the discovery of common regulatory sequence motifs that might be involved in co-ordinated gene expression. By studying promoter sequences of the human ribosomal protein genes we recently identified a novel highly specific Localized Tandem Sequence Motif (LTSM. In this work we sought to identify additional genes and LTSM-binding proteins to elucidate potential regulatory mechanisms. Results Genome-wide analyses allowed finding a considerable number of additional LTSM-positive genes, the products of which are involved in translation, among them, translation initiation and elongation factors, and 5S rRNA. Electromobility shift assays then showed specific signals demonstrating the binding of protein complexes to LTSM in ribosomal protein gene promoters. Pull-down assays with LTSM-containing oligonucleotides and subsequent mass spectrometric analysis identified the related multifunctional nucleotide binding proteins NonO and SFPQ in the binding complex. Functional characterization then revealed that LTSM enhances the transcriptional activity of the promoters in dependency of the distance from the transcription start site. Conclusions Our data demonstrate the power of bioinformatic analyses for the identification of biologically relevant sequence motifs. LTSM and the here found LTSM-binding proteins NonO and SFPQ were discovered through a synergistic combination of bioinformatic and biochemical methods and are regulators of the expression of a set of genes of the translational apparatus in a distance-dependent manner.

Ginseng gintonin activates the human cardiac delayed rectifier K+ channel: involvement of Ca2+/calmodulin binding sites.

Science.gov (United States)

Choi, Sun-Hye; Lee, Byung-Hwan; Kim, Hyeon-Joong; Jung, Seok-Won; Kim, Hyun-Sook; Shin, Ho-Chul; Lee, Jun-Hee; Kim, Hyoung-Chun; Rhim, Hyewhon; Hwang, Sung-Hee; Ha, Tal Soo; Kim, Hyun-Ji; Cho, Hana; Nah, Seung-Yeol

2014-09-01

Gintonin, a novel, ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand, elicits [Ca(2+)]i transients in neuronal and non-neuronal cells via pertussis toxin-sensitive and pertussis toxin-insensitive G proteins. The slowly activating delayed rectifier K(+) (I(Ks)) channel is a cardiac K(+) channel composed of KCNQ1 and KCNE1 subunits. The C terminus of the KCNQ1 channel protein has two calmodulin-binding sites that are involved in regulating I(Ks) channels. In this study, we investigated the molecular mechanisms of gintonin-mediated activation of human I(Ks) channel activity by expressing human I(Ks) channels in Xenopus oocytes. We found that gintonin enhances IKs channel currents in concentration- and voltage-dependent manners. The EC50 for the I(Ks) channel was 0.05 ± 0.01 μg/ml. Gintonin-mediated activation of the I(Ks) channels was blocked by an LPA1/3 receptor antagonist, an active phospholipase C inhibitor, an IP3 receptor antagonist, and the calcium chelator BAPTA. Gintonin-mediated activation of both the I(Ks) channel was also blocked by the calmodulin (CaM) blocker calmidazolium. Mutations in the KCNQ1 [Ca(2+)]i/CaM-binding IQ motif sites (S373P, W392R, or R539W)blocked the action of gintonin on I(Ks) channel. However, gintonin had no effect on hERG K(+) channel activity. These results show that gintonin-mediated enhancement of I(Ks) channel currents is achieved through binding of the [Ca(2+)]i/CaM complex to the C terminus of KCNQ1 subunit.

Arabidopsis IQM4, a Novel Calmodulin-Binding Protein, Is Involved With Seed Dormancy and Germination in Arabidopsis

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Yu Ping Zhou

2018-06-01

Full Text Available Seed dormancy and germination are regulated by complex mechanisms controlled by diverse hormones and environmental cues. Abscisic acid (ABA promotes seed dormancy and inhibits seed germination and post-germination growth. Calmodulin (CaM signals are involved with the inhibition of ABA during seed germination and seedling growth. In this study, we showed that Arabidopsis thaliana IQM4 could bind with calmodulin 5 (CaM5 both in vitro and in vivo, and that the interaction was the Ca2+-independent type. The IQM4 protein was localized in the chloroplast and the IQM4 gene was expressed in most tissues, especially the embryo and germinated seedlings. The T-DNA insertion mutants of IQM4 exhibited the reduced primary seed dormancy and lower ABA levels compared with wild type seeds. Moreover, IQM4 plays key roles in modulating the responses to ABA, salt, and osmotic stress during seed germination and post-germination growth. T-DNA insertion mutants exhibited ABA-insensitive and salt-hypersensitive phenotypes during seed germination and post-germination growth, whereas IQM4-overexpressing lines had ABA- and osmotic-hypersensitive, and salt-insensitive phenotypes. Gene expression analyses showed that mutation of IQM4 inhibited the expression of ABA biosynthetic genes NCED6 and NCED9, and seed maturation regulators LEC1, LEC2, ABI3, and ABI5 during the silique development, as well as promoted the expression of WRKY40 and inhibited that of ABI5 in ABA-regulated seed germination. These observations suggest that IQM4 is a novel Ca2+-independent CaM-binding protein, which is positively involved with seed dormancy and germination in Arabidopsis.

A highly Conserved Aspartic Acid Residue of the Chitosanase from Bacillus Sp. TS Is Involved in the Substrate Binding.

Science.gov (United States)

Zhou, Zhanping; Zhao, Shuangzhi; Liu, Yang; Chang, Zhengying; Ma, Yanhe; Li, Jian; Song, Jiangning

2016-11-01

The chitosanase from Bacillus sp. TS (CsnTS) is an enzyme belonging to the glycoside hydrolase family 8. The sequence of CsnTS shares 98 % identity with the chitosanase from Bacillus sp. K17. Crystallography analysis and site-direct mutagenesis of the chitosanase from Bacillus sp. K17 identified the important residues involved in the catalytic interaction and substrate binding. However, despite progress in understanding the catalytic mechanism of the chitosanase from the family GH8, the functional roles of some residues that are highly conserved throughout this family have not been fully elucidated. This study focused on one of these residues, i.e., the aspartic acid residue at position 318. We found that apart from asparagine, mutation of Asp318 resulted in significant loss of enzyme activity. In-depth investigations showed that mutation of this residue not only impaired enzymatic activity but also affected substrate binding. Taken together, our results showed that Asp318 plays an important role in CsnTS activity.

Interaction of the amyloid precursor protein-like protein 1 (APLP1) E2 domain with heparan sulfate involves two distinct binding modes

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Dahms, Sven O., E-mail: sdahms@fli-leibniz.de [Leibniz Institute for Age Research (FLI), Beutenbergstrasse 11, 07745 Jena (Germany); Mayer, Magnus C. [Freie Universität Berlin, Thielallee 63, 14195 Berlin (Germany); Miltenyi Biotec GmbH, Robert-Koch-Strasse 1, 17166 Teterow (Germany); Roeser, Dirk [Leibniz Institute for Age Research (FLI), Beutenbergstrasse 11, 07745 Jena (Germany); Multhaup, Gerd [McGill University Montreal, Montreal, Quebec H3G 1Y6 (Canada); Than, Manuel E., E-mail: sdahms@fli-leibniz.de [Leibniz Institute for Age Research (FLI), Beutenbergstrasse 11, 07745 Jena (Germany)

2015-03-01

Two X-ray structures of APLP1 E2 with and without a heparin dodecasaccharide are presented, revealing two distinct binding modes of the protein to heparan sulfate. The data provide a mechanistic explanation of how APP-like proteins bind to heparan sulfates and how they specifically recognize nonreducing structures of heparan sulfates. Beyond the pathology of Alzheimer’s disease, the members of the amyloid precursor protein (APP) family are essential for neuronal development and cell homeostasis in mammals. APP and its paralogues APP-like protein 1 (APLP1) and APP-like protein 2 (APLP2) contain the highly conserved heparan sulfate (HS) binding domain E2, which effects various (patho)physiological functions. Here, two crystal structures of the E2 domain of APLP1 are presented in the apo form and in complex with a heparin dodecasaccharide at 2.5 Å resolution. The apo structure of APLP1 E2 revealed an unfolded and hence flexible N-terminal helix αA. The (APLP1 E2){sub 2}–(heparin){sub 2} complex structure revealed two distinct binding modes, with APLP1 E2 explicitly recognizing the heparin terminus but also interacting with a continuous heparin chain. The latter only requires a certain register of the sugar moieties that fits to a positively charged surface patch and contributes to the general heparin-binding capability of APP-family proteins. Terminal binding of APLP1 E2 to heparin specifically involves a structure of the nonreducing end that is very similar to heparanase-processed HS chains. These data reveal a conserved mechanism for the binding of APP-family proteins to HS and imply a specific regulatory role of HS modifications in the biology of APP and APP-like proteins.

Modification of opiate agonist binding by pertussis toxin

Energy Technology Data Exchange (ETDEWEB)

Abood, M.E.; Lee, N.M.; Loh, H.H.

1986-03-05

Opiate agonist binding is decreased by GTP, suggesting the possible involvement of GTP binding proteins in regulation of opiate receptor binding. This possibility was addressed by asking whether pertussis toxin treatment, which results in ADP-ribosylation and modification of G proteins, would alter opiate agonist binding. The striatum was chosen for the initial brain area to be studied, since regulation of opiate action in this area had been shown to be modified by pertussis toxin. Treatment of striatal membranes with pertussis toxin results in up to a 55% decrease in /sup 3/(H)-DADLE binding as compared with membranes treated identically without toxin. This corresponds to a near complete ADP-ribosylation of both G proteins in the striatal membrane. The decrease in agonist binding appears to be due to an altered affinity of the receptor for agonist as opposed to a decrease in the number of sites. This effect of pertussis toxin on opiate agonist binding demonstrates the actual involvement of G proteins in regulation of opiate receptor binding.

Modification of opiate agonist binding by pertussis toxin

International Nuclear Information System (INIS)

Abood, M.E.; Lee, N.M.; Loh, H.H.

1986-01-01

Opiate agonist binding is decreased by GTP, suggesting the possible involvement of GTP binding proteins in regulation of opiate receptor binding. This possibility was addressed by asking whether pertussis toxin treatment, which results in ADP-ribosylation and modification of G proteins, would alter opiate agonist binding. The striatum was chosen for the initial brain area to be studied, since regulation of opiate action in this area had been shown to be modified by pertussis toxin. Treatment of striatal membranes with pertussis toxin results in up to a 55% decrease in 3 (H)-DADLE binding as compared with membranes treated identically without toxin. This corresponds to a near complete ADP-ribosylation of both G proteins in the striatal membrane. The decrease in agonist binding appears to be due to an altered affinity of the receptor for agonist as opposed to a decrease in the number of sites. This effect of pertussis toxin on opiate agonist binding demonstrates the actual involvement of G proteins in regulation of opiate receptor binding

Pictorial binding: endeavor to classify

Directory of Open Access Journals (Sweden)

Zinchenko S.

2015-01-01

Full Text Available The article is devoted to the classification of bindings of the 1-19th centuries with a unique and untypical book binding decoration technique (encaustic, tempera and oil paintings. Analysis of design features, materials and techniques of art decoration made it possible to identify them as a separate type - pictorial bindings and divide them into four groups. The first group consists of Coptic bindings, decorated with icon-painting images in encaustic technique. The second group is made up of leather Western bindings of the 13-14th centuries, which have the decoration and technique of ornamentation close to iconography. The third group involves parchment bindings, ornamentation technique of which is closer to the miniature. The last group comprises bindings of East Slavic origin of the 15-19th centuries, decorated with icon-painting pictures made in the technique of tempera or oil painting. The proposed classification requires further basic research as several specific kinds of bindings have not yet been investigated

A New Metal Binding Domain Involved in Cadmium, Cobalt and Zinc Transport

Energy Technology Data Exchange (ETDEWEB)

Smith, Aaron T. [Northwestern Univ., Evanston, IL (United States); Barupala, Dulmini [Wayne State Univ., Detroit, MI (United States); Stemmler, Timothy L. [Wayne State Univ., Detroit, MI (United States); Rosenzweig, Amy C. [Northwestern Univ., Evanston, IL (United States)

2015-07-20

In the P1B-ATPases, which couple cation transport across membranes to ATP hydrolysis, are central to metal homeostasis in all organisms. An important feature of P1B-ATPases is the presence of soluble metal binding domains (MBDs) that regulate transport activity. Only one type of MBD has been characterized extensively, but bioinformatics analyses indicate that a diversity of MBDs may exist in nature. Here we report the biochemical, structural and functional characterization of a new MBD from the Cupriavidus metallidurans P_1B-4-ATPase CzcP (CzcP MBD). The CzcP MBD binds two Cd²⁺, Co²⁺ or Zn²⁺ ions in distinct and unique sites and adopts an unexpected fold consisting of two fused ferredoxin-like domains. Both in vitro and in vivo activity assays using full-length CzcP, truncated CzcP and several variants indicate a regulatory role for the MBD and distinct functions for the two metal binding sites. Moreover, these findings elucidate a previously unknown MBD and suggest new regulatory mechanisms for metal transport by P_1B-ATPases.

Computational analysis of protein-protein interfaces involving an alpha helix: insights for terphenyl-like molecules binding.

Science.gov (United States)

Isvoran, Adriana; Craciun, Dana; Martiny, Virginie; Sperandio, Olivier; Miteva, Maria A

2013-06-14

Protein-Protein Interactions (PPIs) are key for many cellular processes. The characterization of PPI interfaces and the prediction of putative ligand binding sites and hot spot residues are essential to design efficient small-molecule modulators of PPI. Terphenyl and its derivatives are small organic molecules known to mimic one face of protein-binding alpha-helical peptides. In this work we focus on several PPIs mediated by alpha-helical peptides. We performed computational sequence- and structure-based analyses in order to evaluate several key physicochemical and surface properties of proteins known to interact with alpha-helical peptides and/or terphenyl and its derivatives. Sequence-based analysis revealed low sequence identity between some of the analyzed proteins binding alpha-helical peptides. Structure-based analysis was performed to calculate the volume, the fractal dimension roughness and the hydrophobicity of the binding regions. Besides the overall hydrophobic character of the binding pockets, some specificities were detected. We showed that the hydrophobicity is not uniformly distributed in different alpha-helix binding pockets that can help to identify key hydrophobic hot spots. The presence of hydrophobic cavities at the protein surface with a more complex shape than the entire protein surface seems to be an important property related to the ability of proteins to bind alpha-helical peptides and low molecular weight mimetics. Characterization of similarities and specificities of PPI binding sites can be helpful for further development of small molecules targeting alpha-helix binding proteins.

A sequential binding mechanism in a PDZ domain

DEFF Research Database (Denmark)

Chi, Celestine N; Bach, Anders; Engström, Åke

2009-01-01

that ligand binding involves at least a two-step process. By using an ultrarapid continuous-flow mixer, we then detected a hyperbolic dependence of binding rate constants on peptide concentration, corroborating the two-step binding mechanism. Furthermore, we found a similar dependence of the rate constants...

Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals a functional heparin binding motif involved in binding to the Duffy antigen receptor for chemokines

OpenAIRE

Bolton, Michael J; Garry, Robert F

2011-01-01

Abstract Background The HIV surface glycoprotein gp120 (SU, gp120) and the Plasmodium vivax Duffy binding protein (PvDBP) bind to chemokine receptors during infection and have a site of amino acid sequence similarity in their binding domains that often includes a heparin binding motif (HBM). Infection by either pathogen has been found to be inhibited by polyanions. Results Specific polyanions that inhibit HIV infection and bind to the V3 loop of X4 strains also inhibited DBP-mediated infectio...

APC/C-mediated degradation of dsRNA-binding protein 4 (DRB4 involved in RNA silencing.

Directory of Open Access Journals (Sweden)

Katia Marrocco

Full Text Available Selective protein degradation via the ubiquitin-26S proteasome is a major mechanism underlying DNA replication and cell division in all Eukaryotes. In particular, the APC/C (Anaphase Promoting Complex or Cyclosome is a master ubiquitin protein ligase (E3 that targets regulatory proteins for degradation allowing sister chromatid separation and exit from mitosis. Interestingly, recent work also indicates that the APC/C remains active in differentiated animal and plant cells. However, its role in post-mitotic cells remains elusive and only a few substrates have been characterized.In order to identify novel APC/C substrates, we performed a yeast two-hybrid screen using as the bait Arabidopsis APC10/DOC1, one core subunit of the APC/C, which is required for substrate recruitment. This screen identified DRB4, a double-stranded RNA binding protein involved in the biogenesis of different classes of small RNA (sRNA. This protein interaction was further confirmed in vitro and in plant cells. Moreover, APC10 interacts with DRB4 through the second dsRNA binding motif (dsRBD2 of DRB4, which is also required for its homodimerization and binding to its Dicer partner DCL4. We further showed that DRB4 protein accumulates when the proteasome is inactivated and, most importantly, we found that DRB4 stability depends on APC/C activity. Hence, depletion of Arabidopsis APC/C activity by RNAi leads to a strong accumulation of endogenous DRB4, far beyond its normal level of accumulation. However, we could not detect any defects in sRNA production in lines where DRB4 was overexpressed.Our work identified a first plant substrate of the APC/C, which is not a regulator of the cell cycle. Though we cannot exclude that APC/C-dependent degradation of DRB4 has some regulatory roles under specific growth conditions, our work rather points to a housekeeping function of APC/C in maintaining precise cellular-protein concentrations and homeostasis of DRB4.

The binding cavity of mouse major urinary protein is optimised for a variety of ligand binding modes

Energy Technology Data Exchange (ETDEWEB)

Pertinhez, Thelma A.; Ferrari, Elena; Casali, Emanuela [Department of Experimental Medicine, University of Parma, Via Volturno, 39, 43100 Parma (Italy); Patel, Jital A. [Department of Chemistry, University of Oxford, Inorganic Chemistry Laboratory, South Parks Road, Oxford OX1 3QR (United Kingdom); Spisni, Alberto, E-mail: alberto.spisni@unipr.it [Department of Experimental Medicine, University of Parma, Via Volturno, 39, 43100 Parma (Italy); Smith, Lorna J., E-mail: lorna.smith@chem.ox.ac.uk [Department of Chemistry, University of Oxford, Inorganic Chemistry Laboratory, South Parks Road, Oxford OX1 3QR (United Kingdom)

2009-12-25

{sup 15}N and {sup 1}HN chemical shift data and {sup 15}N relaxation studies have been used to characterise the binding of N-phenyl-naphthylamine (NPN) to mouse major urinary protein (MUP). NPN binds in the {beta}-barrel cavity of MUP, hydrogen bonding to Tyr120 and making extensive non-bonded contacts with hydrophobic side chains. In contrast to the natural pheromone 2-sec-butyl-4,5-dihydrothiazole, NPN binding gives no change to the overall mobility of the protein backbone of MUP. Comparison with 11 different ligands that bind to MUP shows a range of binding modes involving 16 different residues in the {beta}-barrel cavity. These finding justify why MUP is able to adapt to allow for many successful binding partners.

A Venom Gland Extracellular Chitin-Binding-Like Protein from Pupal Endoparasitoid Wasps, Pteromalus Puparum, Selectively Binds Chitin

Science.gov (United States)

Chitin-binding proteins (CBPs) existed in various species and involved in different biology processes. In the present study, we cloned a full length cDNA of chitin-binding protein-like (PpCBP-like) from Pteromalus puparum, a pupal endoparasitoid of Pieris rapae. PpCBP-like encoded a 96 putative amin...

Different mechanisms are involved in the antibody mediated inhibition of ligand binding to the urokinase receptor

DEFF Research Database (Denmark)

List, K; Høyer-Hansen, G; Rønne, E

1999-01-01

Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance or interfer......Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance......) can be employed as a highly useful tool to characterize the inhibitory mechanism of specific antagonist antibodies. Two inhibitory antibodies against uPAR, mAb R3 and mAb R5, were shown to exhibit competitive and non-competitive inhibition, respectively, of ligand binding to the receptor. The former...

From simple receptors to complex multimodal percepts: a first global picture on the mechanisms involved in perceptual binding.

Science.gov (United States)

Velik, Rosemarie

2012-01-01

The binding problem in perception is concerned with answering the question how information from millions of sensory receptors, processed by millions of neurons working in parallel, can be merged into a unified percept. Binding in perception reaches from the lowest levels of feature binding up to the levels of multimodal binding of information coming from the different sensor modalities and also from other functional systems. The last 40 years of research have shown that the binding problem cannot be solved easily. Today, it is considered as one of the key questions to brain understanding. To date, various solutions have been suggested to the binding problem including: (1) combination coding, (2) binding by synchrony, (3) population coding, (4) binding by attention, (5) binding by knowledge, expectation, and memory, (6) hardwired vs. on-demand binding, (7) bundling and binding of features, (8) the feature-integration theory of attention, and (9) synchronization through top-down processes. Each of those hypotheses addresses important aspects of binding. However, each of them also suffers from certain weak points and can never give a complete explanation. This article gives a brief overview of the so far suggested solutions of perceptual binding and then shows that those are actually not mutually exclusive but can complement each other. A computationally verified model is presented which shows that, most likely, the different described mechanisms of binding act (1) at different hierarchical levels and (2) in different stages of "perceptual knowledge acquisition." The model furthermore considers and explains a number of inhibitory "filter mechanisms" that suppress the activation of inappropriate or currently irrelevant information.

From simple receptors to complex multimodal percepts: A first global picture on the mechanisms involved in perceptual binding

Directory of Open Access Journals (Sweden)

Rosemarie eVelik

2012-07-01

Full Text Available The binding problem in perception is concerned with answering the question how information from millions of sensory receptors, processed by millions of neurons working in parallel, can be merged into a unified percept. Binding in perception reaches from the lowest levels of feature binding up to the levels of multimodal binding of information coming from the different sensor modalities and also from other functional systems. The last 40 years of research have shown that the binding problem cannot be solved easily. Today, it is considered as one of the key questions to brain understanding. To date, various solutions have been suggested to the binding problem including: (1 combination coding, (2 binding by synchrony, (3 population coding, (4 binding by attention, (5 binding by knowledge, expectation, and memory, (6 hardwired versus on-demand binding, (7 bundling and binding of features, (8 the feature-integration theory of attention, (9 synchronization through top-down processes. Each of those hypotheses addresses important aspects of binding. However, each of them also suffers from certain weak points and can never give a complete explanation. This article gives a brief overview of the so far suggested solutions of perceptual binding and then shows that those are actually not mutually exclusive but can complement each other. A computationally verified model is presented which shows that, most likely, the different described mechanisms of binding act (1 at different hierarchical levels and (2 in different stages of perceptual knowledge acquisition. The model furthermore considers and explains a number of inhibitory filter mechanisms that suppress the activation of inappropriate or currently irrelevant information.

Binding of ethidium to the nucleosome core particle. 2. Internal and external binding modes

International Nuclear Information System (INIS)

McMurray, C.T.; Small, E.W.; van Holde, K.E.

1991-01-01

The authors have previously reported that the binding of ethidium bromide to the nucleosome core particle results in a stepwise dissociation of the structure which involves the initial release of one copy each of H2A and H2B. In this report, they have examined the absorbance and fluorescence properties of intercalated and outside bound forms of ethidium bromide. From these properties, they have measured the extent of external, electrostatic binding of the dye versus internal, intercalation binding to the core particle, free from contribution by linker DNA. They have established that dissociation is induced by the intercalation mode of binding to DNA within the core particle DNA, and not by binding to the histones or by nonintercalative binding to DNA. The covalent binding of [ 3 H]-8-azidoethidium to the core particle clearly shows that < 1.0 adduct is formed per histone octamer over a wide range of input ratios. Simultaneously, analyses of steady-state fluorescence enhancement and fluorescence lifetime data from bound ethidium complexes demonstrate extensive intercalation binding. Combined analyses from steady-state fluorescence intensity with equilibrium dialysis or fluorescence lifetime data revealed that dissociation began when ∼14 ethidium molecules are bound by intercalation to each core particle and < 1.0 nonintercalated ion pair was formed per core particle

Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site.

Science.gov (United States)

Strauss, Mike; Schotte, Lise; Thys, Bert; Filman, David J; Hogle, James M

2016-01-13

Nanobodies, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In this study, we present high-resolution cryo-electron microscopy reconstructions of poliovirus with five neutralizing VHHs. All VHHs bind the capsid in the canyon at sites that extensively overlap the poliovirus receptor-binding site. In contrast, the interaction involves a unique (and surprisingly extensive) surface for each of the five VHHs. Five regions of the capsid were found to participate in binding with all five VHHs. Four of these five regions are known to alter during the expansion of the capsid associated with viral entry. Interestingly, binding of one of the VHHs, PVSS21E, resulted in significant changes of the capsid structure and thus seems to trap the virus in an early stage of expansion. We describe the cryo-electron microscopy structures of complexes of five neutralizing VHHs with the Mahoney strain of type 1 poliovirus at resolutions ranging from 3.8 to 6.3Å. All five VHHs bind deep in the virus canyon at similar sites that overlap extensively with the binding site for the receptor (CD155). The binding surfaces on the VHHs are surprisingly extensive, but despite the use of similar binding surfaces on the virus, the binding surface on the VHHs is unique for each VHH. In four of the five complexes, the virus remains essentially unchanged, but for the fifth there are significant changes reminiscent of but smaller in magnitude than the changes associated with cell entry, suggesting that this VHH traps the virus in a previously undescribed early intermediate state. The neutralizing mechanisms of the VHHs and their potential use as quality control agents for the end game of poliovirus eradication are discussed. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Further biochemical characterization of Mycobacterium leprae laminin-binding proteins

Directory of Open Access Journals (Sweden)

M.A.M. Marques

2001-04-01

Full Text Available It has been demonstrated that the alpha2 chain of laminin-2 present on the surface of Schwann cells is involved in the process of attachment of Mycobacterium leprae to these cells. Searching for M. leprae laminin-binding molecules, in a previous study we isolated and characterized the cationic proteins histone-like protein (Hlp and ribosomal proteins S4 and S5 as potential adhesins involved in M. leprae-Schwann cell interaction. Hlp was shown to bind alpha2-laminins and to greatly enhance the attachment of mycobacteria to ST88-14 Schwann cells. In the present study, we investigated the laminin-binding capacity of the ribosomal proteins S4 and S5. The genes coding for these proteins were PCR amplified and their recombinant products were shown to bind alpha2-laminins in overlay assays. However, when tested in ELISA-based assays and in adhesion assays with ST88-14 cells, in contrast to Hlp, S4 and S5 failed to bind laminin and act as adhesins. The laminin-binding property and adhesin capacity of two basic host-derived proteins were also tested, and only histones, but not cytochrome c, were able to increase bacterial attachment to ST88-14 cells. Our data suggest that the alanine/lysine-rich sequences shared by Hlp and eukaryotic H1 histones might be involved in the binding of these cationic proteins to laminin.

Semi-empirical quantum evaluation of peptide - MHC class II binding

Science.gov (United States)

González, Ronald; Suárez, Carlos F.; Bohórquez, Hugo J.; Patarroyo, Manuel A.; Patarroyo, Manuel E.

2017-01-01

Peptide presentation by the major histocompatibility complex (MHC) is a key process for triggering a specific immune response. Studying peptide-MHC (pMHC) binding from a structural-based approach has potential for reducing the costs of investigation into vaccine development. This study involved using two semi-empirical quantum chemistry methods (PM7 and FMO-DFTB) for computing the binding energies of peptides bonded to HLA-DR1 and HLA-DR2. We found that key stabilising water molecules involved in the peptide binding mechanism were required for finding high correlation with IC50 experimental values. Our proposal is computationally non-intensive, and is a reliable alternative for studying pMHC binding interactions.

Mannan-Binding Lectin Is Involved in the Protection against Renal Ischemia/Reperfusion Injury by Dietary Restriction.

Directory of Open Access Journals (Sweden)

Shushimita Shushimita

Full Text Available Preoperative fasting and dietary restriction offer robust protection against renal ischemia/reperfusion injury (I/RI in mice. We recently showed that Mannan-binding lectin (MBL, the initiator of the lectin pathway of complement activation, plays a pivotal role in renal I/RI. Based on these findings, we investigated the effect of short-term DR (30% reduction of total food intake or three days of water only fasting on MBL in 10-12 weeks old male C57/Bl6 mice. Both dietary regimens significantly reduce the circulating levels of MBL as well as its mRNA expression in liver, the sole production site of MBL. Reconstitution of MBL abolished the protection afforded by dietary restriction, whereas in the fasting group the protection persisted. These data show that modulation of MBL is involved in the protection against renal I/RI induced by dietary restriction, and suggest that the mechanisms of protection induced by dietary restriction and fasting may be different.

ThrR, a DNA-binding transcription factor involved in controlling threonine biosynthesis in Bacillus subtilis.

Science.gov (United States)

Rosenberg, Jonathan; Müller, Peter; Lentes, Sabine; Thiele, Martin J; Zeigler, Daniel R; Tödter, Dominik; Paulus, Henry; Brantl, Sabine; Stülke, Jörg; Commichau, Fabian M

2016-09-01

The threonine dehydratase IlvA is part of the isoleucine biosynthesis pathway in the Gram-positive model bacterium Bacillus subtilis. Consequently, deletion of ilvA causes isoleucine auxotrophy. It has been reported that ilvA pseudo-revertants having a derepressed hom-thrCB operon appear in the presence of threonine. Here we have characterized two classes of ilvA pseudo-revertants. In the first class the hom-thrCB operon was derepressed unmasking the threonine dehydratase activity of the threonine synthase ThrC. In the second class of mutants, threonine biosynthesis was more broadly affected. The first class of ilvA pseudo-revertants had a mutation in the Phom promoter (P*hom ), resulting in constitutive expression of the hom-thrCB operon. In the second class of ilvA pseudo-revertants, the thrR gene encoding a putative DNA-binding protein was inactivated, also resulting in constitutive expression of the hom-thrCB operon. Here we demonstrate that ThrR is indeed a DNA-binding transcription factor that regulates the hom-thrCB operon and the thrD aspartokinase gene. DNA binding assays uncovered the DNA-binding site of ThrR and revealed that the repressor competes with the RNA polymerase for DNA binding. This study also revealed that ThrR orthologs are ubiquitous in genomes from the Gram-positive phylum Firmicutes and in some Gram-negative bacteria. © 2016 John Wiley & Sons Ltd.

Singular Value Decomposition and Ligand Binding Analysis

Directory of Open Access Journals (Sweden)

André Luiz Galo

2013-01-01

Full Text Available Singular values decomposition (SVD is one of the most important computations in linear algebra because of its vast application for data analysis. It is particularly useful for resolving problems involving least-squares minimization, the determination of matrix rank, and the solution of certain problems involving Euclidean norms. Such problems arise in the spectral analysis of ligand binding to macromolecule. Here, we present a spectral data analysis method using SVD (SVD analysis and nonlinear fitting to determine the binding characteristics of intercalating drugs to DNA. This methodology reduces noise and identifies distinct spectral species similar to traditional principal component analysis as well as fitting nonlinear binding parameters. We applied SVD analysis to investigate the interaction of actinomycin D and daunomycin with native DNA. This methodology does not require prior knowledge of ligand molar extinction coefficients (free and bound, which potentially limits binding analysis. Data are acquired simply by reconstructing the experimental data and by adjusting the product of deconvoluted matrices and the matrix of model coefficients determined by the Scatchard and McGee and von Hippel equation.

Molecular switches for pheromone release from a moth pheromone-binding protein

International Nuclear Information System (INIS)

Xu Wei; Leal, Walter S.

2008-01-01

Pheromone-binding proteins (PBPs) are involved in the uptake of pheromones from pores on the antennae, transport through an aqueous environment surrounding the olfactory receptor neurons, and fast delivery to pheromone receptors. We tested the hypothesis that a C-terminal segment and a flexible loop are involved in the release of pheromones to membrane-bound receptors. We expressed in Escherichia coli 11 mutants of the PBP from the silkworm moth, BmorPBP, taking into consideration structural differences between the forms with high and low binding affinity. The N-terminus was truncated and His-69, His-70 and His-95 at the base of a flexible loop, and a cluster of acidic residues at the C-terminus were mutated. Binding assays and circular dichroism analyses support a mechanism involving protonation of acidic residues Asp-132 and Glu-141 at the C-terminus and histidines, His-70 and His-95, in the base of a loop covering the binding pocket. The former leads to the formation of a new α-helix, which competes with pheromone for the binding pocket, whereas positive charge repulsion of the histidines opens the opposite side of the binding pocket

The Fragile X Protein binds mRNAs involved in cancer progression and modulates metastasis formation

Science.gov (United States)

Lucá, Rossella; Averna, Michele; Zalfa, Francesca; Vecchi, Manuela; Bianchi, Fabrizio; Fata, Giorgio La; Del Nonno, Franca; Nardacci, Roberta; Bianchi, Marco; Nuciforo, Paolo; Munck, Sebastian; Parrella, Paola; Moura, Rute; Signori, Emanuela; Alston, Robert; Kuchnio, Anna; Farace, Maria Giulia; Fazio, Vito Michele; Piacentini, Mauro; De Strooper, Bart; Achsel, Tilmann; Neri, Giovanni; Neven, Patrick; Evans, D Gareth; Carmeliet, Peter; Mazzone, Massimiliano; Bagni, Claudia

2013-01-01

The role of the fragile X mental retardation protein (FMRP) is well established in brain, where its absence leads to the fragile X syndrome (FXS). FMRP is almost ubiquitously expressed, suggesting that, in addition to its effects in brain, it may have fundamental roles in other organs. There is evidence that FMRP expression can be linked to cancer. FMR1 mRNA, encoding FMRP, is overexpressed in hepatocellular carcinoma cells. A decreased risk of cancer has been reported in patients with FXS while a patient-case with FXS showed an unusual decrease of tumour brain invasiveness. However, a role for FMRP in regulating cancer biology, if any, remains unknown. We show here that FMRP and FMR1 mRNA levels correlate with prognostic indicators of aggressive breast cancer, lung metastases probability and triple negative breast cancer (TNBC). We establish that FMRP overexpression in murine breast primary tumours enhances lung metastasis while its reduction has the opposite effect regulating cell spreading and invasion. FMRP binds mRNAs involved in epithelial mesenchymal transition (EMT) and invasion including E-cadherin and Vimentin mRNAs, hallmarks of EMT and cancer progression. PMID:24092663

Structural and mutational analyses of the receptor binding domain of botulinum D/C mosaic neurotoxin: Insight into the ganglioside binding mechanism

Energy Technology Data Exchange (ETDEWEB)

Nuemket, Nipawan [Graduate School of Life Sciences, Hokkaido University, Sapporo 060-0810 (Japan); Tanaka, Yoshikazu [Creative Research Institution ' Sousei,' Hokkaido University, Sapporo 001-0021 (Japan); Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810 (Japan); Tsukamoto, Kentaro; Tsuji, Takao [Department of Microbiology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192 (Japan); Nakamura, Keiji; Kozaki, Shunji [Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka 598-8531 (Japan); Yao, Min [Graduate School of Life Sciences, Hokkaido University, Sapporo 060-0810 (Japan); Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810 (Japan); Tanaka, Isao, E-mail: tanaka@castor.sci.hokudai.ac.jp [Graduate School of Life Sciences, Hokkaido University, Sapporo 060-0810 (Japan); Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810 (Japan)

2011-07-29

Highlights: {yields} We determined the crystal structure of the receptor binding domain of BoNT in complex with 3'-sialyllactose. {yields} An electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. {yields} Alanine site-directed mutagenesis showed that GBS and GBL are important for ganglioside binding. {yields} A cell binding mechanism, which involves cooperative contribution of two sites, was proposed. -- Abstract: Clostridium botulinum type D strain OFD05, which produces the D/C mosaic neurotoxin, was isolated from cattle killed by the recent botulism outbreak in Japan. The D/C mosaic neurotoxin is the most toxic of the botulinum neurotoxins (BoNT) characterized to date. Here, we determined the crystal structure of the receptor binding domain of BoNT from strain OFD05 in complex with 3'-sialyllactose at a resolution of 3.0 A. In the structure, an electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. Alanine site-directed mutagenesis showed the significant contribution of the residues surrounding the cleft to ganglioside recognition. In addition, a loop adjoining the cleft also plays an important role in ganglioside recognition. In contrast, little effect was observed when the residues located around the surface previously identified as the protein receptor binding site in other BoNTs were substituted. The results of cell binding analysis of the mutants were significantly correlated with the ganglioside binding properties. Based on these observations, a cell binding mechanism of BoNT from strain OFD05 is proposed, which involves cooperative contribution of two ganglioside binding sites.

Neural correlates of shape-color binding in visual working memory.

Science.gov (United States)

Parra, Mario A; Della Sala, Sergio; Logie, Robert H; Morcom, Alexa M

2014-01-01

The present study addressed an outstanding issue regarding feature binding in working memory (WM): whether this function engages specific resources relative to those required to process individual features. We investigated the brain regions supporting the encoding and maintenance of features and bindings in a change detection task, in which 22 healthy young volunteers remembered visual arrays of abstract shapes, colors or shape-color bindings while undergoing fMRI. After an unfilled delay they saw a second array and judged whether the features or combination of features presented across the two arrays were the same or different. Temporary retention of feature bindings was found to involve additional cortical regions compared with retaining single features, regardless of whether the number of objects or the number of features differed between feature-only and binding conditions. This binding-specific activation is consistent with the involvement of different neural generators that collectively support visual temporary memory for features and for feature bindings. Regions within the parietal, temporal and occipital cortex, but not within the prefrontal cortex or the medial temporal lobe, appear to support the integrated object binding function investigated in this study. Our findings suggest that both individual features and their binding within integrated objects are used to represent complex objects in WM. © 2013 Elsevier Ltd. All rights reserved.

Disruption of visual feature binding in working memory.

Science.gov (United States)

Ueno, Taiji; Allen, Richard J; Baddeley, Alan D; Hitch, Graham J; Saito, Satoru

2011-01-01

In a series of five experiments, we studied the effect of a visual suffix on the retention in short-term visual memory of both individual visual features and objects involving the binding of two features. Experiments 1A, 1B, and 2 involved suffixes consisting of features external to the to-be-remembered set and revealed a modest but equivalent disruption on individual and bound feature conditions. Experiments 3A and 3B involved suffixes comprising features that could potentially have formed part of the to-be-remembered set (but did not on that trial). Both experiments showed greater disruption of retention for objects comprising bound features than for their individual features. The results are interpreted as differentiating two components of suffix interference, one affecting memory for features and bindings equally, the other affecting memory for bindings. The general component is tentatively identified with the attentional cost of operating a filter to prevent the suffix from entering visual working memory, whereas the specific component is attributed to the particular fragility of bound representations when the filter fails.

Characterization of the receptor-binding domain of Ebola glycoprotein in viral entry.

Science.gov (United States)

Wang, Jizhen; Manicassamy, Balaji; Caffrey, Michael; Rong, Lijun

2011-06-01

Ebola virus infection causes severe hemorrhagic fever in human and non-human primates with high mortality. Viral entry/infection is initiated by binding of glycoprotein GP protein on Ebola virion to host cells, followed by fusion of virus-cell membrane also mediated by GP. Using an human immunodeficiency virus (HIV)-based pseudotyping system, the roles of 41 Ebola GP1 residues in the receptor-binding domain in viral entry were studied by alanine scanning substitutions. We identified that four residues appear to be involved in protein folding/structure and four residues are important for viral entry. An improved entry interference assay was developed and used to study the role of these residues that are important for viral entry. It was found that R64 and K95 are involved in receptor binding. In contrast, some residues such as I170 are important for viral entry, but do not play a major role in receptor binding as indicated by entry interference assay and/or protein binding data, suggesting that these residues are involved in post-binding steps of viral entry. Furthermore, our results also suggested that Ebola and Marburg viruses share a common cellular molecule for entry.

Pressure locking and thermal binding of gate valves

Energy Technology Data Exchange (ETDEWEB)

Kelly, E.M.

1996-12-01

Pressure locking and thermal binding represent potential common mode failure mechanisms that can cause safety-related power-operated gate valves to fail in the closed position, thus rendering redundant safety-related systems incapable of performing their safety functions. Supplement 6 to Generic Letter 89-10, {open_quotes}Safety-Related Motor-Operated Gate Valve Testing and Surveillance,{close_quotes} provided an acceptable approach to addressing pressure locking and thermal binding of gate valves. More recently, the NRC has issued Generic Letter 95-07, {open_quotes}Pressure Locking and Thermal Binding of Safety-Related Power-Operated Gate Valves,{close_quotes} to request that licensees take certain actions to ensure that safety-related power-operated gate valves that are susceptible to pressure locking or thermal binding are capable of performing their safety functions within the current licensing bases. Over the past two years, several plants in Region I determined that valves in certain systems were potentially susceptible to pressure locking and thermal binding, and have taken various corrective actions. The NRC Region I Systems Engineering Branch has been actively involved in the inspection of licensee actions in response to the pressure locking and thermal binding issue. Region I continues to maintain an active involvement in this area, including participation with the Office of Nuclear Reactor Regulation in reviewing licensee responses to Generic Letter 95-07.

DnaA protein DNA-binding domain binds to Hda protein to promote inter-AAA+ domain interaction involved in regulatory inactivation of DnaA.

Science.gov (United States)

Keyamura, Kenji; Katayama, Tsutomu

2011-08-19

Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis.

DnaA Protein DNA-binding Domain Binds to Hda Protein to Promote Inter-AAA+ Domain Interaction Involved in Regulatory Inactivation of DnaA*

Science.gov (United States)

Keyamura, Kenji; Katayama, Tsutomu

2011-01-01

Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis. PMID:21708944

Involvement of histidine residues in the pH-dependent β-galactoside binding activity of human galectin-1.

Science.gov (United States)

Hiramatsu, Hirotsugu; Takeuchi, Katsuyuki; Takeuchi, Hideo

2013-04-02

The pH dependence of the β-galactoside binding activity of human galectin-1 (hGal-1) was investigated by fluorescence spectroscopy using lactose as a ligand. The obtained binding constant Kb was 2.94 ± 0.10 mM(-1) at pH 7.5. The Kb value decreased at acidic pH with a midpoint of transition at pH 6.0 ± 0.1. To elucidate the molecular mechanism of the pH dependence, we investigated the structures of hGal-1 and its two His mutants (H44Q and H52Q) using fluorescence, circular dichroism, UV absorption, and UV resonance Raman spectroscopy. Analysis of the spectra has shown that the pKa values of His44 and His52 are 5.7 ± 0.2 and 6.3 ± 0.1, respectively. The protonation of His52 below pH 6.3 induces a small change in secondary structure and partly reduces the galactoside binding activity. On the other hand, the protonation of His44 below pH 5.7 exerts a cation-π interaction with Trp68 and largely diminishes the galactoside binding activity. With reference to the literature X-ray structures at pH 7.0 and 5.6, protonated His52 is proposed to move slightly away from the galactoside-binding region with a partial unfolding of the β-strand containing His52. On the other hand, protonated His44 becomes unable to form a hydrogen bond with galactoside and additionally induces a reorientation and/or displacement of Trp68 through cation-π interaction, leading to a loosening of the galactoside-binding pocket. These structural changes associated with His protonation are likely to be the origin of the pH dependence of the galactoside binding activity of hGal-1.

Specific DNA-binding proteins and DNA sequences involved in steroid hormone regulation of gene expression

International Nuclear Information System (INIS)

Spelsberg, T.; Hora, J.; Horton, M.; Goldberger, A.; Littlefield, B.; Seelke, R.; Toyoda, H.

1987-01-01

Steroid hormones circulate in the blood and are taken by target cells via complexes with intracellular binding proteins termed receptors, that are hormone and tissue specific. Each receptor binds it specific steroid with very high affinity, having an equilibrium dissociation constant (K/sub d/) in the range of 10 -9 to 10 -10 M. Once bound by their specific steroid hormones, the steroid receptors undergo a conformational change which allows them to bind with high affinity to sites on chromatin, termed nuclear acceptor sites. There are estimated 5,000 to 10,000 of these sites expressed with an equal number not expressed (''masked'') in intact chromatin. The result of the binding to nuclear acceptor sites is an alteration of gene transcription or, in some cases, gene expression as measured by the changing levels of specific RNAs and proteins in that target tissue. Each steroid regulates specific effects on the RNA and protein profiles. The chronology of the above mechanism of action after injection of radiolabelled steroid as is follows: Steroid-receptor complex formation (1 minute), nuclear acceptor sites (2 minutes), effects on RNA synthesis (10 to 30 minutes), and finally the changing protein profiles via changes in protein synthesis and protein turnover (1 to 6 hours). Thus steroid receptors represent one of the first identified intracellular gene regulation proteins. The receptor molecules themselves are regulated by the presence or absence of the steroid molecule

Alpine ski bindings and injuries. Current findings.

Science.gov (United States)

Natri, A; Beynnon, B D; Ettlinger, C F; Johnson, R J; Shealy, J E

1999-07-01

In spite of the fact that the overall incidence of alpine ski injuries has decreased during the last 25 years, the incidence of serious knee sprains usually involving the anterior cruciate ligament (ACL) has risen dramatically since the late 1970s. This trend runs counter to a dramatic reduction in lower leg injuries that began in the early 1970s and to date has lowered the risk of injury below the knee by almost 90%. One of the primary design objectives of modern ski boots and bindings has been to protect the skier from tibia and ankle fractures. So, in that sense, they have done an excellent job. However, despite advances in equipment design, modern ski bindings have not protected the knee from serious ligament trauma. At the present time, we are unaware of any binding design, settings or function that can protect both the knee and lower extremities from serious ligament sprains. No innovative change in binding design appears to be on the horizon that has the potential to reduce the risk of these severe knee injuries. Indeed, only 1 study has demonstrated a means to help reduce this risk of serious knee sprains, and this study involved education of skiers, not ski equipment. Despite the inability of bindings to reduce the risk of severe knee injuries there can be no doubt that improvement in ski bindings has been the most important factor in the marked reduction in incidence of lower leg and ankle injuries during the last 25 years. The authors strongly endorse the application of present International Standards Organisation (ISO) and American Society for Testing and Materials (ASTM) standards concerning mounting, setting and maintaining modern 'state of the art' bindings.

A photoaffinity scan maps regions of the p85 SH2 domain involved in phosphoprotein binding.

Science.gov (United States)

Williams, K P; Shoelson, S E

1993-03-15

Src homology 2 (SH2) domains are modular phosphotyrosine binding pockets found within a wide variety of cytoplasmic signaling molecules. Here we develop a new approach to analyzing protein-protein interfaces termed photoaffinity scanning, and apply the method to map regions of the phosphatidylinositol 3-kinase p85 SH2 domain that participate in phospho-protein binding. Each residue except phosphotyrosine (pY) within a tightly binding, IRS-1-derived phosphopeptide (GNGDpYMPMSPKS) was substituted with the photoactive amino acid, benzoylphenylalanine (Bpa). Whereas most substitutions had little effect on binding affinity, Bpa substitution of either Met (+1 and +3 with respect to pY) reduced affinity 50-100-fold to confirm their importance in the pYMXM recognition motif. In three cases photolysis of SH2 domain/Bpa phosphopeptide complexes led to cross-linking of > 50% of the SH2 domain; cross-link positions were identified by microsequence, amino acid composition, and electrospray mass spectrometric analyses. Bpa-1 cross-links within alpha-helix I, whereas Bpa+1 and Bpa+4 cross-link the SH2 domain within the flexible loop C-terminal to alpha-helix II. Moreover, cross-linking at any position prevents SH2 domain cleavage at a trypsin-sensitive site within the flexible loop between beta-strands 1 and 2. Therefore, at least three distinct SH2 regions in addition to the beta-sheet participate in phosphoprotein binding; the loop cross-linked by phosphopeptide residues C-terminal to pY appears to confer specificity to the phosphoprotein/SH2 domain interaction.

Zinc Binding by Lactic Acid Bacteria

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Jasna Mrvčić

2009-01-01

Full Text Available Zinc is an essential trace element in all organisms. A common method for the prevention of zinc deficiency is pharmacological supplementation, especially in a highly available form of a metalloprotein complex. The potential of different microbes to bind essential and toxic heavy metals has recently been recognized. In this work, biosorption of zinc by lactic acid bacteria (LAB has been investigated. Specific LAB were assessed for their ability to bind zinc from a water solution. Significant amount of zinc ions was bound, and this binding was found to be LAB species-specific. Differences among the species in binding performance at a concentration range between 10–90 mg/L were evaluated with Langmuir model for biosorption. Binding of zinc was a fast process, strongly influenced by ionic strength, pH, biomass concentration, and temperature. The most effective metal-binding LAB species was Leuconostoc mesenteroides (27.10 mg of Zn2+ per gram of dry mass bound at pH=5 and 32 °C, during 24 h. FT-IR spectroscopy analysis and electron microscopy demonstrated that passive adsorption and active uptake of the zinc ions were involved.

Structures of Orf Virus Chemokine Binding Protein in Complex with Host Chemokines Reveal Clues to Broad Binding Specificity.

Science.gov (United States)

Couñago, Rafael M; Knapp, Karen M; Nakatani, Yoshio; Fleming, Stephen B; Corbett, Michael; Wise, Lyn M; Mercer, Andrew A; Krause, Kurt L

2015-07-07

The chemokine binding protein (CKBP) from orf virus (ORFV) binds with high affinity to chemokines from three classes, C, CC, and CXC, making it unique among poxvirus CKBPs described to date. We present its crystal structure alone and in complex with three CC chemokines, CCL2, CCL3, and CCL7. ORFV CKBP possesses a β-sandwich fold that is electrostatically and sterically complementary to its binding partners. Chemokines bind primarily through interactions involving the N-terminal loop and a hydrophobic recess on the ORFV CKBP β-sheet II surface, and largely polar interactions between the chemokine 20s loop and a negatively charged surface groove located at one end of the CKBP β-sheet II surface. ORFV CKBP interacts with leukocyte receptor and glycosaminoglycan binding sites found on the surface of bound chemokines. SEC-MALLS and chromatographic evidence is presented supporting that ORFV CKBP is a dimer in solution over a broad range of protein concentrations. Copyright © 2015 Elsevier Ltd. All rights reserved.

The fragile X protein binds mRNAs involved in cancer progression and modulates metastasis formation.

Science.gov (United States)

Lucá, Rossella; Averna, Michele; Zalfa, Francesca; Vecchi, Manuela; Bianchi, Fabrizio; La Fata, Giorgio; Del Nonno, Franca; Nardacci, Roberta; Bianchi, Marco; Nuciforo, Paolo; Munck, Sebastian; Parrella, Paola; Moura, Rute; Signori, Emanuela; Alston, Robert; Kuchnio, Anna; Farace, Maria Giulia; Fazio, Vito Michele; Piacentini, Mauro; De Strooper, Bart; Achsel, Tilmann; Neri, Giovanni; Neven, Patrick; Evans, D Gareth; Carmeliet, Peter; Mazzone, Massimiliano; Bagni, Claudia

2013-10-01

The role of the fragile X mental retardation protein (FMRP) is well established in brain, where its absence leads to the fragile X syndrome (FXS). FMRP is almost ubiquitously expressed, suggesting that, in addition to its effects in brain, it may have fundamental roles in other organs. There is evidence that FMRP expression can be linked to cancer. FMR1 mRNA, encoding FMRP, is overexpressed in hepatocellular carcinoma cells. A decreased risk of cancer has been reported in patients with FXS while a patient-case with FXS showed an unusual decrease of tumour brain invasiveness. However, a role for FMRP in regulating cancer biology, if any, remains unknown. We show here that FMRP and FMR1 mRNA levels correlate with prognostic indicators of aggressive breast cancer, lung metastases probability and triple negative breast cancer (TNBC). We establish that FMRP overexpression in murine breast primary tumours enhances lung metastasis while its reduction has the opposite effect regulating cell spreading and invasion. FMRP binds mRNAs involved in epithelial mesenchymal transition (EMT) and invasion including E-cadherin and Vimentin mRNAs, hallmarks of EMT and cancer progression. © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.

Factors Affecting the Binding of a Recombinant Heavy Metal-Binding Domain (CXXC motif Protein to Heavy Metals

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Kamala Boonyodying

2012-06-01

Full Text Available A number of heavy metal-binding proteins have been used to study bioremediation. CXXC motif, a metal binding domain containing Cys-X-X-Cys motif, has been identified in various organisms. These proteins are capable of binding various types of heavy metals. In this study, heavy metal binding domain (CXXC motif recombinant protein encoded from mcsA gene of S. aureus were cloned and overexpressed in Escherichia coli. The factors involved in the metal-binding activity were determined in order to analyze the potential of recombinant protein for bioremediation. A recombinant protein can be bound to Cd2+, Co2+, Cu2+ and Zn2+. The thermal stability of a recombinant protein was tested, and the results showed that the metal binding activity to Cu2+ and Zn2+ still exist after treating the protein at 85ºC for 30 min. The temperature and pH that affected the metal binding activity was tested and the results showed that recombinant protein was still bound to Cu2+ at 65ºC, whereas a pH of 3-7 did not affect the metal binding E. coli harboring a pRset with a heavy metal-binding domain CXXC motif increased the resistance of heavy metals against CuCl2 and CdCl2. This study shows that metal binding domain (CXXC motif recombinant protein can be effectively bound to various types of heavy metals and may be used as a potential tool for studying bioremediation.

The peripheral binding of 14-3-3γ to membranes involves isoform-specific histidine residues.

Directory of Open Access Journals (Sweden)

Helene J Bustad

Full Text Available Mammalian 14-3-3 protein scaffolds include seven conserved isoforms that bind numerous phosphorylated protein partners and regulate many cellular processes. Some 14-3-3-isoforms, notably γ, have elevated affinity for membranes, which might contribute to modulate the subcellular localization of the partners and substantiate the importance of investigating molecular mechanisms of membrane interaction. By applying surface plasmon resonance we here show that the binding to phospholipid bilayers is stimulated when 14-3-3γ is complexed with its partner, a peptide corresponding to the Ser19-phosphorylated N-terminal region of tyrosine hydroxylase. Moreover, membrane interaction is dependent on salts of kosmotropic ions, which also stabilize 14-3-3γ. Electrostatic analysis of available crystal structures of γ and of the non-membrane-binding ζ-isoform, complemented with molecular dynamics simulations, indicate that the electrostatic potential distribution of phosphopeptide-bound 14-3-3γ is optimal for interaction with the membrane through amphipathic helices at the N-terminal dimerization region. In addition, His158, and especially His195, both specific to 14-3-3γ and located at the convex lateral side, appeared to be pivotal for the ligand induced membrane interaction, as corroborated by site-directed mutagenesis. The participation of these histidine residues might be associated to their increased protonation upon membrane binding. Overall, these results reveal membrane-targeting motifs and give insights on mechanisms that furnish the 14-3-3γ scaffold with the capacity for tuned shuffling from soluble to membrane-bound states.

Characterization of Novel Calmodulin Binding Domains within IQ Motifs of IQGAP1

Science.gov (United States)

Jang, Deok-Jin; Ban, Byungkwan; Lee, Jin-A

2011-01-01

IQ motif-containing GTPase-activating protein 1 (IQGAP1), which is a well-known calmodulin (CaM) binding protein, is involved in a wide range of cellular processes including cell proliferation, tumorigenesis, adhesion, and migration. Interaction of IQGAP1 with CaM is important for its cellular functions. Although each IQ domain of IQGAP1 for CaM binding has been characterized in a Ca2+-dependent or -independent manner, it was not clear which IQ motifs are physiologically relevant for CaM binding in the cells. In this study, we performed immunoprecipitation using 3xFLAGhCaM in mammalian cell lines to characterize the domains of IQGAP1 that are key for CaM binding under physiological conditions. Interestingly, using this method, we identified two novel domains, IQ(2.7-3) and IQ(3.5-4.4), within IQGAP1 that were involved in Ca2+-independent or -dependent CaM binding, respectively. Mutant analysis clearly showed that the hydrophobic regions within IQ(2.7-3) were mainly involved in apoCaM binding, while the basic amino acids and hydrophobic region of IQ(3.5-4.4) were required for Ca2+/CaM binding. Finally, we showed that IQ(2.7-3) was the main apoCaM binding domain and both IQ(2.7-3) and IQ(3.5-4.4) were required for Ca2+/CaM binding within IQ(1- 2-3-4). Thus, we identified and characterized novel direct CaM binding motifs essential for IQGAP1. This finding indicates that IQGAP1 plays a dynamic role via direct interactions with CaM in a Ca2+-dependent or -independent manner. PMID:22080369

Penicillin-binding proteins in Actinobacteria.

Science.gov (United States)

Ogawara, Hiroshi

2015-04-01

Because some Actinobacteria, especially Streptomyces species, are β-lactam-producing bacteria, they have to have some self-resistant mechanism. The β-lactam biosynthetic gene clusters include genes for β-lactamases and penicillin-binding proteins (PBPs), suggesting that these are involved in self-resistance. However, direct evidence for the involvement of β-lactamases does not exist at the present time. Instead, phylogenetic analysis revealed that PBPs in Streptomyces are distinct in that Streptomyces species have much more PBPs than other Actinobacteria, and that two to three pairs of similar PBPs are present in most Streptomyces species examined. Some of these PBPs bind benzylpenicillin with very low affinity and are highly similar in their amino-acid sequences. Furthermore, other low-affinity PBPs such as SCLAV_4179 in Streptomyces clavuligerus, a β-lactam-producing Actinobacterium, may strengthen further the self-resistance against β-lactams. This review discusses the role of PBPs in resistance to benzylpenicillin in Streptomyces belonging to Actinobacteria.

Effect of Ca2+ on the promiscuous target-protein binding of calmodulin.

Directory of Open Access Journals (Sweden)

Annie M Westerlund

2018-04-01

Full Text Available Calmodulin (CaM is a calcium sensing protein that regulates the function of a large number of proteins, thus playing a crucial part in many cell signaling pathways. CaM has the ability to bind more than 300 different target peptides in a Ca2+-dependent manner, mainly through the exposure of hydrophobic residues. How CaM can bind a large number of targets while retaining some selectivity is a fascinating open question. Here, we explore the mechanism of CaM selective promiscuity for selected target proteins. Analyzing enhanced sampling molecular dynamics simulations of Ca2+-bound and Ca2+-free CaM via spectral clustering has allowed us to identify distinct conformational states, characterized by interhelical angles, secondary structure determinants and the solvent exposure of specific residues. We searched for indicators of conformational selection by mapping solvent exposure of residues in these conformational states to contacts in structures of CaM/target peptide complexes. We thereby identified CaM states involved in various binding classes arranged along a depth binding gradient. Binding Ca2+ modifies the accessible hydrophobic surface of the two lobes and allows for deeper binding. Apo CaM indeed shows shallow binding involving predominantly polar and charged residues. Furthermore, binding to the C-terminal lobe of CaM appears selective and involves specific conformational states that can facilitate deep binding to target proteins, while binding to the N-terminal lobe appears to happen through a more flexible mechanism. Thus the long-ranged electrostatic interactions of the charged residues of the N-terminal lobe of CaM may initiate binding, while the short-ranged interactions of hydrophobic residues in the C-terminal lobe of CaM may account for selectivity. This work furthers our understanding of the mechanism of CaM binding and selectivity to different target proteins and paves the way towards a comprehensive model of CaM selectivity.

Involvement of a bifunctional, paired-like DNA-binding domain and a transpositional enhancer in Sleeping Beauty transposition.

Science.gov (United States)

Izsvák, Zsuzsanna; Khare, Dheeraj; Behlke, Joachim; Heinemann, Udo; Plasterk, Ronald H; Ivics, Zoltán

2002-09-13

Sleeping Beauty (SB) is the most active Tc1/mariner-like transposon in vertebrate species. Each of the terminal inverted repeats (IRs) of SB contains two transposase-binding sites (DRs). This feature, termed the IR/DR structure, is conserved in a group of Tc1-like transposons. The DNA-binding region of SB transposase, similar to the paired domain of Pax proteins, consists of two helix-turn-helix subdomains (PAI + RED = PAIRED). The N-terminal PAI subdomain was found to play a dominant role in contacting the DRs. Transposase was able to bind to mutant sites retaining the 3' part of the DRs; thus, primary DNA binding is not sufficient to determine the specificity of the transposition reaction. The PAI subdomain was also found to bind to a transpositional enhancer-like sequence within the left IR of SB, and to mediate protein-protein interactions between transposase subunits. A tetrameric form of the transposase was detected in solution, consistent with an interaction between the IR/DR structure and a transposase tetramer. We propose a model in which the transpositional enhancer and the PAI subdomain stabilize complexes formed by a transposase tetramer bound at the IR/DR. These interactions may result in enhanced stability of synaptic complexes, which might explain the efficient transposition of Sleeping Beauty in vertebrate cells.

What Happened to the IGF Binding Proteins?

Science.gov (United States)

Bach, Leon A

2018-02-01

Insulinlike growth factor (IGF) binding proteins (IGFBPs) 1 to 6 are high-affinity regulators of IGF activity. They generally inhibit IGF actions by preventing binding to the IGF-I receptor but can also enhance their actions under some conditions. Posttranslational modifications such as glycosylation and phosphorylation modulate IGFBP properties, and IGFBP proteolysis results in IGF release. IGFBPs have more recently been shown to have IGF-independent actions. A number of mechanisms are involved, including modulation of other growth factor pathways, nuclear localization and transcriptional regulation, interaction with the sphingolipid pathway, and binding to non-IGF biomolecules in the extracellular space and matrix, on the cell surface and intracellularly. IGFBPs modulate important biological processes, including cell proliferation, survival, migration, senescence, autophagy, and angiogenesis. Their actions have been implicated in growth, metabolism, cancer, stem cell maintenance and differentiation, and immune regulation. Recent studies have shown that epigenetic mechanisms are involved in the regulation of IGFBP abundance. A more complete understanding of IGFBP biology is necessary to further define their cellular roles and determine their therapeutic potential. Copyright © 2018 Endocrine Society.

FRET-based binding assay between a fluorescent cAMP analogue and a cyclic nucleotide-binding domain tagged with a CFP.

Science.gov (United States)

Romero, Francisco; Santana-Calvo, Carmen; Sánchez-Guevara, Yoloxochitl; Nishigaki, Takuya

2017-09-01

The cyclic nucleotide-binding domain (CNBD) functions as a regulatory domain of many proteins involved in cyclic nucleotide signalling. We developed a straightforward and reliable binding assay based on intermolecular fluorescence resonance energy transfer (FRET) between an adenosine-3', 5'-cyclic monophosphate analogue labelled with fluorescein and a recombinant CNBD of human EPAC1 tagged with a cyan fluorescence protein (CFP). The high FRET efficiency of this method (~ 80%) allowed us to perform several types of binding experiments with nanomolar range of sample using conventional equipment. In addition, the CFP tag on the CNBD enabled us to perform a specific binding experiment using an unpurified protein. Considering these advantages, this technique is useful to study poorly characterized CNBDs. © 2017 Federation of European Biochemical Societies.

Adaptive evolution of transcription factor binding sites

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Berg Johannes

2004-10-01

Full Text Available Abstract Background The regulation of a gene depends on the binding of transcription factors to specific sites located in the regulatory region of the gene. The generation of these binding sites and of cooperativity between them are essential building blocks in the evolution of complex regulatory networks. We study a theoretical model for the sequence evolution of binding sites by point mutations. The approach is based on biophysical models for the binding of transcription factors to DNA. Hence we derive empirically grounded fitness landscapes, which enter a population genetics model including mutations, genetic drift, and selection. Results We show that the selection for factor binding generically leads to specific correlations between nucleotide frequencies at different positions of a binding site. We demonstrate the possibility of rapid adaptive evolution generating a new binding site for a given transcription factor by point mutations. The evolutionary time required is estimated in terms of the neutral (background mutation rate, the selection coefficient, and the effective population size. Conclusions The efficiency of binding site formation is seen to depend on two joint conditions: the binding site motif must be short enough and the promoter region must be long enough. These constraints on promoter architecture are indeed seen in eukaryotic systems. Furthermore, we analyse the adaptive evolution of genetic switches and of signal integration through binding cooperativity between different sites. Experimental tests of this picture involving the statistics of polymorphisms and phylogenies of sites are discussed.

Thermodynamic fingerprints of ligand binding to human telomeric G-quadruplexes

OpenAIRE

Bon?ina, Matja?; Podlipnik, ?rtomir; Piantanida, Ivo; Eilmes, Julita; Teulade-Fichou, Marie-Paule; Vesnaver, Gorazd; Lah, Jurij

2015-01-01

Thermodynamic studies of ligand binding to human telomere (ht) DNA quadruplexes, as a rule, neglect the involvement of various ht-DNA conformations in the binding process. Therefore, the thermodynamic driving forces and the mechanisms of ht-DNA G-quadruplex-ligand recognition remain poorly understood. In this work we characterize thermodynamically and structurally binding of netropsin (Net), dibenzotetraaza[14]annulene derivatives (DP77, DP78), cationic porphyrin (TMPyP4) and two bisquinolini...

Different types of working memory binding in epilepsy patients with unilateral anterior temporal lobectomy.

Science.gov (United States)

van Geldorp, Bonnie; Bouman, Zita; Hendriks, Marc P H; Kessels, Roy P C

2014-03-01

The medial temporal lobe is an important structure for long-term memory formation, but its role in working memory is less clear. Recent studies have shown hippocampal involvement during working memory tasks requiring binding of information. It is yet unclear whether this is limited to tasks containing spatial features. The present study contrasted three binding conditions and one single-item condition in patients with unilateral anterior temporal lobectomy. A group of 43 patients with temporal lobectomy (23 left; 20 right) and 20 matched controls were examined with a working memory task assessing spatial relational binding (object-location), non-spatial relational binding (object-object), conjunctive binding (object-colour) and working memory for single items. We varied the delay period (3 or 6s), as there is evidence showing that delay length may modulate working memory performance. The results indicate that performance was worse for patients than for controls in both relational binding conditions, whereas patients were unimpaired in conjunctive binding. Single-item memory was found to be marginally impaired, due to a deficit on long-delay trials only. In conclusion, working memory binding deficits are found in patients with unilateral anterior temporal lobectomy. The role of the medial temporal lobe in working memory is not limited to tasks containing spatial features. Rather, it seems to be involved in relational binding, but not in conjunctive binding. The medial temporal lobe gets involved when working memory capacity does not suffice, for example when relations have to be maintained or when the delay period is long. Copyright © 2014 Elsevier Inc. All rights reserved.

Binding of ethyl pyruvate to bovine serum albumin: Calorimetric, spectroscopic and molecular docking studies

Energy Technology Data Exchange (ETDEWEB)

Pathak, Mallika [Department of Chemistry, Miranda House, University of Delhi, Delhi 11007 (India); Mishra, Rashmi; Agarwala, Paban K. [Department of Radiation Genetics and Epigenetics, Division of Radioprotective Drug Development Research, Institute of Nuclear Medicine and Allied Sciences, Delhi 110054 (India); Ojha, Himanshu, E-mail: himanshu.drdo@gmail.com [Department of Radiation Genetics and Epigenetics, Division of Radioprotective Drug Development Research, Institute of Nuclear Medicine and Allied Sciences, Delhi 110054 (India); Singh, Bhawna [Department of Radiation Genetics and Epigenetics, Division of Radioprotective Drug Development Research, Institute of Nuclear Medicine and Allied Sciences, Delhi 110054 (India); Singh, Anju; Kukreti, Shrikant [Nucleic Acid Research Laboratory, Department of Chemistry, University of Delhi, Delhi 11007 (India)

2016-06-10

Highlights: • ITC study showed binding of ethyl pyruvate with BSA with high binding affinity. • Ethyl pyruvate binding caused conformation alteration of BSA. • Fluorescence quenching mechanism is static in nature. • Electrostatic, hydrogen bonding and hydrophobic forces involved in binding. • Docking confirmed role of electrostatic, hydrogen bonding and hydrophobic forces. - Abstract: Various in vitro and in vivo studies have shown the anti-inflammatory and anticancer potential role of ethyl pyruvate. Bio-distribution of drugs is significantly influenced by the drug-serum protein binding. Therefore, the binding mechanism of the ethyl pyruvate with bovine serum albumin was investigated using UV–vis absorption, fluorescence, circular dichroism, isothermal titration calorimetry and molecular docking techniques. Absorption and fluorescence quenching studies indicated the binding of ethyl pyruvate with protein. Circular dichroism spectra of bovine serum albumin confirmed significant change in the conformation of protein upon binding. Thermodynamic data confirmed that ethyl pyruvate binds to bovine serum albumin at the two different sites with high affinity. Binding of ethyl pyruvate to bovine serum albumin involves hydrogen bonding, van der Waal and hydrophobic interactions. Further, docking studies indicated that ethyl pyruvate could bind significantly at the three binding sites. The results will definitely contribute to the development of ethyl pyruvate as drug.

Identification and therapeutic potential of a vitronectin binding region of meningococcal msf.

Science.gov (United States)

Hill, Darryl J; Griffiths, Natalie J; Borodina, Elena; Andreae, Clio A; Sessions, Richard B; Virji, Mumtaz

2015-01-01

The human pathogen Neisseria meningitides (Nm) attains serum resistance via a number of mechanisms, one of which involves binding to the host complement regulator protein vitronectin. We have shown previously that the Meningococcal surface fibril (Msf), a trimeric autotransporter, binds to the activated form of vitronectin (aVn) to increase Nm survival in human serum. In this study, we aimed to identify the aVn-binding region of Msf to assess its potential as an antigen which can elicit antibodies that block aVn binding and/or possess bactericidal properties. Using several recombinant Msf fragments spanning its surface-exposed region, the smallest aVn-binding recombinants were found to span residues 1-86 and 39-124. The use of further deletion constructs and overlapping recombinant Msf fragments suggested that a region of Msf comprising residues 39-82 may be primarily important for aVn binding and that other regions may also be involved but to a lesser extent. Molecular modelling implicated K66 and K68, conserved in all available Msf sequences, to be involved in the interaction. Recombinant fragments which bound to aVn were able to reduce the survival advantage conveyed by aVn-interaction in serum bactericidal assays. Antibodies raised against one such fragment inhibited aVn binding to Msf. In addition, the antibodies enhanced specific killing of Msf-expressing Nm in a dose-dependent manner. Overall, this study identifies an aVn-binding region of Msf, an adhesin known to impart serum resistance properties to the pathogen; and shows that this region of Msf can elicit antibodies with dual properties which reduce pathogen survival within the host and thus has potential as a vaccine antigen.

Spectroscopic studies on Titanium ion binding to the apo lactoferrin

International Nuclear Information System (INIS)

Moshtaghie, A.A.; Ani, M.; Arabi, M.H.

2006-01-01

Titanium is a relatively abundant element that has found growing applications in medical science and recently some of Titanium compounds are introduced as anticancer drugs. In spite of very limited data which exist on the Titanium metabolism, some proteins might be involved in the mechanism of action of Titanium up to our knowledge, there is not any report in the literature concerning binding of Titanium to apo lactoferrin. Binding of apo lactoferrin with Ti(IV)-citrate was studied by spectroflourimeterey and spectrophotometery techniques under physiological conditions. The spectroflourimeteric studies revealed a significant fluorescence quenching, that indicated binding of apo lactoferrin with Ti(IV). The same reaction was monitored through spectrophotometry technique; this represents a characteristic UV difference band at 267 nm, which is different from lac-Fe (III). Titration studies how that lactoferrin specifically binds two moles Ti(IV) as complex with citrate per mol protein. Spectroflourimeterey and spectrophotometery techniques indicated that Ti(IV) ions cause a reduction (13%-14%) in binding of Fe(III) to lactoferrin. In overall, we may come to this conclusion that this element might be involved in the iron metabolism

Theoretical Study of Molecular Determinants Involved in Signal Binding to the TraR Protein of Agrobacterium tumefaciens

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N. Kumar

2005-10-01

Full Text Available N-acylated homoserine lactone (AHL mediated cell-cell communication in bacteria is dependent on the recognition of the cognate signal by its receptor. This interaction allows the receptor-ligand complex to act as a transcriptional activator, controlling the expression of a range of bacterial phenotypes, including virulence factor expression and biofilm formation. One approach to determine the key features of signal- binding is to model the intermolecular interactions between the receptor and ligand using computational-based modeling software (LigandFit. In this communication, we have modeled the crystal structure of the AHL receptor protein TraR and its AHL signal N-(3- oxooctanoyl-homoserine lactone from Agrobacterium tumefaciens and compared it to the previously reported antagonist behaviour of a number of AHL analogues, in an attempt to determine structural constraints for ligand binding. We conclude that (i a common conformation of the AHL in the hydrophobic and hydrophilic region exists for ligand-binding, (ii a tail chain length threshold of 8 carbons is most favourable for ligand-binding affinity, (iii the positive correlation in the docking studies could be used a virtual screening tool.

Clinical relevance of drug binding to plasma proteins

Science.gov (United States)

Ascenzi, Paolo; Fanali, Gabriella; Fasano, Mauro; Pallottini, Valentina; Trezza, Viviana

2014-12-01

Binding to plasma proteins highly influences drug efficacy, distribution, and disposition. Serum albumin, the most abundant protein in plasma, is a monomeric multi-domain macromolecule that displays an extraordinary ligand binding capacity, providing a depot and carrier for many endogenous and exogenous compounds, such as fatty acids and most acidic drugs. α-1-Acid glycoprotein, the second main plasma protein, is a glycoprotein physiologically involved in the acute phase reaction and is the main carrier for basic and neutral drugs. High- and low-density lipoproteins play a limited role in drug binding and are natural drug delivery system only for few lipophilic drugs or lipid-based formulations. Several factors influence drug binding to plasma proteins, such as pathological conditions, concurrent administration of drugs, sex, and age. Any of these factors, in turn, influences drug efficacy and toxicity. Here, biochemical, biomedical, and biotechnological aspects of drug binding to plasma proteins are reviewed.

Species Differences in the Carbohydrate Binding Preferences of Surfactant Protein D

DEFF Research Database (Denmark)

Crouch, Erika C.; Smith, Kelly; McDonald, Barbara

2006-01-01

Interactions of surfactant protein D (SP-D) with micro-organisms and organic antigens involve binding to the trimeric neck plus carbohydrate recognition domain (neck+CRD). In these studies, we compared the ligand binding of homologous human, rat, and mouse trimeric neck+CRD fusion proteins, each ...

HemeBIND: a novel method for heme binding residue prediction by combining structural and sequence information

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Hu Jianjun

2011-05-01

Full Text Available Abstract Background Accurate prediction of binding residues involved in the interactions between proteins and small ligands is one of the major challenges in structural bioinformatics. Heme is an essential and commonly used ligand that plays critical roles in electron transfer, catalysis, signal transduction and gene expression. Although much effort has been devoted to the development of various generic algorithms for ligand binding site prediction over the last decade, no algorithm has been specifically designed to complement experimental techniques for identification of heme binding residues. Consequently, an urgent need is to develop a computational method for recognizing these important residues. Results Here we introduced an efficient algorithm HemeBIND for predicting heme binding residues by integrating structural and sequence information. We systematically investigated the characteristics of binding interfaces based on a non-redundant dataset of heme-protein complexes. It was found that several sequence and structural attributes such as evolutionary conservation, solvent accessibility, depth and protrusion clearly illustrate the differences between heme binding and non-binding residues. These features can then be separately used or combined to build the structure-based classifiers using support vector machine (SVM. The results showed that the information contained in these features is largely complementary and their combination achieved the best performance. To further improve the performance, an attempt has been made to develop a post-processing procedure to reduce the number of false positives. In addition, we built a sequence-based classifier based on SVM and sequence profile as an alternative when only sequence information can be used. Finally, we employed a voting method to combine the outputs of structure-based and sequence-based classifiers, which demonstrated remarkably better performance than the individual classifier alone

Identification and therapeutic potential of a vitronectin binding region of meningococcal msf.

Directory of Open Access Journals (Sweden)

Darryl J Hill

Full Text Available The human pathogen Neisseria meningitides (Nm attains serum resistance via a number of mechanisms, one of which involves binding to the host complement regulator protein vitronectin. We have shown previously that the Meningococcal surface fibril (Msf, a trimeric autotransporter, binds to the activated form of vitronectin (aVn to increase Nm survival in human serum. In this study, we aimed to identify the aVn-binding region of Msf to assess its potential as an antigen which can elicit antibodies that block aVn binding and/or possess bactericidal properties. Using several recombinant Msf fragments spanning its surface-exposed region, the smallest aVn-binding recombinants were found to span residues 1-86 and 39-124. The use of further deletion constructs and overlapping recombinant Msf fragments suggested that a region of Msf comprising residues 39-82 may be primarily important for aVn binding and that other regions may also be involved but to a lesser extent. Molecular modelling implicated K66 and K68, conserved in all available Msf sequences, to be involved in the interaction. Recombinant fragments which bound to aVn were able to reduce the survival advantage conveyed by aVn-interaction in serum bactericidal assays. Antibodies raised against one such fragment inhibited aVn binding to Msf. In addition, the antibodies enhanced specific killing of Msf-expressing Nm in a dose-dependent manner. Overall, this study identifies an aVn-binding region of Msf, an adhesin known to impart serum resistance properties to the pathogen; and shows that this region of Msf can elicit antibodies with dual properties which reduce pathogen survival within the host and thus has potential as a vaccine antigen.

Probing binding hot spots at protein-RNA recognition sites.

Science.gov (United States)

Barik, Amita; Nithin, Chandran; Karampudi, Naga Bhushana Rao; Mukherjee, Sunandan; Bahadur, Ranjit Prasad

2016-01-29

We use evolutionary conservation derived from structure alignment of polypeptide sequences along with structural and physicochemical attributes of protein-RNA interfaces to probe the binding hot spots at protein-RNA recognition sites. We find that the degree of conservation varies across the RNA binding proteins; some evolve rapidly compared to others. Additionally, irrespective of the structural class of the complexes, residues at the RNA binding sites are evolutionary better conserved than those at the solvent exposed surfaces. For recognitions involving duplex RNA, residues interacting with the major groove are better conserved than those interacting with the minor groove. We identify multi-interface residues participating simultaneously in protein-protein and protein-RNA interfaces in complexes where more than one polypeptide is involved in RNA recognition, and show that they are better conserved compared to any other RNA binding residues. We find that the residues at water preservation site are better conserved than those at hydrated or at dehydrated sites. Finally, we develop a Random Forests model using structural and physicochemical attributes for predicting binding hot spots. The model accurately predicts 80% of the instances of experimental ΔΔG values in a particular class, and provides a stepping-stone towards the engineering of protein-RNA recognition sites with desired affinity. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Deconstructing the DGAT1 enzyme: membrane interactions at substrate binding sites.

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Jose L S Lopes

Full Text Available Diacylglycerol acyltransferase 1 (DGAT1 is a key enzyme in the triacylglyceride synthesis pathway. Bovine DGAT1 is an endoplasmic reticulum membrane-bound protein associated with the regulation of fat content in milk and meat. The aim of this study was to evaluate the interaction of DGAT1 peptides corresponding to putative substrate binding sites with different types of model membranes. Whilst these peptides are predicted to be located in an extramembranous loop of the membrane-bound protein, their hydrophobic substrates are membrane-bound molecules. In this study, peptides corresponding to the binding sites of the two substrates involved in the reaction were examined in the presence of model membranes in order to probe potential interactions between them that might influence the subsequent binding of the substrates. Whilst the conformation of one of the peptides changed upon binding several types of micelles regardless of their surface charge, suggesting binding to hydrophobic domains, the other peptide bound strongly to negatively-charged model membranes. This binding was accompanied by a change in conformation, and produced leakage of the liposome-entrapped dye calcein. The different hydrophobic and electrostatic interactions observed suggest the peptides may be involved in the interactions of the enzyme with membrane surfaces, facilitating access of the catalytic histidine to the triacylglycerol substrates.

Nonspecific DNA Binding and Bending by HUαβ: Interfaces of the Three Binding Modes Characterized by Salt Dependent Thermodynamics

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Koh, Junseock; Shkel, Irina; Saecker, Ruth M.; Record, M. Thomas

2011-01-01

Previous ITC and FRET studies demonstrated that Escherichia coli HUαβ binds nonspecifically to duplex DNA in three different binding modes: a tighter-binding 34 bp mode which interacts with DNA in large (>34 bp) gaps between bound proteins, reversibly bending it 140° and thereby increasing its flexibility, and two weaker, modestly cooperative small-site-size modes (10 bp, 6 bp) useful for filling gaps between bound proteins shorter than 34 bp. Here we use ITC to determine the thermodynamics of these binding modes as a function of salt concentration, and deduce that DNA in the 34 bp mode is bent around but not wrapped on the body of HU, in contrast to specific binding of IHF. Analyses of binding isotherms (8, 15, 34 bp DNA) and initial binding heats (34, 38, 160 bp DNA) reveal that all three modes have similar log-log salt concentration derivatives of the binding constants (Ski) even though their binding site sizes differ greatly; most probable values of Ski on 34 bp or larger DNA are − 7.5 ± 0.5. From the similarity of Ski values, we conclude that binding interfaces of all three modes involve the same region of the arms and saddle of HU. All modes are entropy-driven, as expected for nonspecific binding driven by the polyelectrolyte effect. The bent-DNA 34 bp mode is most endothermic, presumably because of the cost of HU-induced DNA bending, while the 6 bp mode is modestly exothermic at all salt concentrations examined. Structural models consistent with the observed Ski values are proposed. PMID:21513716

Relationship of frontal D2/3 binding potentials to cognition

DEFF Research Database (Denmark)

Fagerlund, Birgitte; Pinborg, Lars H; Mortensen, Erik Lykke

2013-01-01

for set shifting. The main findings indicated a relation between D2/3 receptor binding in the frontal cortex and set shifting, planning and attention, but also support a differential involvement of cortical dopamine D2/3 receptor binding in at least some cognitive functions, perhaps particularly attention......Studies of in vivo dopamine receptors in schizophrenia have mostly focused on D2 receptors in striatal areas or on D1 receptors in cortex. No previous study has examined the correlation between cortical dopamine D2/3 receptor binding potentials and cognition in schizophrenia patients. The objective......, in schizophrenia patients compared to healthy people. The results suggest that cortical D2/3 receptor function may be more involved in some cognitive functions (i.e. attention, fluency and planning) in patients with schizophrenia than in healthy people, suggesting that information processing in schizophrenia may...

Functional interaction of the DNA-binding transcription factor Sp1 through its DNA-binding domain with the histone chaperone TAF-I.

Science.gov (United States)

Suzuki, Toru; Muto, Shinsuke; Miyamoto, Saku; Aizawa, Kenichi; Horikoshi, Masami; Nagai, Ryozo

2003-08-01

Transcription involves molecular interactions between general and regulatory transcription factors with further regulation by protein-protein interactions (e.g. transcriptional cofactors). Here we describe functional interaction between DNA-binding transcription factor and histone chaperone. Affinity purification of factors interacting with the DNA-binding domain of the transcription factor Sp1 showed Sp1 to interact with the histone chaperone TAF-I, both alpha and beta isoforms. This interaction was specific as Sp1 did not interact with another histone chaperone CIA nor did other tested DNA-binding regulatory factors (MyoD, NFkappaB, p53) interact with TAF-I. Interaction of Sp1 and TAF-I occurs both in vitro and in vivo. Interaction with TAF-I results in inhibition of DNA-binding, and also likely as a result of such, inhibition of promoter activation by Sp1. Collectively, we describe interaction between DNA-binding transcription factor and histone chaperone which results in negative regulation of the former. This novel regulatory interaction advances our understanding of the mechanisms of eukaryotic transcription through DNA-binding regulatory transcription factors by protein-protein interactions, and also shows the DNA-binding domain to mediate important regulatory interactions.

Development of a Novel Tetravalent Synthetic Peptide That Binds to Phosphatidic Acid.

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Rina Ogawa

Full Text Available We employed a multivalent peptide-library screening technique to identify a peptide motif that binds to phosphatidic acid (PA, but not to other phospholipids such as phosphatidylcholine (PC, phosphatidylethanolamine (PE, and phosphatidylserine (PS. A tetravalent peptide with the sequence motif of MARWHRHHH, designated as PAB-TP (phosphatidic acid-binding tetravalent peptide, was shown to bind as low as 1 mol% of PA in the bilayer membrane composed of PC and cholesterol. Kinetic analysis of the interaction between PAB-TP and the membranes containing 10 mol% of PA showed that PAB-TP associated with PA with a low dissociation constant of KD = 38 ± 5 nM. Coexistence of cholesterol or PE with PA in the membrane enhanced the PAB-TP binding to PA by increasing the ionization of the phosphomonoester head group as well as by changing the microenvironment of PA molecules in the membrane. Amino acid replacement analysis demonstrated that the tryptophan residue at position 4 of PAB-TP was involved in the interaction with PA. Furthermore, a series of amino acid substitutions at positions 5 to 9 of PAB-TP revealed the involvement of consecutive histidine and arginine residues in recognition of the phosphomonoester head group of PA. Our results demonstrate that the recognition of PA by PAB-TP is achieved by a combination of hydrophobic, electrostatic and hydrogen-bond interactions, and that the tetravalent structure of PAB-TP contributes to the high affinity binding to PA in the membrane. The novel PA-binding tetravalent peptide PAB-TP will provide insight into the molecular mechanism underlying the recognition of PA by PA-binding proteins that are involved in various cellular events.

Involvement of a putative substrate binding site in the biogenesis and assembly of phosphatidylserine decarboxylase 1 from Saccharomyces cerevisiae.

Science.gov (United States)

Di Bartolomeo, Francesca; Doan, Kim Nguyen; Athenstaedt, Karin; Becker, Thomas; Daum, Günther

2017-07-01

In the yeast Saccharomyces cerevisiae, the mitochondrial phosphatidylserine decarboxylase 1 (Psd1p) produces the largest amount of cellular phosphatidylethanolamine (PE). Psd1p is synthesized as a larger precursor on cytosolic ribosomes and then imported into mitochondria in a three-step processing event leading to the formation of an α-subunit and a β-subunit. The α-subunit harbors a highly conserved motif, which was proposed to be involved in phosphatidylserine (PS) binding. Here, we present a molecular analysis of this consensus motif for the function of Psd1p by using Psd1p variants bearing either deletions or point mutations in this region. Our data show that mutations in this motif affect processing and stability of Psd1p, and consequently the enzyme's activity. Thus, we conclude that this consensus motif is essential for structural integrity and processing of Psd1p. Copyright © 2017 Elsevier B.V. All rights reserved.

Diagnostic utility of leptin and insulin-like growth factor binding ...

African Journals Online (AJOL)

Serum levels of leptin, insulin growth factor binding protein-2 (IGFBP-2) and alpha fetoprotein (AFP) were measured. ... renewal in response to nutrients. IGF pathway is not only involved in cell growth in tissue culture, but it is also involved in ...

The Role of Pectin in Pb Binding by Carrot Peel Biosorbents: Isoterm Adsorption Study

Science.gov (United States)

Hastuti, B.; Totiana, F.; Winiasih, R.

2018-04-01

Cheaply and abundantly biosorption available materials such as carrot peels can be a cost-efficient method for removing heavy metals from wastewater. To investigate the role pectin plays in metal binding by carrot peels, commerce pectin was compared. FTIR spectra confirmed the presence of carboxyl and hydroxyl groups in commerce pectin and carrot pectin. Isoterm experiments showed that all materials could remove Pb (II) ion. All of materials binding Pb (II) follow Freundlich models adsorption. The commerce pectin bindsPb (II) by involving energy 16.6 KJ/mole whereas pectin from carrot peel involves energy 21.09 KJ/mole. It indicates that commerce pectin binds the Pb (II) by physics adsorption whereas pectin from carrot peel by physics and chemical adsorption.

Pheromone Binding Protein EhipPBP1 Is Highly Enriched in the Male Antennae of the Seabuckthorn Carpenterworm and Is Binding to Sex Pheromone Components

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Ping Hu

2018-04-01

Full Text Available The seabuckthorn carpenterworm moth Eogystia hippophaecolus is a major threat to seabuckthorn plantations, causing considerable ecological and economic losses in China. Transcriptomic analysis of E. hippophaecolus previously identified 137 olfactory proteins, including three pheromone-binding proteins (PBPs. We investigated the function of E. hippophaecolus PBP1 by studying its mRNA and protein expression profiles and its binding ability with different compounds. The highest levels of expression were in the antennae, particularly in males, with much lower levels of expression in the legs and external genitals. Recombinant PBP1 showed strong binding to sex-pheromone components, suggesting that antennal EhipPBP1 is involved in binding sex-pheromone components during pheromone communication.

NADP+ binding to the regulatory subunit of methionine adenosyltransferase II increases intersubunit binding affinity in the hetero-trimer.

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Beatriz González

Full Text Available Mammalian methionine adenosyltransferase II (MAT II is the only hetero-oligomer in this family of enzymes that synthesize S-adenosylmethionine using methionine and ATP as substrates. Binding of regulatory β subunits and catalytic α2 dimers is known to increase the affinity for methionine, although scarce additional information about this interaction is available. This work reports the use of recombinant α2 and β subunits to produce oligomers showing kinetic parameters comparable to MAT II purified from several tissues. According to isothermal titration calorimetry data and densitometric scanning of the stained hetero-oligomer bands on denatured gels, the composition of these oligomers is that of a hetero-trimer with α2 dimers associated to single β subunits. Additionally, the regulatory subunit is able to bind NADP(+ with a 1:1 stoichiometry, the cofactor enhancing β to α2-dimer binding affinity. Mutants lacking residues involved in NADP(+ binding and N-terminal truncations of the β subunit were able to oligomerize with α2-dimers, although the kinetic properties appeared altered. These data together suggest a role for both parts of the sequence in the regulatory role exerted by the β subunit on catalysis. Moreover, preparation of a structural model for the hetero-oligomer, using the available crystal data, allowed prediction of the regions involved in β to α2-dimer interaction. Finally, the implications that the presence of different N-terminals in the β subunit could have on MAT II behavior are discussed in light of the recent identification of several splicing forms of this subunit in hepatoma cells.

Penicillin-binding site on the Escherichia coli cell envelope

International Nuclear Information System (INIS)

Amaral, L.; Lee, Y.; Schwarz, U.; Lorian, V.

1986-01-01

The binding of 35 S-labeled penicillin to distinct penicillin-binding proteins (PBPs) of the cell envelope obtained from the sonication of Escherichia coli was studied at different pHs ranging from 4 to 11. Experiments distinguishing the effect of pH on penicillin binding by PBP 5/6 from its effect on beta-lactamase activity indicated that although substantial binding occurred at the lowest pH, the amount of binding increased with pH, reaching a maximum at pH 10. Based on earlier studies, it is proposed that the binding at high pH involves the formation of a covalent bond between the C-7 of penicillin and free epsilon amino groups of the PBPs. At pHs ranging from 4 to 8, position 1 of penicillin, occupied by sulfur, is considered to be the site that establishes a covalent bond with the sulfhydryl groups of PBP 5. The use of specific blockers of free epsilon amino groups or sulfhydryl groups indicated that wherever the presence of each had little or no effect on the binding of penicillin by PBP 5, the presence of both completely prevented binding. The specific blocker of the hydroxyl group of serine did not affect the binding of penicillin

The effectiveness of ski bindings and their professional adjustment for preventing alpine skiing injuries.

Science.gov (United States)

Finch, C F; Kelsall, H L

1998-06-01

This article presents a critical review of the extent to which alpine ski bindings and their adjustment have been formally demonstrated to prevent injuries. It considers a range of evidence, from anecdotal evidence and informed opinion to biomechanical studies, testing of equipment, epidemiological studies and controlled field evaluations. A total of 15 published studies examining the effectiveness of bindings and their adjustment were identified. All of these included anecdotal or informed opinion, and all but one focused on equipment design. Seven studies involved the testing of bindings or binding prototypes, 2 studies presented biomechanical models of the forces involved in binding operation, 6 reported an epidemiological evaluation of ski bindings and 2 considered skiers' behaviours towards binding adjustment. Some of the reviewed articles relate to the study of the biomechanics of ski bindings and their release in response to various loads and loading patterns. Other studies examined the contribution of bindings and binding-release to lower extremity, equipment-related injuries, the effect of various methods of binding adjustment on injury risk and the determinants of skiers' behaviour relating to professional binding adjustment. Most of the evidence suggests that currently used bindings are insufficient for the multidirectional release required to reduce the risk of injury to the lower limb, especially at the knee. This evidence suggests that further technical developments and innovations are required. The standard of the manufacture of bindings and boots also needs to be considered. The optimal adjustment of bindings using a testing device has been shown to be associated with a reduced risk of lower extremity injury. Generally, however, the adjustment of bindings has been shown to be inadequate, especially for children's bindings. Recommendations for further research, development and implementation with respect to ski binding and their adjustment are given

Echinococcus granulosus fatty acid binding proteins subcellular localization.

Science.gov (United States)

Alvite, Gabriela; Esteves, Adriana

2016-05-01

Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes. Copyright © 2016 Elsevier Inc. All rights reserved.

Non-canonical binding interactions of the RNA recognition motif (RRM) domains of P34 protein modulate binding within the 5S ribonucleoprotein particle (5S RNP).

Science.gov (United States)

Kamina, Anyango D; Williams, Noreen

2017-01-01

RNA binding proteins are involved in many aspects of RNA metabolism. In Trypanosoma brucei, our laboratory has identified two trypanosome-specific RNA binding proteins P34 and P37 that are involved in the maturation of the 60S subunit during ribosome biogenesis. These proteins are part of the T. brucei 5S ribonucleoprotein particle (5S RNP) and P34 binds to 5S ribosomal RNA (rRNA) and ribosomal protein L5 through its N-terminus and its RNA recognition motif (RRM) domains. We generated truncated P34 proteins to determine these domains' interactions with 5S rRNA and L5. Our analyses demonstrate that RRM1 of P34 mediates the majority of binding with 5S rRNA and the N-terminus together with RRM1 contribute the most to binding with L5. We determined that the consensus ribonucleoprotein (RNP) 1 and 2 sequences, characteristic of canonical RRM domains, are not fully conserved in the RRM domains of P34. However, the aromatic amino acids previously described to mediate base stacking interactions with their RNA target are conserved in both of the RRM domains of P34. Surprisingly, mutation of these aromatic residues did not disrupt but instead enhanced 5S rRNA binding. However, we identified four arginine residues located in RRM1 of P34 that strongly impact L5 binding. These mutational analyses of P34 suggest that the binding site for 5S rRNA and L5 are near each other and specific residues within P34 regulate the formation of the 5S RNP. These studies show the unique way that the domains of P34 mediate binding with the T. brucei 5S RNP.

Determining Membrane Protein-Lipid Binding Thermodynamics Using Native Mass Spectrometry.

Science.gov (United States)

Cong, Xiao; Liu, Yang; Liu, Wen; Liang, Xiaowen; Russell, David H; Laganowsky, Arthur

2016-04-06

Membrane proteins are embedded in the biological membrane where the chemically diverse lipid environment can modulate their structure and function. However, the thermodynamics governing the molecular recognition and interaction of lipids with membrane proteins is poorly understood. Here, we report a method using native mass spectrometry (MS), to determine thermodynamics of individual ligand binding events to proteins. Unlike conventional methods, native MS can resolve individual ligand binding events and, coupled with an apparatus to control the temperature, determine binding thermodynamic parameters, such as for protein-lipid interactions. We validated our approach using three soluble protein-ligand systems (maltose binding protein, lysozyme, and nitrogen regulatory protein) and obtained similar results to those using isothermal titration calorimetry and surface plasmon resonance. We also determined for the first time the thermodynamics of individual lipid binding to the ammonia channel (AmtB), an integral membrane protein from Escherichia coli. Remarkably, we observed distinct thermodynamic signatures for the binding of different lipids and entropy-enthalpy compensation for binding lipids of variable chain length. Additionally, using a mutant form of AmtB that abolishes a specific phosphatidylglycerol (PG) binding site, we observed distinct changes in the thermodynamic signatures for binding PG, implying these signatures can identify key residues involved in specific lipid binding and potentially differentiate between specific lipid binding sites.

The Collagen-Binding Adhesin Is a Virulence Factor in Staphylococcus aureus Keratitis

Science.gov (United States)

Rhem, Marcus N.; Lech, Elizabeth M.; Patti, Joseph M.; McDevitt, Damien; Höök, Magnus; Jones, Dan B.; Wilhelmus, Kirk R.

2000-01-01

A collagen-binding strain of Staphylococcus aureus produced suppurative inflammation in a rabbit model of soft contact lens-associated bacterial keratitis more often than its collagen-binding-negative isogenic mutant. Reintroduction of the cna gene on a multicopy plasmid into the mutant helped it regain its corneal adherence and infectivity. The topical application of a collagen-binding peptide before bacterial challenge decreased S. aureus adherence to deepithelialized corneas. These data suggest that the collagen-binding adhesin is involved in the pathogenesis of S. aureus infection of the cornea. PMID:10816547

Characterization of the dextran-binding domain in the glucan-binding protein C of Streptococcus mutans.

Science.gov (United States)

Takashima, Y; Fujita, K; Ardin, A C; Nagayama, K; Nomura, R; Nakano, K; Matsumoto-Nakano, M

2015-10-01

Streptococcus mutans produces multiple glucan-binding proteins (Gbps), among which GbpC encoded by the gbpC gene is known to be a cell-surface-associated protein involved in dextran-induced aggregation. The purpose of the present study was to characterize the dextran-binding domain of GbpC using bioinformatics analysis and molecular techniques. Bioinformatics analysis specified five possible regions containing molecular binding sites termed GB1 through GB5. Next, truncated recombinant GbpC (rGbpC) encoding each region was produced using a protein expression vector and five deletion mutant strains were generated, termed CDGB1 through CDGB5 respectively. The dextran-binding rates of truncated rGbpC that included the GB1, GB3, GB4 and GB5 regions in the upstream sequences were higher than that of the construct containing GB2 in the downstream region. In addition, the rates of dextran-binding for strains CDGB4 and CD1, which was entire gbpC deletion mutant, were significantly lower than for the other strains, while those of all other deletion mutants were quite similar to that of the parental strain MT8148. Biofilm structures formed by CDGB4 and CD1 were not as pronounced as that of MT8148, while those formed by other strains had greater density as compared to that of CD1. Our results suggest that the dextran-binding domain may be located in the GB4 region in the interior of the gbpC gene. Bioinformatics analysis is useful for determination of functional domains in many bacterial species. © 2015 The Society for Applied Microbiology.

Gonadal cell surface receptor for plasma retinol-binding protein

International Nuclear Information System (INIS)

Krishna Bhat, M.; Cama, H.R.

1979-01-01

A specific membrane receptor for plasma retinol-binding protein has been demonstrated in testicular cells. Prealbumin-2 did not show any specific binding to the membrane. The affinity of retinol-binding protein for receptor drastically decreases upon delivery of retinol and the retinol-binding protein does not enter the cell. The mechanism of delivery of retinol to the target cell by plasma retinol-binding protein has been investigated. The process involves two steps; direct binding of retinol-binding protein to the receptor and uptake of retinol by the target cell with a concomitant drastic reduction in the affinity of the retinol-binding protein to the receptor. Probably the second step of the process needs a cytosolic factor, possibly the cellular retinol-binding protein or an enzyme. The binding of retinol-binding protein to the receptor is saturable and reversible. The interaction shows a Ksub(d) value of 2.1x10 -10 . The specific binding of a retinol-binding protein with great affinity has been employed in the development of a method for radioassay of the receptor. The receptor level of the gonadal cell has been found to vary with the stage of differentiation. The receptor concentrations in 11-week-old birds and adult birds are comparable. Testosterone treatment of 11-week-old birds produced a substantial increase in the receptor concentration over control, while the protein content increased marginally, indicating that, probably, synthesis of the receptor is specifcally induced by testosterone during spermatogenesis, and the concentration of receptor is relatively higher before the formation of the acrosome. (Auth.)

Binding of ReO4- with an engineered MoO42--binding protein: towards a new approach in radiopharmaceutical applications

International Nuclear Information System (INIS)

Aryal, Baikuntha P.; Brugarolas, Pedro; He, Chuan

2012-01-01

Radiolabeled biomolecules are routinely used for clinical diagnostics. 99m Tc is the most commonly used radioactive tracer in radiopharmaceuticals. 188 Re and 186 Re are also commonly used as radioactive tracers in medicine. However, currently available methods for radiolabeling are lengthy and involve several steps in bioconjugation processes. In this work we present a strategy to engineer proteins that may selectively recognize the perrhenate (ReO 4 - ) ion as a new way to label proteins. We found that a molybdate (MoO 4 2- )-binding protein (ModA) from Escherichia coli can bind perrhenate with high affinity. Using fluorescence and isothermal titration calorimetry measurements, we determined the dissociation constant of ModA for ReO 4 - to be 541 nM and we solved a crystal structure of ModA with a bound ReO 4 - . On the basis of the structure we created a mutant protein containing a disulfide linkage, which exhibited increased affinity for perrhenate (K d = 104 nM). High-resolution crystal structures of ModA (1.7 (angstrom)) and A11C/R153C mutant (2.0 (angstrom)) were solved with bound perrhenate. Both structures show that a perrhenate ion occupies the molybdate binding site using the same amino acid residues that are involved in molybdate binding. The overall structure of the perrhenate-bound ModA is unchanged compared with that of the molybdate-bound form. In the mutant protein, the bound perrhenate is further stabilized by the engineered disulfide bond.

Low 5-HT1B receptor binding in the migraine brain

DEFF Research Database (Denmark)

Deen, Marie; Hansen, Hanne D; Hougaard, Anders

2018-01-01

Background The pathophysiology of migraine may involve dysfunction of serotonergic signaling. In particular, the 5-HT1B receptor is considered a key player due to the efficacy of 5-HT1B receptor agonists for treatment of migraine attacks. Aim To examine the cerebral 5-HT1B receptor binding....... Patients who reported migraine brain regions involved in pain modulation as regions of interest and applied a latent variable model (LVM) to assess the group effect on binding across these regions. Results Our data...... support a model wherein group status predicts the latent variable ( p = 0.038), with migraine patients having lower 5-HT1B receptor binding across regions compared to controls. Further, in a whole-brain voxel-based analysis, time since last migraine attack correlated positively with 5-HT1B receptor...

Effects of common anesthetic agents on [(18)F]flumazenil binding to the GABAA receptor

DEFF Research Database (Denmark)

Palner, Mikael; Beinat, Corinne; Banister, Sam

2016-01-01

in preclinical imaging studies and clinical imaging studies involving patient populations that do not tolerate relatively longer scan times. The objective of this study was to examine the effects of anesthesia on the binding of [(18)F]flumazenil to GABAA receptors in mice. METHODS: Brain and whole blood...... mice. CONCLUSIONS: Anesthesia has pronounced effects on the binding and blood-brain distribution of [(18)F]flumazenil. Consequently, considerable caution must be exercised in the interpretation of preclinical and clinical PET studies of GABAA receptors involving the use of anesthesia.......BACKGROUND: The availability of GABAA receptor binding sites in the brain can be assessed by positron emission tomography (PET) using the radioligand, [(18)F]flumazenil. However, the brain uptake and binding of this PET radioligand are influenced by anesthetic drugs, which are typically needed...

Secbase: database module to retrieve secondary structure elements with ligand binding motifs.

Science.gov (United States)

Koch, Oliver; Cole, Jason; Block, Peter; Klebe, Gerhard

2009-10-01

Secbase is presented as a novel extension module of Relibase. It integrates the information about secondary structure elements into the retrieval facilities of Relibase. The data are accessible via the extended Relibase user interface, and integrated retrieval queries can be addressed using an extended version of Reliscript. The primary information about alpha-helices and beta-sheets is used as provided by the PDB. Furthermore, a uniform classification of all turn families, based on recent clustering methods, and a new helix assignment that is based on this turn classification has been included. Algorithms to analyze the geometric features of helices and beta-strands were also implemented. To demonstrate the performance of the Secbase implementation, some application examples are given. They provide new insights into the involvement of secondary structure elements in ligand binding. A survey of water molecules detected next to the N-terminus of helices is analyzed to show their involvement in ligand binding. Additionally, the parallel oriented NH groups at the alpha-helix N-termini provide special binding motifs to bind particular ligand functional groups with two adjacent oxygen atoms, e.g., as found in negatively charged carboxylate or phosphate groups, respectively. The present study also shows that the specific structure of the first turn of alpha-helices provides a suitable explanation for stabilizing charged structures. The magnitude of the overall helix macrodipole seems to have no or only a minor influence on binding. Furthermore, an overview of the involvement of secondary structure elements with the recognition of some important endogenous ligands such as cofactors shows some distinct preference for particular binding motifs and amino acids.

Tetherin restricts productive HIV-1 cell-to-cell transmission.

Directory of Open Access Journals (Sweden)

Nicoletta Casartelli

2010-06-01

Full Text Available The IFN-inducible antiviral protein tetherin (or BST-2/CD317/HM1.24 impairs release of mature HIV-1 particles from infected cells. HIV-1 Vpu antagonizes the effect of tetherin. The fate of virions trapped at the cell surface remains poorly understood. Here, we asked whether tetherin impairs HIV cell-to-cell transmission, a major means of viral spread. Tetherin-positive or -negative cells, infected with wild-type or DeltaVpu HIV, were used as donor cells and cocultivated with target lymphocytes. We show that tetherin inhibits productive cell-to-cell transmission of DeltaVpu to targets and impairs that of WT HIV. Tetherin accumulates with Gag at the contact zone between infected and target cells, but does not prevent the formation of virological synapses. In the presence of tetherin, viruses are then mostly transferred to targets as abnormally large patches. These viral aggregates do not efficiently promote infection after transfer, because they accumulate at the surface of target cells and are impaired in their fusion capacities. Tetherin, by imprinting virions in donor cells, is the first example of a surface restriction factor limiting viral cell-to-cell spread.

Plant RNA binding proteins for control of RNA virus infection

Directory of Open Access Journals (Sweden)

Sung Un eHuh

2013-12-01

Full Text Available Plant RNA viruses have effective strategies to infect host plants through either direct or indirect interactions with various host proteins, thus suppressing the host immune system. When plant RNA viruses enter host cells exposed RNAs of viruses are recognized by the host immune system through processes such as siRNA-dependent silencing. Interestingly, some host RNA binding proteins have been involved in the inhibition of RNA virus replication, movement, and translation through RNA-specific binding. Host plants intensively use RNA binding proteins for defense against viral infections in nature. In this mini review, we will summarize the function of some host RNA binding proteins which act in a sequence-specific binding manner to the infecting virus RNA. It is important to understand how plants effectively suppresses RNA virus infections via RNA binding proteins, and this defense system can be potentially developed as a synthetic virus defense strategy for use in crop engineering.

Functional connectivity supporting the selective maintenance of feature-location binding in visual working memory

Directory of Open Access Journals (Sweden)

Sachiko eTakahama

2014-06-01

Full Text Available Information on an object’s features bound to its location is very important for maintaining object representations in visual working memory. Interactions with dynamic multi-dimensional objects in an external environment require complex cognitive control, including the selective maintenance of feature-location binding. Here, we used event-related functional magnetic resonance imaging to investigate brain activity and functional connectivity related to the maintenance of complex feature-location binding. Participants were required to detect task-relevant changes in feature-location binding between objects defined by color, orientation, and location. We compared a complex binding task requiring complex feature-location binding (color-orientation-location with a simple binding task in which simple feature-location binding, such as color-location, was task-relevant and the other feature was task-irrelevant. Univariate analyses showed that the dorsolateral prefrontal cortex (DLPFC, hippocampus, and frontoparietal network were activated during the maintenance of complex feature-location binding. Functional connectivity analyses indicated cooperation between the inferior precentral sulcus (infPreCS, DLPFC, and hippocampus during the maintenance of complex feature-location binding. In contrast, the connectivity for the spatial updating of simple feature-location binding determined by reanalyzing the data from Takahama et al. (2010 demonstrated that the superior parietal lobule (SPL cooperated with the DLPFC and hippocampus. These results suggest that the connectivity for complex feature-location binding does not simply reflect general memory load and that the DLPFC and hippocampus flexibly modulate the dorsal frontoparietal network, depending on the task requirements, with the infPreCS involved in the maintenance of complex feature-location binding and the SPL involved in the spatial updating of simple feature-location binding.

An overview of the prediction of protein DNA-binding sites.

Science.gov (United States)

Si, Jingna; Zhao, Rui; Wu, Rongling

2015-03-06

Interactions between proteins and DNA play an important role in many essential biological processes such as DNA replication, transcription, splicing, and repair. The identification of amino acid residues involved in DNA-binding sites is critical for understanding the mechanism of these biological activities. In the last decade, numerous computational approaches have been developed to predict protein DNA-binding sites based on protein sequence and/or structural information, which play an important role in complementing experimental strategies. At this time, approaches can be divided into three categories: sequence-based DNA-binding site prediction, structure-based DNA-binding site prediction, and homology modeling and threading. In this article, we review existing research on computational methods to predict protein DNA-binding sites, which includes data sets, various residue sequence/structural features, machine learning methods for comparison and selection, evaluation methods, performance comparison of different tools, and future directions in protein DNA-binding site prediction. In particular, we detail the meta-analysis of protein DNA-binding sites. We also propose specific implications that are likely to result in novel prediction methods, increased performance, or practical applications.

Residues in the H+ Translocation Site Define the pKa for Sugar Binding to LacY†

Science.gov (United States)

Smirnova, Irina; Kasho, Vladimir; Sugihara, Junichi; Choe, Jun-Yong; Kaback, H. Ronald

2009-01-01

A remarkably high pKa of approximately 10.5 has been determined for sugar-binding affinity to the lactose permease of Escherichia coli (LacY), indicating that, under physiological conditions, substrate binds to fully protonated LacY. We have now systematically tested site-directed replacements for the residues involved in sugar binding, as well as H+ translocation and coupling, in order to determine which residues may be responsible for this alkaline pKa. Mutations in the sugar-binding site (Glu126, Trp151, Glu269) markedly decrease affinity for sugar but do not alter the pKa for binding. In contrast, replacements for residues involved in H+ translocation (Arg302, Tyr236, His322, Asp240, Glu325, Lys319) exhibit pKa values for sugar binding that are either shifted toward neutral pH or independent of pH. Values for the apparent dissociation constant for sugar binding (Kdapp) increase greatly for all mutants except neutral replacements for Glu325 or Lys319, which are characterized by remarkably high affinity sugar binding (i.e., low Kdapp) from pH 5.5 to pH 11. The pH dependence of the on- and off-rate constants for sugar binding measured directly by stopped-flow fluorometry implicates koff as a major factor for the affinity change at alkaline pH and confirms the effects of pH on Kdapp inferred from steady-state fluorometry. These results indicate that the high pKa for sugar binding by wild-type LacY cannot be ascribed to any single amino acid residue but appears to reside within a complex of residues involved in H+ translocation. There is structural evidence for water bound in this complex, and the water could be the site of protonation responsible for the pH dependence of sugar binding. PMID:19689129

Participation of Water in the Binding of Estrogen Receptor with Estrogen Responsive Element in vitro.

Science.gov (United States)

Zhu, Guo-Zhang; Tang, Guo-Qing; Ruan, Kang-Cheng; Gong, Yue-Ting; Zhang, Yong-Lian

1998-01-01

Many reports have showed that bound water was involved in the interaction between/among the macromolecules. However, it has not been reported whether bound water is also involved in the binding of trans-factors and cis-elements in the regulation of the eukaryotic gene trans-cription or not. Preliminary studies have been made on the effect of bound water on the binding of estrogen receptor with estrogen responsive element in vitro. In the gel retardation assay using the cytosol extract of rat uterus as the supplier of estrogen receptor and 32 bp oligonucleotide containing a concensus vitellogenin A(2) ERE as the probe, various cosolvents, such as glycerol, sucrose, N-dimethylformamide and dimethylsulfoxide, were added respectively to the reaction mixture in varying concentrations to regulate the osmotic pressure. The results indicated that the binding of ER-ERE was enhanced with the increase in the final concentration of these individual cosolvents. On the other hand, when the reaction was carried out under an increasing hydrostatic pressure, the ER-ERE binding was decreased sharply. After decompression the binding of ER-ERE was gradually restored to the normal level with the lapse of time. These results suggested that bound water was directly involved in the binding of ER-ERE and may play an important role in the regulation of the eukaryotic gene transcription.

Predictions of RNA-binding ability and aggregation propensity of proteins

OpenAIRE

Agostini, Federico, 1985-

2014-01-01

RNA-binding proteins (RBPs) control the fate of a multitude of coding and non-coding transcripts. Formation of ribonucleoprotein (RNP) complexes fine-tunes regulation of post-transcriptional events and influences gene expression. Recently, it has been observed that non-canonical proteins with RNA-binding ability are enriched in structurally disordered and low-complexity regions that are generally involved in functional and dysfunctional associations. Therefore, it is possible that interaction...

Characterization of 6-mercaptopurine binding to bovine serum albumin and its displacement from the binding sites by quercetin and rutin

Energy Technology Data Exchange (ETDEWEB)

Ehteshami, Mehdi [Nutrition Research Center, School of Health and Nutrition, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of); Rasoulzadeh, Farzaneh [Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of); Mahboob, Soltanali [Nutrition Research Center, School of Health and Nutrition, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of); Rashidi, Mohammad-Reza, E-mail: rashidi@tbzmed.ac.ir [Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of)

2013-03-15

Binding of a drug to the serum albumins as major serum transport proteins can be influenced by other ligands leading to alteration of its pharmacological properties. In the present study, binding characteristics of 6-mercaptopurine (6-MP) with bovine serum albumin (BSA) together with its displacement from its binding site by quercetin and rutin have been investigated by the spectroscopic method. According to the binding parameters, a static quenching component in overall dynamic quenching process is operative in the interaction between 6-MP and BSA. The binding of 6-MP to BSA occurred spontaneously due to entropy-driven hydrophobic interactions. The synchronous fluorescence spectroscopy study revealed that the secondary structure of BSA is changed in the presence of 6-MP and both Tyr and Trp residues participate in the interaction between 6-MP and BSA with the later one being more dominant. The binding constant value of 6-MP-BSA in the presence of quercetin and rutin increased. 6-MP was displaced by ibuprofen indicating that the binding site of 6-MP on albumin is site II. Therefore, the change of the pharmacokinetic and pharmacodynamic properties of 6-MP by quercetin and rutin through alteration of binding capacity of 6-MP to the serum albumin cannot be ruled out. In addition, the displacement study showed that 6-MP is located in site II of BSA. - Highlights: Black-Right-Pointing-Pointer Participation of both Tyr and particularly Trp residues in the interaction between 6-MP and BSA. Black-Right-Pointing-Pointer Involvement of a static quenching component in an overall dynamic quenching process. Black-Right-Pointing-Pointer Ability of quercetin and rutin to change the binding constants of 6-MP-BSA complex. Black-Right-Pointing-Pointer Binding of 6-MP to BSA through entropy-driven hydrophobic interactions.

Characterization of a 105-kDa plasma membrane associated glycoprotein that is involved in West Nile virus binding and infection

International Nuclear Information System (INIS)

Chu, J.J.H.; Ng, M.L.

2003-01-01

This study attempts to isolate and characterize West Nile virus-binding molecules on the plasma membrane of Vero and murine neuroblastoma cells that is responsible for virus entry. Pretreatment of Vero cells with proteases, glycosidases (endoglycosidase H, α-mannosidase), and sodium periodate strongly inhibited West Nile virus infection, whereas treatments with phospholipases and heparinases had no effect. The virus overlay protein blot detected a 105-kDa molecule on the plasma membrane extract of Vero and murine neuroblastoma cells that bind to WN virus. Treatment of the 105-kDa molecules with β-mercaptoethanol resulted in the virus binding to a series of lower molecular weight bands ranging from 30 to 40 kDa. The disruption of disulfide-linked subunits did not affect virus binding. N-linked sugars with mannose residues on the 105-kDa membrane proteins were found to be important in virus binding. Specific antibodies against the 105-kDa glycoprotein were highly effective in blocking virus entry. These results strongly supported the possibility that the 105-kDa protease-sensitive glycoprotein with complex N-linked sugars could be the putative receptor for WN virus

The interrelationship between ligand binding and thermal unfolding of the folate binding protein. The role of self-association and pH

DEFF Research Database (Denmark)

Holm, Jan; Babol, Linnea N.; Markova, Natalia

2014-01-01

The present study utilized a combination of DLS (dynamic light scattering) and DSC (differential scanning calorimetry) to address thermostability of high-affinity folate binding protein (FBP), a transport protein and cellular receptor for the vitamin folate. At pH7.4 (pI=7-8) ligand binding......, intermolecular forces involved in concentration-dependent multimerization thus contribute to the thermostability of holo-FBP. Hence, thermal unfolding and dissociation of holo-FBP multimers occur simultaneously consistent with a gradual decrease from octameric to monomeric holo-FBP (10μM) in DLS after a step-wise...

Distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases of Actinobacteria.

Science.gov (United States)

Ogawara, Hiroshi

2016-09-01

PASTA domains (penicillin-binding protein and serine/threonine kinase-associated domains) have been identified in penicillin-binding proteins and serine/threonine kinases of Gram-positive Firmicutes and Actinobacteria. They are believed to bind β-lactam antibiotics, and be involved in peptidoglycan metabolism, although their biological function is not definitively clarified. Actinobacteria, especially Streptomyces species, are distinct in that they undergo complex cellular differentiation and produce various antibiotics including β-lactams. This review focuses on the distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases in Actinobacteria. In Actinobacteria, PASTA domains are detectable exclusively in class A but not in class B penicillin-binding proteins, in sharp contrast to the cases in other bacteria. In penicillin-binding proteins, PASTA domains distribute independently from taxonomy with some distribution bias. Particularly interesting thing is that no Streptomyces species have penicillin-binding protein with PASTA domains. Protein kinases in Actinobacteria possess 0 to 5 PASTA domains in their molecules. Protein kinases in Streptomyces can be classified into three groups: no PASTA domain, 1 PASTA domain and 4 PASTA domain-containing groups. The 4 PASTA domain-containing groups can be further divided into two subgroups. The serine/threonine kinases in different groups may perform different functions. The pocket region in one of these subgroup is more dense and extended, thus it may be involved in binding of ligands like β-lactams more efficiently.

Identification of Inhibitors of Biological Interactions Involving Intrinsically Disordered Proteins

Directory of Open Access Journals (Sweden)

Daniela Marasco

2015-04-01

Full Text Available Protein–protein interactions involving disordered partners have unique features and represent prominent targets in drug discovery processes. Intrinsically Disordered Proteins (IDPs are involved in cellular regulation, signaling and control: they bind to multiple partners and these high-specificity/low-affinity interactions play crucial roles in many human diseases. Disordered regions, terminal tails and flexible linkers are particularly abundant in DNA-binding proteins and play crucial roles in the affinity and specificity of DNA recognizing processes. Protein complexes involving IDPs are short-lived and typically involve short amino acid stretches bearing few “hot spots”, thus the identification of molecules able to modulate them can produce important lead compounds: in this scenario peptides and/or peptidomimetics, deriving from structure-based, combinatorial or protein dissection approaches, can play a key role as hit compounds. Here, we propose a panoramic review of the structural features of IDPs and how they regulate molecular recognition mechanisms focusing attention on recently reported drug-design strategies in the field of IDPs.

An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase

DEFF Research Database (Denmark)

Wong, Mei Mei Jaslyn Elizabeth; Midtgaard, Søren Roi; Gysel, Kira

2015-01-01

of multiple LysM domains in substrate binding has so far lacked support from high-resolution structures of ligand-bound complexes. Here, a structural study of the Thermus thermophilus NlpC/P60 endopeptidase containing two LysM domains is presented. The crystal structure and small-angle X-ray scattering...

Reduced post-synaptic serotonin type 1A receptor binding in bipolar depression

Science.gov (United States)

Nugent, Allison C.; Bain, Earle E.; Carlson, Paul J.; Neumeister, Alexander; Bonne, Omer; Carson, Richard E.; Eckelman, William; Herscovitch, Peter; Zarate, Carlos A.; Charney, Dennis S.; Drevets, Wayne C.

2013-01-01

Multiple lines of evidence suggest that serotonin type 1A (5-HT1A) receptor dysfunction is involved in the pathophysiology of mood disorders, and that alterations in 5-HT1A receptor function play a role in the mechanisms of antidepressant and mood stabilizer treatment. The literature is in disagreement, however, as to whether 5-HT1A receptor binding abnormalities exist in bipolar disorder (BD). We acquired PET images of 5-HT1A receptor binding in 26 unmedicated BD subjects and 37 healthy controls using [18F]FCWAY, a highly selective 5-HT1A receptor radio-ligand. The mean 5-HT1A receptor binding potential (BPP) was significantly lower in BD subjects compared to controls in cortical regions where 5-HT1A receptors are expressed post-synaptically, most prominently in the mesiotemporal cortex. Post-hoc assessments involving other receptor specific binding parameters suggested that this difference particularly affected the females with BD. The mean BPP did not differ between groups in the raphe nucleus, however, where 5-HT1A receptors are predominantly expressed pre-synaptically. Across subjects the BPP in the mesiotemporal cortex was inversely correlated with trough plasma cortisol levels, consistent with preclinical literature indicating that hippocampal 5-HT1A receptor expression is inhibited by glucocorticoid receptor stimulation. These findings suggest that 5-HT1A receptor binding is abnormally reduced in BD, and this abnormality may particularly involve the postsynaptic 5-HT1A receptor system of individuals with a tendency toward cortisol hypersecretion. PMID:23434290

RNA-binding properties and mapping of the RNA-binding domain from the movement protein of Prunus necrotic ringspot virus.

Science.gov (United States)

Herranz, M Carmen; Pallás, Vicente

2004-03-01

The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is involved in intercellular virus transport. In this study, putative RNA-binding properties of the PNRSV MP were studied. The PNRSV MP was produced in Escherichia coli using an expression vector. Electrophoretic mobility shift assays (EMSAs) using DIG-labelled riboprobes demonstrated that PNRSV MP bound ssRNA cooperatively without sequence specificity. Two different ribonucleoprotein complexes were found to be formed depending on the molar MP : PNRSV RNA ratio. The different responses of the complexes to urea treatment strongly suggested that they have different structural properties. Deletion mutagenesis followed by Northwestern analysis allowed location of a nucleic acid binding domain to aa 56-88. This 33 aa RNA-binding motif is the smallest region delineated among members of the family Bromoviridae for which RNA-binding properties have been demonstrated. This domain is highly conserved within all phylogenetic subgroups previously described for PNRSV isolates. Interestingly, the RNA-binding domain described here and the one described for Alfamovirus are located at the N terminus of their corresponding MPs, whereas similar domains previously characterized in members of the genera Bromovirus and Cucumovirus are present at the C terminus, strongly reflecting their corresponding phylogenetic relationships. The evolutionary implications of this observation are discussed.

Identification of FUSE-binding proteins as interacting partners of TIA proteins

International Nuclear Information System (INIS)

Rothe, Francoise; Gueydan, Cyril; Bellefroid, Eric; Huez, Georges; Kruys, Veronique

2006-01-01

TIA-1 and TIAR are closely related RNA-binding proteins involved in several mechanisms of RNA metabolism, including alternative hnRNA splicing and mRNA translation regulation. In particular, TIA-1 represses tumor necrosis factor (TNF) mRNA translation by binding to the AU-rich element (ARE) present in the mRNA 3' untranslated region. Here, we demonstrate that TIA proteins interact with FUSE-binding proteins (FBPs) and that fbp genes are co-expressed with tia genes during Xenopus embryogenesis. FBPs participate in various steps of RNA processing and degradation. In Cos cells, FBPs co-localize with TIA proteins in the nucleus and migrate into TIA-enriched cytoplasmic granules upon oxidative stress. Overexpression of FBP2-KH3 RNA-binding domain fused to EGFP induces the specific sequestration of TIA proteins in cytoplasmic foci, thereby precluding their nuclear accumulation. In cytosolic RAW 264.7 macrophage extracts, FBPs are found associated in EMSA to the TIA-1/TNF-ARE complex. Together, our results indicate that TIA and FBP proteins may thus be relevant biological involved in common events of RNA metabolism occurring both in the nucleus and the cytoplasm

Calcium-dependent binding of Escherichia coli alpha-hemolysin to erythrocytes

International Nuclear Information System (INIS)

Boehm, D.F.

1989-01-01

Alpha hemolysin (AH), a protein secreted by certain strains of Escherichia coli, causes lysis of erythrocytes (RBCs) and is cytotoxic for other cells. The primary structure of AH contains an eight amino acid sequence tandemly repeated 13 times near the C-terminus. These repeated sequences are essential for hemolytic activity. AH also requires an unknown modification by an accessory protein, Hly C, for hemolytic activity. The role of calcium in the interaction of Ah with RBCs was investigated using recombinant strains which produced active and inactive forms of the toxin. Hemolytic activity was calcium-dependent. Osmotic protection experiments and immunoblots of SDS-PAGE separated proteins from washed, toxin-treated RBCs showed that the binding of active AH to RBCs was calcium-dependent. Binding of active AH to RBCs increased the calcium permeability of RBC membranes and resulted in changes in membrane protein profiles. The changes in membrane proteins did not cause the lysis of the cells. These results were consistent with a mechanism of lysis involving the formation of cation-selective pores in the membranes of target cells. 45 Ca-autoradiography of the recombinant hemolysins separated by SDS-PAGE and transferred to nitrocellulose showed that active AH bound calcium. The domain involved in binding calcium was identified as the tandemly repeated sequences since a deletion hemolysin missing 11 of the 13 repeated sequences did not bind calcium. This deletion hemolysin was non-hemolytic and did not bind to RBC membranes. Hemolysin lacking the Hly C modification was also non-hemolytic and did not bind to RBC membranes. This unmodified AH contained the repeated sequences and bound calcium as efficiently as active AH

Identification of potential nuclear reprogramming and differentiation factors by a novel selection method for cloning chromatin-binding proteins

International Nuclear Information System (INIS)

Wang Liu; Zheng Aihua; Yi Ling; Xu Chongren; Ding Mingxiao; Deng Hongkui

2004-01-01

Nuclear reprogramming is critical for animal cloning and stem cell creation through nuclear transfer, which requires extensive remodeling of chromosomal architecture involving dramatic changes in chromatin-binding proteins. To understand the mechanism of nuclear reprogramming, it is critical to identify chromatin-binding factors specify the reprogramming process. In this report, we have developed a high-throughput selection method, based on T7 phage display and chromatin immunoprecipitation, to isolate chromatin-binding factors expressed in mouse embryonic stem cells using primary mouse embryonic fibroblast chromatin. Seven chromatin-binding proteins have been isolated by this method. We have also isolated several chromatin-binding proteins involved in hepatocyte differentiation. Our method provides a powerful tool to rapidly and selectively identify chromatin-binding proteins. The method can be used to study epigenetic modification of chromatin during nuclear reprogramming, cell differentiation, and transdifferentiation

Detection of secondary binding sites in proteins using fragment screening.

Science.gov (United States)

Ludlow, R Frederick; Verdonk, Marcel L; Saini, Harpreet K; Tickle, Ian J; Jhoti, Harren

2015-12-29

Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets.

The binding of cytochrome c to neuroglobin: A docking and surface plasmon resonance study

DEFF Research Database (Denmark)

Bønding, Signe Helbo; Henty, K.; Dingley, A.J.

2008-01-01

is associated with a small unfavourable enthalpy change (1.9 kcal mol-1) and a moderately large, favourable entropy change (14.8 cal mol-1 deg-1). The sensitivity of the binding constant to the presence of salt suggests that the complex formation involves electrostatic interactions....... one major binding site for cytochrome c to neuroglobin. The results yield a plausible structure for the most likely complex structure in which the hemes of each protein are in close contact. NMR analysis identifies the formation of a weak complex in which the heme group of cytochrome c is involved....... surface plasmon resonance studies provide a value of 45 μM for the equilibrium constant for cytochrome c binding to neuroglobin, which increases significantly as the ionic strength of the solution increases. The temperature dependence of the binding constant indicates that the complex formation...

Nucleic acids encoding phloem small RNA-binding proteins and transgenic plants comprising them

Science.gov (United States)

Lucas, William J.; Yoo, Byung-Chun; Lough, Tony J.; Varkonyi-Gasic, Erika

2007-03-13

The present invention provides a polynucleotide sequence encoding a component of the protein machinery involved in small RNA trafficking, Cucurbita maxima phloem small RNA-binding protein (CmPSRB 1), and the corresponding polypeptide sequence. The invention also provides genetic constructs and transgenic plants comprising the polynucleotide sequence encoding a phloem small RNA-binding protein to alter (e.g., prevent, reduce or elevate) non-cell autonomous signaling events in the plants involving small RNA metabolism. These signaling events are involved in a broad spectrum of plant physiological and biochemical processes, including, for example, systemic resistance to pathogens, responses to environmental stresses, e.g., heat, drought, salinity, and systemic gene silencing (e.g., viral infections).

Structural and functional characterization of an orphan ATP-binding cassette ATPase involved in manganese utilization and tolerance in Leptospira spp.

Science.gov (United States)

Benaroudj, Nadia; Saul, Frederick; Bellalou, Jacques; Miras, Isabelle; Weber, Patrick; Bondet, Vincent; Murray, Gerald L; Adler, Ben; Ristow, Paula; Louvel, Hélène; Haouz, Ahmed; Picardeau, Mathieu

2013-12-01

Pathogenic Leptospira species are the etiological agents of the widespread zoonotic disease leptospirosis. Most organisms, including Leptospira, require divalent cations for proper growth, but because of their high reactivity, these metals are toxic at high concentrations. Therefore, bacteria have acquired strategies to maintain metal homeostasis, such as metal import and efflux. By screening Leptospira biflexa transposon mutants for their ability to use Mn(2+), we have identified a gene encoding a putative orphan ATP-binding cassette (ABC) ATPase of unknown function. Inactivation of this gene in both L. biflexa and L. interrogans strains led to mutants unable to grow in medium in which iron was replaced by Mn(2+), suggesting an involvement of this ABC ATPase in divalent cation uptake. A mutation in this ATPase-coding gene increased susceptibility to Mn(2+) toxicity. Recombinant ABC ATPase of the pathogen L. interrogans exhibited Mg(2+)-dependent ATPase activity involving a P-loop motif. The structure of this ATPase was solved from a crystal containing two monomers in the asymmetric unit. Each monomer adopted a canonical two-subdomain organization of the ABC ATPase fold with an α/β subdomain containing the Walker motifs and an α subdomain containing the ABC signature motif (LSSGE). The two monomers were arranged in a head-to-tail orientation, forming a V-shaped particle with all the conserved ABC motifs at the dimer interface, similar to functional ABC ATPases. These results provide the first structural and functional characterization of a leptospiral ABC ATPase.

A unique bivalent binding and inhibition mechanism by the yatapoxvirus interleukin 18 binding protein.

Directory of Open Access Journals (Sweden)

Brian Krumm

Full Text Available Interleukin 18 (IL18 is a cytokine that plays an important role in inflammation as well as host defense against microbes. Mammals encode a soluble inhibitor of IL18 termed IL18 binding protein (IL18BP that modulates IL18 activity through a negative feedback mechanism. Many poxviruses encode homologous IL18BPs, which contribute to virulence. Previous structural and functional studies on IL18 and IL18BPs revealed an essential binding hot spot involving a lysine on IL18 and two aromatic residues on IL18BPs. The aromatic residues are conserved among the very diverse mammalian and poxviruses IL18BPs with the notable exception of yatapoxvirus IL18BPs, which lack a critical phenylalanine residue. To understand the mechanism by which yatapoxvirus IL18BPs neutralize IL18, we solved the crystal structure of the Yaba-Like Disease Virus (YLDV IL18BP and IL18 complex at 1.75 Å resolution. YLDV-IL18BP forms a disulfide bonded homo-dimer engaging IL18 in a 2∶2 stoichiometry, in contrast to the 1∶1 complex of ectromelia virus (ECTV IL18BP and IL18. Disruption of the dimer interface resulted in a functional monomer, however with a 3-fold decrease in binding affinity. The overall architecture of the YLDV-IL18BP:IL18 complex is similar to that observed in the ECTV-IL18BP:IL18 complex, despite lacking the critical lysine-phenylalanine interaction. Through structural and mutagenesis studies, contact residues that are unique to the YLDV-IL18BP:IL18 binding interface were identified, including Q67, P116 of YLDV-IL18BP and Y1, S105 and D110 of IL18. Overall, our studies show that YLDV-IL18BP is unique among the diverse family of mammalian and poxvirus IL-18BPs in that it uses a bivalent binding mode and a unique set of interacting residues for binding IL18. However, despite this extensive divergence, YLDV-IL18BP binds to the same surface of IL18 used by other IL18BPs, suggesting that all IL18BPs use a conserved inhibitory mechanism by blocking a putative receptor-binding

Item-location binding in working memory: is it hippocampus-dependent?

Science.gov (United States)

Allen, Richard J; Vargha-Khadem, Faraneh; Baddeley, Alan D

2014-07-01

A general consensus is emerging that the hippocampus has an important and active role in the creation of new long-term memory representations of associations or bindings between elements. However, it is less clear whether this contribution can be extended to the creation of temporary bound representations in working memory, involving the retention of small numbers of items over short delays. We examined this by administering a series of recognition and recall tests of working memory for colour-location binding and object-location binding to a patient with highly selective hippocampal damage (Jon), and groups of control participants. Jon achieved high levels of accuracy in all working memory tests of recognition and recall binding across retention intervals of up to 10s. In contrast, Jon performed at chance on an unexpected delayed test of the same object-location binding information. These findings indicate a clear dissociation between working memory and long-term memory, with no evidence for a critical hippocampal contribution to item-location binding in working memory. Copyright © 2014 Elsevier Ltd. All rights reserved.

An Overview of the Prediction of Protein DNA-Binding Sites

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Jingna Si

2015-03-01

Full Text Available Interactions between proteins and DNA play an important role in many essential biological processes such as DNA replication, transcription, splicing, and repair. The identification of amino acid residues involved in DNA-binding sites is critical for understanding the mechanism of these biological activities. In the last decade, numerous computational approaches have been developed to predict protein DNA-binding sites based on protein sequence and/or structural information, which play an important role in complementing experimental strategies. At this time, approaches can be divided into three categories: sequence-based DNA-binding site prediction, structure-based DNA-binding site prediction, and homology modeling and threading. In this article, we review existing research on computational methods to predict protein DNA-binding sites, which includes data sets, various residue sequence/structural features, machine learning methods for comparison and selection, evaluation methods, performance comparison of different tools, and future directions in protein DNA-binding site prediction. In particular, we detail the meta-analysis of protein DNA-binding sites. We also propose specific implications that are likely to result in novel prediction methods, increased performance, or practical applications.

Expression of an expansin carbohydrate-binding module affects ...

African Journals Online (AJOL)

Expansins are believed to be involved in disrupting the non-covalent adhesion of cellulose to matrix polysaccharides, thereby promoting wall creep. We have targeted a putative potato expansin (EXPA) carbohydrate-binding module (CBM) to the cell walls of tobacco plants. Histological examinations and electron ...

Evolutionarily conserved bias of amino-acid usage refines the definition of PDZ-binding motif

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Launey Thomas

2011-06-01

Full Text Available Abstract Background The interactions between PDZ (PSD-95, Dlg, ZO-1 domains and PDZ-binding motifs play central roles in signal transductions within cells. Proteins with PDZ domains bind to PDZ-binding motifs almost exclusively when the motifs are located at the carboxyl (C- terminal ends of their binding partners. However, it remains little explored whether PDZ-binding motifs show any preferential location at the C-terminal ends of proteins, at genome-level. Results Here, we examined the distribution of the type-I (x-x-S/T-x-I/L/V or type-II (x-x-V-x-I/V PDZ-binding motifs in proteins encoded in the genomes of five different species (human, mouse, zebrafish, fruit fly and nematode. We first established that these PDZ-binding motifs are indeed preferentially present at their C-terminal ends. Moreover, we found specific amino acid (AA bias for the 'x' positions in the motifs at the C-terminal ends. In general, hydrophilic AAs were favored. Our genomics-based findings confirm and largely extend the results of previous interaction-based studies, allowing us to propose refined consensus sequences for all of the examined PDZ-binding motifs. An ontological analysis revealed that the refined motifs are functionally relevant since a large fraction of the proteins bearing the motif appear to be involved in signal transduction. Furthermore, co-precipitation experiments confirmed two new protein interactions predicted by our genomics-based approach. Finally, we show that influenza virus pathogenicity can be correlated with PDZ-binding motif, with high-virulence viral proteins bearing a refined PDZ-binding motif. Conclusions Our refined definition of PDZ-binding motifs should provide important clues for identifying functional PDZ-binding motifs and proteins involved in signal transduction.

Functional Advantages of Conserved Intrinsic Disorder in RNA-Binding Proteins.

Science.gov (United States)

Varadi, Mihaly; Zsolyomi, Fruzsina; Guharoy, Mainak; Tompa, Peter

2015-01-01

Proteins form large macromolecular assemblies with RNA that govern essential molecular processes. RNA-binding proteins have often been associated with conformational flexibility, yet the extent and functional implications of their intrinsic disorder have never been fully assessed. Here, through large-scale analysis of comprehensive protein sequence and structure datasets we demonstrate the prevalence of intrinsic structural disorder in RNA-binding proteins and domains. We addressed their functionality through a quantitative description of the evolutionary conservation of disordered segments involved in binding, and investigated the structural implications of flexibility in terms of conformational stability and interface formation. We conclude that the functional role of intrinsically disordered protein segments in RNA-binding is two-fold: first, these regions establish extended, conserved electrostatic interfaces with RNAs via induced fit. Second, conformational flexibility enables them to target different RNA partners, providing multi-functionality, while also ensuring specificity. These findings emphasize the functional importance of intrinsically disordered regions in RNA-binding proteins.

Functional Advantages of Conserved Intrinsic Disorder in RNA-Binding Proteins.

Directory of Open Access Journals (Sweden)

Mihaly Varadi

Full Text Available Proteins form large macromolecular assemblies with RNA that govern essential molecular processes. RNA-binding proteins have often been associated with conformational flexibility, yet the extent and functional implications of their intrinsic disorder have never been fully assessed. Here, through large-scale analysis of comprehensive protein sequence and structure datasets we demonstrate the prevalence of intrinsic structural disorder in RNA-binding proteins and domains. We addressed their functionality through a quantitative description of the evolutionary conservation of disordered segments involved in binding, and investigated the structural implications of flexibility in terms of conformational stability and interface formation. We conclude that the functional role of intrinsically disordered protein segments in RNA-binding is two-fold: first, these regions establish extended, conserved electrostatic interfaces with RNAs via induced fit. Second, conformational flexibility enables them to target different RNA partners, providing multi-functionality, while also ensuring specificity. These findings emphasize the functional importance of intrinsically disordered regions in RNA-binding proteins.

A novel cell binding site in the coiled‐coil domain of laminin involved in capillary morphogenesis

DEFF Research Database (Denmark)

Sanz, Laura; García-Bermejo, Laura; Blanco, Francisco J

2003-01-01

Recently, we reported the isolation and characterization of an anti‐laminin antibody that modulates the extracellular matrix‐dependent morphogenesis of endothelial cells. Here we use this antibody to precisely map the binding site responsible for mediating this biologically important interaction....

Binding of ReO4(-) with an engineered MoO4(2-)-binding protein: towards a new approach in radiopharmaceutical applications.

Science.gov (United States)

Aryal, Baikuntha P; Brugarolas, Pedro; He, Chuan

2012-01-01

Radiolabeled biomolecules are routinely used for clinical diagnostics. (99m)Tc is the most commonly used radioactive tracer in radiopharmaceuticals. (188)Re and (186)Re are also commonly used as radioactive tracers in medicine. However, currently available methods for radiolabeling are lengthy and involve several steps in bioconjugation processes. In this work we present a strategy to engineer proteins that may selectively recognize the perrhenate (ReO(4)(-)) ion as a new way to label proteins. We found that a molybdate (MoO(4)(2-))-binding protein (ModA) from Escherichia coli can bind perrhenate with high affinity. Using fluorescence and isothermal titration calorimetry measurements, we determined the dissociation constant of ModA for ReO(4)(-) to be 541 nM and we solved a crystal structure of ModA with a bound ReO(4)(-). On the basis of the structure we created a mutant protein containing a disulfide linkage, which exhibited increased affinity for perrhenate (K(d) = 104 nM). High-resolution crystal structures of ModA (1.7 Å) and A11C/R153C mutant (2.0 Å) were solved with bound perrhenate. Both structures show that a perrhenate ion occupies the molybdate binding site using the same amino acid residues that are involved in molybdate binding. The overall structure of the perrhenate-bound ModA is unchanged compared with that of the molybdate-bound form. In the mutant protein, the bound perrhenate is further stabilized by the engineered disulfide bond. © SBIC 2011

Proadifen-sensitive high affinity binding of 3H-alaproclate to liver membranes

International Nuclear Information System (INIS)

Ross, S.B.

1987-01-01

3 H-alaproclate, a selective 5 h ydroxytryptamine uptake inhibitor, was found to bind to microsomal membranes from the rat liver with high affinity (K D -=3 nM) and large capacity (B max about 2 nmol/g liver). This binding was stereoselective since S-( - )-alaproclate was 30 times more potent than the R-( + )-enantiomer to displace the 3 H-labelled racemate. Proadifen (SKF 525A), an inhibitor of cytochrome P-450, displaced the 3 H-alaproclate binding with the same, high affinity (K i =3 nM) as alaproclate itself. Repeated treatment with phenobarbital sodium (5x75 mg/kg intraperitoneally) increased the number of alaproclate binding sites 7-8 times without changing the affinity. However, most of the phenobarbital induced 3 H-alaproclate binding was not displaceable by proadifen, showing the presence of at least two different high affinity binding sites. The possible involvement of cytochrome P-450 in the alaproclate binding is discussed. (author)

Binding of host-selective toxin analogs to mitochondria from normal and Texas male sterile cytoplasm maize

International Nuclear Information System (INIS)

Frantzen, K.A.; Daly, J.M.; Knoche, H.W.

1987-01-01

Tritium-labeled toxin analogs were prepared by reduction with NaB 3 H 4 of either the toxin from Helminthosporium maydis race T or a toxin component from Phyllosticta maydis. These reduced analogs had high radiochemical specific activities, high biological activities, and plant specificities identical to the native toxins. A filtration assay was developed to test the binding of these labeled analogs to isolated mitochondria. Binding was not energy dependent nor was there measurable matrical uptake. The analogs were shown to be lipophilic, a characteristic which gave rise to considerable nondisplaceable binding. Under conditions limiting nondisplaceable binding, the displaceable binding was shown to be linear with respect to toxin concentration and unsaturable. No significant differences were observed in the binding characteristics between the mitochondria from normal and male-sterile (Texas) cytoplasm maize. The findings suggest that, at physiologically relevant concentrations, these toxin analogs permeate the membranes of susceptible and resistant mitochondria alike. The lack of demonstrable specific binding does not rule out the involvement of a classical receptor site but does indicate that other kinds of molecular interactions may be involved in the mechanisms for toxicity and specificity

Stanniocalcin 1 binds hemin through a partially conserved heme regulatory motif

International Nuclear Information System (INIS)

Westberg, Johan A.; Jiang, Ji; Andersson, Leif C.

2011-01-01

Highlights: → Stanniocalcin 1 (STC1) binds heme through novel heme binding motif. → Central iron atom of heme and cysteine-114 of STC1 are essential for binding. → STC1 binds Fe 2+ and Fe 3+ heme. → STC1 peptide prevents oxidative decay of heme. -- Abstract: Hemin (iron protoporphyrin IX) is a necessary component of many proteins, functioning either as a cofactor or an intracellular messenger. Hemoproteins have diverse functions, such as transportation of gases, gas detection, chemical catalysis and electron transfer. Stanniocalcin 1 (STC1) is a protein involved in respiratory responses of the cell but whose mechanism of action is still undetermined. We examined the ability of STC1 to bind hemin in both its reduced and oxidized states and located Cys 114 as the axial ligand of the central iron atom of hemin. The amino acid sequence differs from the established (Cys-Pro) heme regulatory motif (HRM) and therefore presents a novel heme binding motif (Cys-Ser). A STC1 peptide containing the heme binding sequence was able to inhibit both spontaneous and H 2 O 2 induced decay of hemin. Binding of hemin does not affect the mitochondrial localization of STC1.

New Mechanisms of Mercury Binding to Peat

Science.gov (United States)

Nagy, K. L.; Manceau, A.; Gasper, J. D.; Ryan, J. N.; Aiken, G. R.

2007-12-01

Mercury can be immobilized in the aquatic environment by binding to peat, a solid form of natural organic matter. Binding mechanisms can vary in strength and reversibility, and therefore will control concentrations of bioreactive mercury, may explain rates of mercury methylation, and are important for designing approaches to improve water quality using natural wetlands or engineered phytoremediation schemes. In addition, strong binding between mercury and peat is likely to result in the fixation of mercury that ultimately resides in coal. The mechanisms by which aqueous mercury at low concentrations reacts with both dissolved and solid natural organic matter remain incompletely understood, despite recent efforts. We have identified three distinct binding mechanisms of divalent cationic mercury to solid peats from the Florida Everglades using EXAFS spectroscopic data (FAME beamline, European Synchrotron Radiation Facility (ESRF)) obtained on experimental samples as compared to relevant references including mercury-bearing solids and mercury bound to various organic molecules. The proportions of the three molecular configurations vary with Hg concentration, and two new configurations that involve sulfur ligands occur at Hg concentrations up to about 4000 ppm. The binding mechanism at the lowest experimental Hg concentration (60-80 ppm) elucidates published reports on the inhibition of metacinnabar formation in the presence of Hg-bearing solutions and dissolved natural organic matter, and also, the differences in extent of mercury methylation in distinct areas of the Florida Everglades.

Comprehensive human transcription factor binding site map for combinatory binding motifs discovery.

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Arnoldo J Müller-Molina

Full Text Available To know the map between transcription factors (TFs and their binding sites is essential to reverse engineer the regulation process. Only about 10%-20% of the transcription factor binding motifs (TFBMs have been reported. This lack of data hinders understanding gene regulation. To address this drawback, we propose a computational method that exploits never used TF properties to discover the missing TFBMs and their sites in all human gene promoters. The method starts by predicting a dictionary of regulatory "DNA words." From this dictionary, it distills 4098 novel predictions. To disclose the crosstalk between motifs, an additional algorithm extracts TF combinatorial binding patterns creating a collection of TF regulatory syntactic rules. Using these rules, we narrowed down a list of 504 novel motifs that appear frequently in syntax patterns. We tested the predictions against 509 known motifs confirming that our system can reliably predict ab initio motifs with an accuracy of 81%-far higher than previous approaches. We found that on average, 90% of the discovered combinatorial binding patterns target at least 10 genes, suggesting that to control in an independent manner smaller gene sets, supplementary regulatory mechanisms are required. Additionally, we discovered that the new TFBMs and their combinatorial patterns convey biological meaning, targeting TFs and genes related to developmental functions. Thus, among all the possible available targets in the genome, the TFs tend to regulate other TFs and genes involved in developmental functions. We provide a comprehensive resource for regulation analysis that includes a dictionary of "DNA words," newly predicted motifs and their corresponding combinatorial patterns. Combinatorial patterns are a useful filter to discover TFBMs that play a major role in orchestrating other factors and thus, are likely to lock/unlock cellular functional clusters.

DNA-Binding Properties of African Swine Fever Virus pA104R, a Histone-Like Protein Involved in Viral Replication and Transcription.

Science.gov (United States)

Frouco, Gonçalo; Freitas, Ferdinando B; Coelho, João; Leitão, Alexandre; Martins, Carlos; Ferreira, Fernando

2017-06-15

African swine fever virus (ASFV) codes for a putative histone-like protein (pA104R) with extensive sequence homology to bacterial proteins that are implicated in genome replication and packaging. Functional characterization of purified recombinant pA104R revealed that it binds to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) over a wide range of temperatures, pH values, and salt concentrations and in an ATP-independent manner, with an estimated binding site size of about 14 to 16 nucleotides. Using site-directed mutagenesis, the arginine located in pA104R's DNA-binding domain, at position 69, was found to be relevant for efficient DNA-binding activity. Together, pA104R and ASFV topoisomerase II (pP1192R) display DNA-supercoiling activity, although none of the proteins by themselves do, indicating that the two cooperate in this process. In ASFV-infected cells, A104R transcripts were detected from 2 h postinfection (hpi) onward, reaching a maximum concentration around 16 hpi. pA104R was detected from 12 hpi onward, localizing with viral DNA replication sites and being found exclusively in the Triton-insoluble fraction. Small interfering RNA (siRNA) knockdown experiments revealed that pA104R plays a critical role in viral DNA replication and gene expression, with transfected cells showing lower viral progeny numbers (up to a reduction of 82.0%), lower copy numbers of viral genomes (-78.3%), and reduced transcription of a late viral gene (-47.6%). Taken together, our results strongly suggest that pA104R participates in the modulation of viral DNA topology, probably being involved in viral DNA replication, transcription, and packaging, emphasizing that ASFV mutants lacking the A104R gene could be used as a strategy to develop a vaccine against ASFV. IMPORTANCE Recently reintroduced in Europe, African swine fever virus (ASFV) causes a fatal disease in domestic pigs, causing high economic losses in affected countries, as no vaccine or treatment is currently

Chlamydial entry involves TARP binding of guanine nucleotide exchange factors.

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B Josh Lane

2008-03-01

Full Text Available Chlamydia trachomatis attachment to cells induces the secretion of the elementary body-associated protein TARP (Translocated Actin Recruiting Protein. TARP crosses the plasma membrane where it is immediately phosphorylated at tyrosine residues by unknown host kinases. The Rac GTPase is also activated, resulting in WAVE2 and Arp2/3-dependent recruitment of actin to the sites of chlamydia attachment. We show that TARP participates directly in chlamydial invasion activating the Rac-dependent signaling cascade to recruit actin. TARP functions by binding two distinct Rac guanine nucleotide exchange factors (GEFs, Sos1 and Vav2, in a phosphotyrosine-dependent manner. The tyrosine phosphorylation profile of the sequence YEPISTENIYESI within TARP, as well as the transient activation of the phosphatidylinositol 3-kinase (PI3-K, appears to determine which GEF is utilized to activate Rac. The first and second tyrosine residues, when phosphorylated, are utilized by the Sos1/Abi1/Eps8 and Vav2, respectively, with the latter requiring the lipid phosphatidylinositol 3,4,5-triphosphate. Depletion of these critical signaling molecules by siRNA resulted in inhibition of chlamydial invasion to varying degrees, owing to a possible functional redundancy of the two pathways. Collectively, these data implicate TARP in signaling to the actin cytoskeleton remodeling machinery, demonstrating a mechanism by which C.trachomatis invades non-phagocytic cells.

Interspecies In Vitro Evaluation of Stereoselective Protein Binding for 3,4-Methylenedioxymethamphetamine

Directory of Open Access Journals (Sweden)

Wan Raihana Wan Aasim

2017-01-01

Full Text Available Abuse of 3,4-methylenedioxymethamphetamine (MDMA is becoming more common worldwide. To date, there is no information available on stereoselectivity of MDMA protein binding in humans, rats, and mice. Since stereoselectivity plays an important role in MDMA’s pharmacokinetics and pharmacodynamics, in this study we investigated its stereoselectivity in protein binding. The stereoselective protein binding of rac-MDMA was investigated using two different concentrations (20 and 200 ng/mL in human plasma and mouse and rat sera using an ultrafiltration technique. No significant stereoselectivity in protein binding was observed in both human plasma and rat serum; however, a significant stereoselective binding (p<0.05 was observed in mouse serum. Since the protein binding of MDMA in mouse serum is considerably lower than in humans and rats, caution should be exercised when using mice for in vitro studies involving MDMA.

GTP-binding proteins in rat liver nuclear envelopes

International Nuclear Information System (INIS)

Rubins, J.B.; Benditt, J.O.; Dickey, B.F.; Riedel, N.

1990-01-01

Nuclear transport as well as reassembly of the nuclear envelope (NE) after completion of mitosis are processes that have been shown to require GTP and ATP. To study the presence and localization of GTP-binding proteins in the NE, we have combined complementary techniques of [alpha-32P]GTP binding to Western-blotted proteins and UV crosslinking of [alpha-32P]GTP with well-established procedures for NE subfractionation. GTP binding to blotted NE proteins revealed five low molecular mass GTP-binding proteins of 26, 25, 24.5, 24, and 23 kDa, and [alpha-32P]GTP photoaffinity labeling revealed major proteins with apparent molecular masses of 140, 53, 47, 33, and 31 kDa. All GTP-binding proteins appear to localize preferentially to the inner nuclear membrane, possibly to the interface between inner nuclear membrane and lamina. Despite the evolutionary conservation between the NE and the rough endoplasmic reticulum, the GTP-binding proteins identified differed between these two compartments. Most notably, the 68- and 30-kDa GTP-binding subunits of the signal recognition particle receptor, which photolabeled with [alpha-32P]GTP in the rough endoplasmic reticulum fraction, were totally excluded from the NE fraction. Conversely, a major 53-kDa photolabeled protein in the NE was absent from rough endoplasmic reticulum. Whereas Western-blotted NE proteins bound GTP specifically, all [alpha-32P]GTP photolabeled proteins could be blocked by competition with ATP, although with a competition profile that differed from that obtained with GTP. In comparative crosslinking studies with [alpha-32P]ATP, we have identified three specific ATP-binding proteins with molecular masses of 160, 78, and 74 kDa. The localization of GTP- and ATP-binding proteins within the NE appears appropriate for their involvement in nuclear transport and in the GTP-dependent fusion of nuclear membranes

Binding of resveratrol with sodium caseinate in aqueous solutions.

Science.gov (United States)

Acharya, Durga P; Sanguansri, Luz; Augustin, Mary Ann

2013-11-15

The interaction between resveratrol (Res) and sodium caseinate (Na-Cas) has been studied by measuring fluorescence quenching of the protein by resveratrol. Quenching constants were determined using Stern-Volmer equation, which suggests that both dynamic and static quenching occur between Na-Cas and Res. Binding constants for the complexation between Na-Cas and Res were determined at different temperatures. The large binding constants (3.7-5.1×10(5)M(-1)) suggest that Res has strong affinity for Na-Cas. This affinity decreases as the temperature is raised from 25 to 37°C. The binding involves both hydrogen bonding and hydrophobic interaction, as suggested by negative enthalpy change and positive entropy change for the binding reaction. The present study indicates that Na-Cas, a common food protein, may be used as a carrier of Res, a bioactive polyphenol which is insoluble in both water and oils. Copyright © 2013 Elsevier Ltd. All rights reserved.

Workshop on gate valve pressure locking and thermal binding

International Nuclear Information System (INIS)

Brown, E.J.

1995-07-01

The purpose of the Workshop on Gate Valve Pressure Locking and Thermal Binding was to discuss pressure locking and thermal binding issues that could lead to inoperable gate valves in both boiling water and pressurized water reactors. The goal was to foster exchange of information to develop the technical bases to understand the phenomena, identify the components that are susceptible, discuss actual events, discuss the safety significance, and illustrate known corrective actions that can prevent or limit the occurrence of pressure locking or thermal binding. The presentations were structured to cover U.S. Nuclear Regulatory Commission staff evaluation of operating experience and planned regulatory activity; industry discussions of specific events, including foreign experience, and efforts to determine causes and alleviate the affects; and valve vendor experience and recommended corrective action. The discussions indicated that identifying valves susceptible to pressure locking and thermal binding was a complex process involving knowledge of components, systems, and plant operations. The corrective action options are varied and straightforward

Extended HSR/CARD domain mediates AIRE binding to DNA

Energy Technology Data Exchange (ETDEWEB)

Maslovskaja, Julia, E-mail: julia.maslovskaja@ut.ee; Saare, Mario; Liiv, Ingrid; Rebane, Ana; Peterson, Pärt

2015-12-25

Autoimmune regulator (AIRE) activates the transcription of many genes in an unusual promiscuous and stochastic manner. The mechanism by which AIRE binds to the chromatin and DNA is not fully understood, and the regulatory elements that AIRE target genes possess are not delineated. In the current study, we demonstrate that AIRE activates the expression of transiently transfected luciferase reporters that lack defined promoter regions, as well as intron and poly(A) signal sequences. Our protein-DNA interaction experiments with mutated AIRE reveal that the intact homogeneously staining region/caspase recruitment domain (HSR/CARD) and amino acids R113 and K114 are key elements involved in AIRE binding to DNA. - Highlights: • Promoter and mRNA processing elements are not important for AIRE to activate gene expression from reporter plasmids. • AIRE protein fragment aa 1–138 mediates direct binding to DNA. • Integrity of the HSR/CARD domain is needed for AIRE binding to DNA.

Extended HSR/CARD domain mediates AIRE binding to DNA

International Nuclear Information System (INIS)

Maslovskaja, Julia; Saare, Mario; Liiv, Ingrid; Rebane, Ana; Peterson, Pärt

2015-01-01

Autoimmune regulator (AIRE) activates the transcription of many genes in an unusual promiscuous and stochastic manner. The mechanism by which AIRE binds to the chromatin and DNA is not fully understood, and the regulatory elements that AIRE target genes possess are not delineated. In the current study, we demonstrate that AIRE activates the expression of transiently transfected luciferase reporters that lack defined promoter regions, as well as intron and poly(A) signal sequences. Our protein-DNA interaction experiments with mutated AIRE reveal that the intact homogeneously staining region/caspase recruitment domain (HSR/CARD) and amino acids R113 and K114 are key elements involved in AIRE binding to DNA. - Highlights: • Promoter and mRNA processing elements are not important for AIRE to activate gene expression from reporter plasmids. • AIRE protein fragment aa 1–138 mediates direct binding to DNA. • Integrity of the HSR/CARD domain is needed for AIRE binding to DNA.

The Rho ADP-ribosylating C3 exoenzyme binds cells via an Arg-Gly-Asp motif.

Science.gov (United States)

Rohrbeck, Astrid; Höltje, Markus; Adolf, Andrej; Oms, Elisabeth; Hagemann, Sandra; Ahnert-Hilger, Gudrun; Just, Ingo

2017-10-27

The Rho ADP-ribosylating C3 exoenzyme (C3bot) is a bacterial protein toxin devoid of a cell-binding or -translocation domain. Nevertheless, C3 can efficiently enter intact cells, including neurons, but the mechanism of C3 binding and uptake is not yet understood. Previously, we identified the intermediate filament vimentin as an extracellular membranous interaction partner of C3. However, uptake of C3 into cells still occurs (although reduced) in the absence of vimentin, indicating involvement of an additional host cell receptor. C3 harbors an Arg-Gly-Asp (RGD) motif, which is the major integrin-binding site, present in a variety of integrin ligands. To check whether the RGD motif of C3 is involved in binding to cells, we performed a competition assay with C3 and RGD peptide or with a monoclonal antibody binding to β1-integrin subunit and binding assays in different cell lines, primary neurons, and synaptosomes with C3-RGD mutants. Here, we report that preincubation of cells with the GRGDNP peptide strongly reduced C3 binding to cells. Moreover, mutation of the RGD motif reduced C3 binding to intact cells and also to recombinant vimentin. Anti-integrin antibodies also lowered the C3 binding to cells. Our results indicate that the RGD motif of C3 is at least one essential C3 motif for binding to host cells and that integrin is an additional receptor for C3 besides vimentin. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Mannan-binding lectin is involved in the protection against renal ischemia/ reperfusion injury by dietary restriction

NARCIS (Netherlands)

Shushimita; P. van der Pol (Pieter); R.W.F. de Bruin (Ron); J.N.M. IJzermans (Jan); C. van Kooten (Cees); F.J.M.F. Dor (Frank)

2015-01-01

textabstractPreoperative fasting and dietary restriction offer robust protection against renal ischemia/ reperfusion injury (I/RI) in mice.We recently showed that Mannan-binding lectin (MBL), the initiator of the lectin pathway of complement activation, plays a pivotal role in renal I/RI. Based on

DNA-Damage Response RNA-Binding Proteins (DDRBPs): Perspectives from a New Class of Proteins and Their RNA Targets.

Science.gov (United States)

Dutertre, Martin; Vagner, Stéphan

2017-10-27

Upon DNA damage, cells trigger an early DNA-damage response (DDR) involving DNA repair and cell cycle checkpoints, and late responses involving gene expression regulation that determine cell fate. Screens for genes involved in the DDR have found many RNA-binding proteins (RBPs), while screens for novel RBPs have identified DDR proteins. An increasing number of RBPs are involved in early and/or late DDR. We propose to call this new class of actors of the DDR, which contain an RNA-binding activity, DNA-damage response RNA-binding proteins (DDRBPs). We then discuss how DDRBPs contribute not only to gene expression regulation in the late DDR but also to early DDR signaling, DNA repair, and chromatin modifications at DNA-damage sites through interactions with both long and short noncoding RNAs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Co-suppression of sterol-regulatory element binding protein ...

African Journals Online (AJOL)

Administrator

2011-06-22

Jun 22, 2011 ... In Arabidopsis,. At5g35220 gene being sterol regulatory element-binding protein site 2, protease and metalloendopeptidase activity were required for chloroplast development and play a role in regulation of endodermal plastid size and number that are involved in ethylene-dependent gravitropism of light-.

Identification of Bacillus thuringiensis Cry3Aa toxin domain II loop 1 as the binding site of Tenebrio molitor cadherin repeat CR12.

Science.gov (United States)

Zúñiga-Navarrete, Fernando; Gómez, Isabel; Peña, Guadalupe; Amaro, Itzel; Ortíz, Ernesto; Becerril, Baltazar; Ibarra, Jorge E; Bravo, Alejandra; Soberón, Mario

2015-04-01

Bacillus thuringiensis Cry toxins exert their toxic effect by specific recognition of larval midgut proteins leading to oligomerization of the toxin, membrane insertion and pore formation. The exposed domain II loop regions of Cry toxins have been shown to be involved in receptor binding. Insect cadherins have shown to be functionally involved in toxin binding facilitating toxin oligomerization. Here, we isolated a VHH (VHHA5) antibody by phage display that binds Cry3Aa loop 1 and competed with the binding of Cry3Aa to Tenebrio molitor brush border membranes. VHHA5 also competed with the binding of Cry3Aa to a cadherin fragment (CR12) that was previously shown to be involved in binding and toxicity of Cry3Aa, indicating that Cry3Aa binds CR12 through domain II loop 1. Moreover, we show that a loop 1 mutant, previously characterized to have increased toxicity to T. molitor, displayed a correlative enhanced binding affinity to T. molitor CR12 and to VHHA5. These results show that Cry3Aa domain II loop 1 is a binding site of CR12 T. molitor cadherin. Copyright © 2015 Elsevier Ltd. All rights reserved.

CARBOHYDRATE-CONTAINING COMPOUNDS WHICH BIND TO CARBOHYDRATE BINDING RECEPTORS

DEFF Research Database (Denmark)

1995-01-01

Carbohydrate-containing compounds which contain saccharides or derivatives thereof and which bind to carbohydrate binding receptors are useful in pharmaceutical products for treatment of inflammatory diseases and other diseases.......Carbohydrate-containing compounds which contain saccharides or derivatives thereof and which bind to carbohydrate binding receptors are useful in pharmaceutical products for treatment of inflammatory diseases and other diseases....

Characterization of breakpoint cluster region kinase and SH2-binding activities.

Science.gov (United States)

Afar, D E; Witte, O N

1995-01-01

BCR is an interesting signaling protein, whose cellular function is currently unknown. Its biochemical properties include serine kinase activity, SH2-binding activity, and a GTPase-activating activity. The SH2-binding activity is particularly interesting because it may link BCR to signaling pathways involving SH2-containing molecules. Since tyrosine phosphorylation of BCR has been detected in CML-derived cell lines and since tyrosine-phosphorylated BCR shows increased affinity toward certain SH2 domains, it seems particularly important to further characterize this activity. This chapter described a simple purification scheme for partial purification of BCR, which can be used to assess in vitro kinase and SH2-binding activities.

Differential Modulation of Annexin I Binding Sites on Monocytes and Neutrophils

Directory of Open Access Journals (Sweden)

H. S. Euzger

1999-01-01

Full Text Available Specific binding sites for the anti-inflammatory protein annexin I have been detected on the surface of human monocytes and polymorphonuclear leukocytes (PMN. These binding sites are proteinaceous in nature and are sensitive to cleavage by the proteolytic enzymes trypsin, collagenase, elastase and cathepsin G. When monocytes and PMN were isolated independently from peripheral blood, only the monocytes exhibited constitutive annexin I binding. However PMN acquired the capacity to bind annexin I following co-culture with monocytes. PMN incubation with sodium azide, but not protease inhibitors, partially blocked this process. A similar increase in annexin I binding capacity was also detected in PMN following adhesion to endothelial monolayers. We propose that a juxtacrine activation rather than a cleavage-mediated transfer is involved in this process. Removal of annexin I binding sites from monocytes with elastase rendered monocytes functionally insensitive to full length annexin I or to the annexin I-derived pharmacophore, peptide Ac2-26, assessed as suppression of the respiratory burst. These data indicate that the annexin I binding site on phagocytic cells may have an important function in the feedback control of the inflammatory response and their loss through cleavage could potentiate such responses.

Expression, purification, crystallization and structure of human adipocyte lipid-binding protein (aP2)

International Nuclear Information System (INIS)

Marr, Eric; Tardie, Mark; Carty, Maynard; Brown Phillips, Tracy; Wang, Ing-Kae; Soeller, Walt; Qiu, Xiayang; Karam, George

2006-01-01

The crystal structure of human adipocyte lipid-binding protein (aP2) with a bound palmitate is reported at 1.5 Å resolution. Human adipocyte lipid-binding protein (aP2) belongs to a family of intracellular lipid-binding proteins involved in the transport and storage of lipids. Here, the crystal structure of human aP2 with a bound palmitate is described at 1.5 Å resolution. Unlike the known crystal structure of murine aP2 in complex with palmitate, this structure shows that the fatty acid is in a folded conformation and that the loop containing Phe57 acts as a lid to regulate ligand binding by excluding solvent exposure to the central binding cavity

Stanniocalcin 1 binds hemin through a partially conserved heme regulatory motif

Energy Technology Data Exchange (ETDEWEB)

Westberg, Johan A., E-mail: johan.westberg@helsinki.fi [Department of Pathology, Haartman Institute, University of Helsinki and HUSLAB, P.O. Box 21, Haartmaninkatu 3, FI-00014 Helsinki (Finland); Jiang, Ji, E-mail: ji.jiang@helsinki.fi [Department of Pathology, Haartman Institute, University of Helsinki and HUSLAB, P.O. Box 21, Haartmaninkatu 3, FI-00014 Helsinki (Finland); Andersson, Leif C., E-mail: leif.andersson@helsinki.fi [Department of Pathology, Haartman Institute, University of Helsinki and HUSLAB, P.O. Box 21, Haartmaninkatu 3, FI-00014 Helsinki (Finland)

2011-06-03

Highlights: {yields} Stanniocalcin 1 (STC1) binds heme through novel heme binding motif. {yields} Central iron atom of heme and cysteine-114 of STC1 are essential for binding. {yields} STC1 binds Fe{sup 2+} and Fe{sup 3+} heme. {yields} STC1 peptide prevents oxidative decay of heme. -- Abstract: Hemin (iron protoporphyrin IX) is a necessary component of many proteins, functioning either as a cofactor or an intracellular messenger. Hemoproteins have diverse functions, such as transportation of gases, gas detection, chemical catalysis and electron transfer. Stanniocalcin 1 (STC1) is a protein involved in respiratory responses of the cell but whose mechanism of action is still undetermined. We examined the ability of STC1 to bind hemin in both its reduced and oxidized states and located Cys{sup 114} as the axial ligand of the central iron atom of hemin. The amino acid sequence differs from the established (Cys-Pro) heme regulatory motif (HRM) and therefore presents a novel heme binding motif (Cys-Ser). A STC1 peptide containing the heme binding sequence was able to inhibit both spontaneous and H{sub 2}O{sub 2} induced decay of hemin. Binding of hemin does not affect the mitochondrial localization of STC1.

Glucostatic regulation of (+)-[3H]amphetamine binding in the hypothalamus: correlation with Na+, K+-ATPase activity

International Nuclear Information System (INIS)

Angel, I.; Hauger, R.L.; Luu, M.D.; Giblin, B.; Skolnick, P.; Paul, S.M.

1985-01-01

Preincubation of rat hypothalamic slices in glucose-free Krebs-Ringer buffer (37 0 C) resulted in a time-dependent decrease in specific (+)-[ 3 H]amphetamine binding in the crude synaptosomal fraction prepared from these slices. The addition of D-glucose resulted in a dose- and time-dependent stimulation of (+)-[ 3 H]amphetamine binding, whereas incubations with L-glucose, 2-deoxy-D-glucose, or 3-O-methyl-D-glucose failed to increase the number of (+)-[ 3 H]amphetamine binding sites. Ouabain potently inhibited the glucose-induced stimulation of (+)-[ 3 H]amphetamine binding, suggesting the involvement of Na + , K + -ATPase. Preincubation of hypothalamic slices with glucose also resulted in an increase in Na + ,K + -ATPase activity and the number of specific high-affinity binding sites for [ 3 H]ouabain, and a good correlation was observed between the glucose-stimulated increase in (+)-[ 3 H]amphetamine and [ 3 H]ouabain binding. These data suggest that the (+)-[ 3 H]amphetamine binding site in hypothalamus, previously linked to the anorectic actions of various phenylethylamines, is regulated both in vitro and in vivo by physiological concentrations of glucose. Glucose and amphetamine appear to interact at common sites in the hypothalamus to stimulate Na + ,K + -ATPase activity, and the latter may be involved in the glucostatic regulation of appetite

Proadifen-sensitive high affinity binding of /sup 3/H-alaproclate to liver membranes

Energy Technology Data Exchange (ETDEWEB)

Ross, S.B.

1987-01-01

/sup 3/H-alaproclate, a selective 5/sub h/ydroxytryptamine uptake inhibitor, was found to bind to microsomal membranes from the rat liver with high affinity (K/sub D/-=3 nM) and large capacity (B/sub max/ about 2 nmol/g liver). This binding was stereoselective since S-( - )-alaproclate was 30 times more potent than the R-( + )-enantiomer to displace the /sup 3/H-labelled racemate. Proadifen (SKF 525A), an inhibitor of cytochrome P-450, displaced the /sup 3/H-alaproclate binding with the same, high affinity (K/sub i/=3 nM) as alaproclate itself. Repeated treatment with phenobarbital sodium (5x75 mg/kg intraperitoneally) increased the number of alaproclate binding sites 7-8 times without changing the affinity. However, most of the phenobarbital induced /sup 3/H-alaproclate binding was not displaceable by proadifen, showing the presence of at least two different high affinity binding sites. The possible involvement of cytochrome P-450 in the alaproclate binding is discussed.

Involvement of individual subsites and secondary substrate binding sites in multiple attack on amylose by barley alpha-amylase

DEFF Research Database (Denmark)

Kramhøft, Birte; Bak-Jensen, Kristian Sass; Mori, Haruhide

2005-01-01

Barley alpha-amylase 1 (AMY1) hydrolyzed amylose with a degree of multiple attack (DMA) of 1.9; that is, on average, 2.9 glycoside bonds are cleaved per productive enzyme-substrate encounter. Six AMY1 mutants, spanning the substrate binding cleft from subsites -6 to +4, and a fusion protein, AMY1...... translocation of substrate in the binding cleft upon the initial cleavage to produce G6-G10, essentially independent of subsite mutations, and short-distance moves resulting in individually very different rates of release of G1-G4. Accordingly, the degree of multiple attack as well as the profile of products...

Characterization of cap binding proteins associated with the nucleus

International Nuclear Information System (INIS)

Patzelt, E.

1986-04-01

Eucaryotic mRNAs a carry 7-methylguanosine triphosphate residue (called cap structure) at their 5' terminus. The cap plays an important role in RNA recognition. Cap binding proteins (CBP) of HeLa cells were identified by photoaffinity labelling using the cap analogue γ-( 32 P)-(4-(benzoyl-phenyl)methylamido)-7-methylguanosine-5'-triphosphate (BP-m 7 GTP). Photoreaction of this cap analogue with HeLa cell initiation factors resulted in specific labelling of two polypeptides of Msub(r) 37000 and 26000. The latter was also labelled in crude initiation factors prepared from reticulocytes and is identical to the cap binding protein CBP I previously identified. These cap binding proteins were also affinity labelled in poliovirus infected cell extracts. Photoaffinity reaction with BP-m 7 GTP of whole HeLa cell homogenate showed three additional polypeptides with Msub(r) 120000, 89000 and 80000. These cap binding proteins were found to be associated with the nucleus and are therefore referred to as nuclear cap binding proteins, i.e. NCBP 1, NCBP 2 and NCBP 3. They were also present in splicing extracts. Photoaffinity labelling in these nuclear extracts was differentially inhibited by various cap analogues and capped mRNAs. Affinity chromatography on immobilized globin mRNA led to a partial separation of the three nuclear cap binding proteins. Chromatography on m 7 GTP-Sepharose resulted in a specific binding of NCBP 3. The different behaviour of the cap binding proteins suggests that they are functionally distinct and that they might be involved in different processes requiring cap recognition. (Author)

Primary Angle Closure and Sequence Variants within MicroRNA Binding Sites of Genes Involved in Eye Development.

Directory of Open Access Journals (Sweden)

Haihong Shi

Full Text Available The formation of primary angle closure (PAC and primary angle closure glaucoma (PACG is regulated by a tissue remodeling pathway that plays a critical role in eye development. MicroRNAs (miRNAs are powerful gene expression regulators and may exert their effects on tissue remodeling genes. This study investigated the associations between gene variants (single-nucleotide polymorphism, SNP in miRNA binding sites in the 3'-UTR region of genes involved in eye development and PAC.The sample consisted of 232 PAC subjects and 306 controls obtained from a population-based cohort in the Funing District of Jiangsu, China. The markers include 9 SNPs in the COL11A1, PCMTD1, ZNRF3, MTHFR, and ALPPL2 genes respectively. SNP genotyping was performed with a TaqMan-MGB probe using an RT-PCR system.Of the 9 SNPs studied, the frequency of the minor A allele of COL11A1 rs1031820 was higher in the PAC group than in the control group in allele analysis (p = 0.047. The genotype analysis indicated that MTHFR rs1537514 is marginally associated with PAC (p = 0.014. The CC genotype of rs1537514 was present solely in the PAC group. However, the differences lost significance after Bonferroni correction.Our study reveals a possible association of COL11A1 and MTHFR with PAC in the Han Chinese population. These results will contribute to an improved understanding of the genetic basis of PACG.

Structural basis for the ligand-binding specificity of fatty acid-binding proteins (pFABP4 and pFABP5) in gentoo penguin.

Science.gov (United States)

Lee, Chang Woo; Kim, Jung Eun; Do, Hackwon; Kim, Ryeo-Ok; Lee, Sung Gu; Park, Hyun Ho; Chang, Jeong Ho; Yim, Joung Han; Park, Hyun; Kim, Il-Chan; Lee, Jun Hyuck

2015-09-11

Fatty acid-binding proteins (FABPs) are involved in transporting hydrophobic fatty acids between various aqueous compartments of the cell by directly binding ligands inside their β-barrel cavities. Here, we report the crystal structures of ligand-unbound pFABP4, linoleate-bound pFABP4, and palmitate-bound pFABP5, obtained from gentoo penguin (Pygoscelis papua), at a resolution of 2.1 Å, 2.2 Å, and 2.3 Å, respectively. The pFABP4 and pFABP5 proteins have a canonical β-barrel structure with two short α-helices that form a cap region and fatty acid ligand binding sites in the hydrophobic cavity within the β-barrel structure. Linoleate-bound pFABP4 and palmitate-bound pFABP5 possess different ligand-binding modes and a unique ligand-binding pocket due to several sequence dissimilarities (A76/L78, T30/M32, underlining indicates pFABP4 residues) between the two proteins. Structural comparison revealed significantly different conformational changes in the β3-β4 loop region (residues 57-62) as well as the flipped Phe60 residue of pFABP5 than that in pFABP4 (the corresponding residue is Phe58). A ligand-binding study using fluorophore displacement assays shows that pFABP4 has a relatively strong affinity for linoleate as compared to pFABP5. In contrast, pFABP5 exhibits higher affinity for palmitate than that for pFABP4. In conclusion, our high-resolution structures and ligand-binding studies provide useful insights into the ligand-binding preferences of pFABPs based on key protein-ligand interactions. Copyright © 2015 Elsevier Inc. All rights reserved.

Induction of novel CD8+ T-cell responses during chronic untreated HIV-1 infection by immunization with subdominant cytotoxic T-lymphocyte epitopes

DEFF Research Database (Denmark)

Kloverpris, Henrik; Karlsson, Ingrid; Bonde, Jesper

2009-01-01

OBJECTIVE:: To investigate the potential to induce additional cytotoxic T-lymphocyte (CTL) immunity during chronic HIV-1 infection. DESIGN:: We selected infrequently targeted or subdominant but conserved HLA-A*0201-binding epitopes in Gag, Pol, Env, Vpu and Vif. These relatively immune silent...... epitopes were modified as anchor-optimized peptides to improve immunogenicity and delivered on autologous monocyte-derived dendritic cells (MDDCs). METHODS:: Twelve treatment-naïve HLA-A*0201 HIV-1-infected Danish individuals received 1 x 10 MDDCs subcutaneously (s.c.) (weeks 0, 2, 4 and 8), pulsed......-cell counts was observed. CONCLUSION:: These data show that it is possible to generate new T-cell responses in treatment-naive HIV-1-infected individuals despite high viral loads, and thereby redirect immunity to target new multiple and rationally selected subdominant CTL epitopes. Further optimization could...

Binding of a novel 12-E2-12 gemini surfactant to xanthine oxidase: Analysis involving tensiometric, spectroscopic, microscopic and molecular docking approach

International Nuclear Information System (INIS)

Akram, Mohd; Bhat, Imtiyaz Ahmad; Kabir-ud-Din

2016-01-01

Binding interaction of a synthesized biodegradable gemini surfactant, ethane-1, 2-diyl bis(N, N-dimethyl-N-dodecylammoniumacetoxy) dichloride (12-E2-12), with bovine milk xanthine oxidase (XO) was studied using tensiometry, fluorescence spectroscopy, UV, CD, FT-IR, TEM and molecular docking. Tensiometry revealed lowering in surface tension (γ) and critical micelle concentration (CMC) of 12-E2-12 upon XO combination, suggesting a significant interaction between XO and 12-E2-12 (both in the bulk as well as at interface). Intrinsic fluorescence studies depict that 12-E2-12 quenches XO fluorescence intensity through static mechanism. The magnitude of binding parameters infers substantial and effective binding of 12-E2-12 to (XO). ANS and pyrene fluorescence demonstrate the exposure of aromatic residues (tyrosine/tryptophan) to a non-polar environment. UV, circular dichroism (CD) and FT-IR results delineate change in the secondary structure of the enzyme XO. Microscopic TEM micrographs confirm the disrupture of enzyme structure at higher concentrations of 12-E2-12. Molecular docking results show that 12-E2-12 binds to XO in the vicinity of both hydrophobic and hydrophilic residues, inferring that binding is governed by both hydrophilic and hydrophobic forces. This study may be of significance in biomedical world to further interpret mechanistic treatment modes of diseases like gout and hyperuricemia. Moreover, this study provides deeper biophysical insight into surfactant–protein interactions. - Highlights: • Binding of biodegradable gemini surfactant 12-E2-12 with xanthine oxidase. • Binding induces conformational changes in the latter. • Conformational change can be useful for biomedical and industrial purposes.

Binding of a novel 12-E2-12 gemini surfactant to xanthine oxidase: Analysis involving tensiometric, spectroscopic, microscopic and molecular docking approach

Energy Technology Data Exchange (ETDEWEB)

Akram, Mohd, E-mail: drmohdakram@rediffmail.com; Bhat, Imtiyaz Ahmad; Kabir-ud-Din

2016-02-15

Binding interaction of a synthesized biodegradable gemini surfactant, ethane-1, 2-diyl bis(N, N-dimethyl-N-dodecylammoniumacetoxy) dichloride (12-E2-12), with bovine milk xanthine oxidase (XO) was studied using tensiometry, fluorescence spectroscopy, UV, CD, FT-IR, TEM and molecular docking. Tensiometry revealed lowering in surface tension (γ) and critical micelle concentration (CMC) of 12-E2-12 upon XO combination, suggesting a significant interaction between XO and 12-E2-12 (both in the bulk as well as at interface). Intrinsic fluorescence studies depict that 12-E2-12 quenches XO fluorescence intensity through static mechanism. The magnitude of binding parameters infers substantial and effective binding of 12-E2-12 to (XO). ANS and pyrene fluorescence demonstrate the exposure of aromatic residues (tyrosine/tryptophan) to a non-polar environment. UV, circular dichroism (CD) and FT-IR results delineate change in the secondary structure of the enzyme XO. Microscopic TEM micrographs confirm the disrupture of enzyme structure at higher concentrations of 12-E2-12. Molecular docking results show that 12-E2-12 binds to XO in the vicinity of both hydrophobic and hydrophilic residues, inferring that binding is governed by both hydrophilic and hydrophobic forces. This study may be of significance in biomedical world to further interpret mechanistic treatment modes of diseases like gout and hyperuricemia. Moreover, this study provides deeper biophysical insight into surfactant–protein interactions. - Highlights: • Binding of biodegradable gemini surfactant 12-E2-12 with xanthine oxidase. • Binding induces conformational changes in the latter. • Conformational change can be useful for biomedical and industrial purposes.

In vitro binding of selenium by rat liver mitochondrial selenium-binding protein

International Nuclear Information System (INIS)

Brian, W.R.; Hoekstra, W.G.

1986-01-01

Last year the authors reported that upon freezing and thawing mitochondria from rats injected with [ 75 Se]Na 2 SeO 3 ( 75 Se-selenite), a 75 Se-binding protein (SeBP) was released. They have studied further in vitro labelling of SeBP. This matrix protein was labelled in vitro when lysed mitochondria (containing non-matrix material) were incubated with 75 Se-selenite but not when matrix material alone was incubated with 75 Se-selenite. Thus, there are one or more promoters of in vitro SeBP labelling in the non-matrix fraction. SeBP was also labelled in vitro when 75 Se-selenite was added to matrix alone and dialyzed. Dialysis tubing, and not the dialysis process, promoted labelling by affecting SeBP and not by affecting 75 Se-selenite. Labelling did not occur when matrix alone and 75 Se-selenite were incubated (not dialyzed) in a glass test tube but did occur in a polystyrene test tube. They hypothesize that non-covalent interactions occur between SeBP and dialysis tubing or polystyrene that expose Se binding sites on the protein. A similar mechanism involving mitochondrial non-matrix material may function in vivo. Non-denaturing disc gel electrophoresis of partially purified SeBP labelled in vivo or in vitro suggested that the same protein was labelled in both conditions. Using in vitro binding techniques, SeBP was also found in sheep liver mitochondrial matrix. This supports the theory that SeBP is important in Se metabolism

Integration Host Factor (IHF binds to the promoter region of the phtD operon involved in phaseolotoxin synthesis in P. syringae pv. phaseolicola NPS3121

Directory of Open Access Journals (Sweden)

Álvarez-Morales Ariel

2011-05-01

Full Text Available Abstract Background Pseudomonas syringae pv. phaseolicola, the causal agent of halo blight disease in beans, produces a toxin known as phaseolotoxin, in whose synthesis participate a group of genes organized within the genome in a region known as the "Pht cluster". This region, which is thought to have been acquired by horizontal gene transfer, includes 5 transcriptional units, two monocistronic (argK, phtL and three polycistronic (phtA, phtD, phtM, whose expression is temperature dependent. So far, the regulatory mechanisms involved in phaseolotoxin synthesis have not been elucidated and the only well-established fact is the requirement of low temperatures for its synthesis. In this work, we searched for regulatory proteins that could be involved in phaseolotoxin synthesis, focusing on the regulation of the phtD operon. Results In this study we identified the global regulator IHF (Integration Host Factor, which binds to the promoter region of the phtD operon, exerting a negative effect on the expression of this operon. This is the first regulatory protein identified as part of the phaseolotoxin synthesis system. Our findings suggest that the Pht cluster was similarly regulated in the ancestral cluster by IHF or similar protein, and integrated into the global regulatory mechanism of P. syringae pv. phaseolicola, after the horizontal gene transfer event by using the host IHF protein. Conclusion This study identifies the IHF protein as one element involved in the regulation of phaseolotoxin synthesis in P. syringae pv. phaseolicola NPS3121 and provides new insights into the regulatory mechanisms involved in phaseolotoxin production.

Ulex europaeus agglutinin-I binds to developing gastrin cells.

Science.gov (United States)

Ge, Z H; Blom, J; Larsson, L I

1998-03-01

We have previously reported that antropyloric gastrin (G) and somatostatin (D) cells derive from precursor (G/D) cells that coexpress both hormones. We have now analyzed this endocrine cell pedigree for binding of Ulex europaeus agglutinin-I (UEA-I), which previously has been reported to represent a useful marker for cell differentiation. Subpopulations of G/D, D, and G cells were all found to express UEA-I binding. Labelling with bromodeoxyuridine showed that UEA-I positive G cells possessed a higher labelling index than UEA-I negative G cells. These data suggest that the UEA-I positive G cells represent maturing cells still involved in DNA synthesis and cell division. Electron microscopically, specific UEA-I binding sites were localized to the secretory granules and the apical cell membrane of G cells. We conclude that UEA-I represents a differentiation marker for G cells. Moreover, the presence of UEA-I binding sites in these cells may be relevant for Helicobacter pylori-mediated disturbances of gastric acid secretion and gastrin hypersecretion.

Experimental and theoretical investigation of bezafibrate binding to serum albumins

Energy Technology Data Exchange (ETDEWEB)

Gałęcki, Krystian, E-mail: kgalecki87@gmail.com [Technical University of Lodz, Lodz (Poland); Hunter, Kelsey [Glasgow Caledonian University, Glasgow, Scotland (United Kingdom); Daňková, Gabriela [Masarykova Univerzita, Brno (Czech Republic); Rivera, Elsy [University of Houston-Downtown, Houston (United States); Tung, Lo Wing [The Hong Kong Polytechnic University (Hong Kong); Mc Sherry, Kenneth [Trinity College Dublin, Dublin (Ireland)

2016-09-15

The purpose of this investigation was to provide insight into the possible mechanism of the intermolecular interactions between antilipemic agent bezafibrate and serum albumins (SAs) including human (HSA) and bovine (BSA). The aim was to indicate the most probable sight of these interactions. Both experimental (spectroscopic) and theoretical methods were applied. It was determined that bezafibrate binds to SAs in one specific binding site, the fatty acid binding site 6. The results obtained from the steady-state and time-resolved fluorescence experiments suggested that existing two distinct stable conformations of the proteins with different exposure to the quencher. The dominate conformer of HSA and BSA characterized by the Stern–Volmer quenching constant (from ratio F{sub 0}/F) equal to 1.24·10{sup 4} and 8.48·10{sup 3} M{sup −1} at 298 K, respectively. The docking results and calculated thermodynamics parameters may be suggested that the binding process is spontaneous and might involve van der Waals and hydrogen bonding forces.

Neural correlates of binding lyrics and melodies for the encoding of new songs.

Science.gov (United States)

Alonso, Irene; Davachi, Lila; Valabrègue, Romain; Lambrecq, Virginie; Dupont, Sophie; Samson, Séverine

2016-02-15

Songs naturally bind lyrics and melody into a unified representation. Using a subsequent memory paradigm, we examined the neural processes associated with binding lyrics and melodies during song encoding. Participants were presented with songs in two conditions: a unified condition (melodies sung with lyrics), and a separate condition (melodies sung with the syllable "la"). In both cases, written lyrics were displayed and participants were instructed to memorize them by repeating them covertly or by generating mental images of the songs. We expected the unified condition to recruit the posterior superior temporal gyrus, known to be involved in perceptual integration of songs, as well as the left inferior frontal gyrus (IFG). Conversely, we hypothesized that the separate condition would engage a larger network including the hippocampus to bind lyrics and melodies of songs, and the basal ganglia and the cerebellum to ensure the correct sequence coupling of verbal and musical information in time. Binding lyrics and melodies in the unified condition revealed activation of the left IFG, bilateral middle temporal gyrus (MTG), and left motor cortex, suggesting a strong linguistic processing for this condition. Binding in the separate compared to the unified condition revealed greater activity in the right hippocampus as well as other areas including the left caudate, left cerebellum, and right IFG. This study provides novel evidence for the role of the right hippocampus in binding lyrics and melodies in songs. Results are discussed in light of studies of binding in the visual domain and highlight the role of regions involved in timing and synchronization such as the basal ganglia and the cerebellum. Copyright © 2015. Published by Elsevier Inc.

Investigation of arc repressor DNA-binding specificity by comparative molecular dynamics simulations.

Science.gov (United States)

Song, Wei; Guo, Jun-Tao

2015-01-01

Transcription factors regulate gene expression through binding to specific DNA sequences. How transcription factors achieve high binding specificity is still not well understood. In this paper, we investigated the role of protein flexibility in protein-DNA-binding specificity by comparative molecular dynamics (MD) simulations. Protein flexibility has been considered as a key factor in molecular recognition, which is intrinsically a dynamic process involving fine structural fitting between binding components. In this study, we performed comparative MD simulations on wild-type and F10V mutant P22 Arc repressor in both free and complex conformations. The F10V mutant has lower DNA-binding specificity though both the bound and unbound main-chain structures between the wild-type and F10V mutant Arc are highly similar. We found that the DNA-binding motif of wild-type Arc is structurally more flexible than the F10V mutant in the unbound state, especially for the six DNA base-contacting residues in each dimer. We demonstrated that the flexible side chains of wild-type Arc lead to a higher DNA-binding specificity through forming more hydrogen bonds with DNA bases upon binding. Our simulations also showed a possible conformational selection mechanism for Arc-DNA binding. These results indicate the important roles of protein flexibility and dynamic properties in protein-DNA-binding specificity.

MANAGING TIGHT BINDING RECEPTORS FOR NEW SPEARATIONS TECHNOLOGIES

Energy Technology Data Exchange (ETDEWEB)

DARYLE H BUSCH RICHARD S GIVENS

2004-12-10

Much of the earth's pollution involves compounds of the metallic elements, including actinides, strontium, cesium, technetium, and RCRA metals. Metal ions bind to molecules called ligands, which are the molecular tools that can manipulate the metal ions under most conditions. This DOE-EMSP sponsored program strives (1) to provide the foundations for using the most powerful ligands in transformational separations technologies and (2) to produce seminal examples of their applications to separations appropriate to the DOE EM mission. These ultra tight-binding ligands can capture metal ions in the most competitive of circumstances (from mineralized sites, lesser ligands, and even extremely dilute solutions), but they react so slowly that they are useless in traditional separations methodologies. Two attacks on this problem are underway. The first accommodates to the challenging molecular lethargy by developing a seminal slow separations methodology termed the soil poultice. The second designs ligands that are only tight-binding while wrapped around the targeted metal ion, but can be put in place by switch-binding and removed by switch-release. We envision a kind of molecular switching process to accelerate the union between metal ion and tight-binding ligand. Molecular switching processes are suggested for overcoming the slow natural equilibration rate with which ultra tight-binding ligands combine with metal ions. Ligands that bind relatively weakly combine with metal ions rapidly, so the trick is to convert a ligand from a weak, rapidly binding species to a powerful, slow releasing ligand--during the binding of the ligand to the metal ion. Such switch-binding ligands must react with themselves, and the reaction must take place under the influence of the metal ion. For example, our generation 1 ligands showed that a well-designed linear ligand with ends that readily combine, forms a cyclic molecule when it wraps around a metal ion. Our generation 2 ligands are

Identification of regions involved in substrate binding and dimer stabilization within the central domains of yeast Hsp40 Sis1.

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Júlio C Borges

Full Text Available Protein folding, refolding and degradation are essential for cellular life and are regulated by protein homeostatic processes such those that involve the molecular chaperone DnaK/Hsp70 and its co-chaperone DnaJ. Hsp70 action is initiated when proteins from the DnaJ family bind an unfolded protein for delivery purposes. In eukaryotes, the DnaJ family can be divided into two main groups, Type I and Type II, represented by yeast cytosolic Ydj1 and Sis1, respectively. Although sharing some unique features both members of the DnaJ family, Ydj1 and Sis1 are structurally and functionally distinct as deemed by previous studies, including the observation that their central domains carry the structural and functional information even in switched chimeras. In this study, we combined several biophysical tools for evaluating the stability of Sis1 and mutants that had the central domains (named Gly/Met rich domain and C-terminal Domain I deleted or switched to those of Ydj1 to gain insight into the role of these regions in the structure and function of Sis1. The mutants retained some functions similar to full length wild-type Sis1, however they were defective in others. We found that: 1 Sis1 unfolds in at least two steps as follows: folded dimer to partially folded monomer and then to an unfolded monomer. 2 The Gly/Met rich domain had intrinsically disordered characteristics and its deletion had no effect on the conformational stability of the protein. 3 The deletion of the C-terminal Domain I perturbed the stability of the dimer. 4 Exchanging the central domains perturbed the conformational stability of the protein. Altogether, our results suggest the existence of two similar subdomains in the C-terminal domain of DnaJ that could be important for stabilizing each other in order to maintain a folded substrate-binding site as well as the dimeric state of the protein.

Localized frustration and binding-induced conformational change in recognition of 5S RNA by TFIIIA zinc finger.

Science.gov (United States)

Tan, Cheng; Li, Wenfei; Wang, Wei

2013-12-19

Protein TFIIIA is composed of nine tandemly arranged Cys2His2 zinc fingers. It can bind either to the 5S RNA gene as a transcription factor or to the 5S RNA transcript as a chaperone. Although structural and biochemical data provided valuable information on the recognition between the TFIIIIA and the 5S DNA/RNA, the involved conformational motions and energetic factors contributing to the binding affinity and specificity remain unclear. In this work, we conducted MD simulations and MM/GBSA calculations to investigate the binding-induced conformational changes in the recognition of the 5S RNA by the central three zinc fingers of TFIIIA and the energetic factors that influence the binding affinity and specificity at an atomistic level. Our results revealed drastic interdomain conformational changes between these three zinc fingers, involving the exposure/burial of several crucial DNA/RNA binding residues, which can be related to the competition between DNA and RNA for the binding of TFIIIA. We also showed that the specific recognition between finger 4/finger 6 and the 5S RNA introduces frustrations to the nonspecific interactions between finger 5 and the 5S RNA, which may be important to achieve optimal binding affinity and specificity.

Identification of Arsenic Direct-Binding Proteins in Acute Promyelocytic Leukaemia Cells

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Tao Zhang

2015-11-01

Full Text Available The identification of arsenic direct-binding proteins is essential for determining the mechanism by which arsenic trioxide achieves its chemotherapeutic effects. At least two cysteines close together in the amino acid sequence are crucial to the binding of arsenic and essential to the identification of arsenic-binding proteins. In the present study, arsenic binding proteins were pulled down with streptavidin and identified using a liquid chromatograph-mass spectrometer (LC-MS/MS. More than 40 arsenic-binding proteins were separated, and redox-related proteins, glutathione S-transferase P1 (GSTP1, heat shock 70 kDa protein 9 (HSPA9 and pyruvate kinase M2 (PKM2, were further studied using binding assays in vitro. Notably, PKM2 has a high affinity for arsenic. In contrast to PKM2, GSTP1and HSPA9 did not combine with arsenic directly in vitro. These observations suggest that arsenic-mediated acute promyelocytic leukaemia (APL suppressive effects involve PKM2. In summary, we identified several arsenic binding proteins in APL cells and investigated the therapeutic mechanisms of arsenic trioxide for APL. Further investigation into specific signal pathways by which PKM2 mediates APL developments may lead to a better understanding of arsenic effects on APL.

Using sequence-specific chemical and structural properties of DNA to predict transcription factor binding sites.

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Amy L Bauer

2010-11-01

Full Text Available An important step in understanding gene regulation is to identify the DNA binding sites recognized by each transcription factor (TF. Conventional approaches to prediction of TF binding sites involve the definition of consensus sequences or position-specific weight matrices and rely on statistical analysis of DNA sequences of known binding sites. Here, we present a method called SiteSleuth in which DNA structure prediction, computational chemistry, and machine learning are applied to develop models for TF binding sites. In this approach, binary classifiers are trained to discriminate between true and false binding sites based on the sequence-specific chemical and structural features of DNA. These features are determined via molecular dynamics calculations in which we consider each base in different local neighborhoods. For each of 54 TFs in Escherichia coli, for which at least five DNA binding sites are documented in RegulonDB, the TF binding sites and portions of the non-coding genome sequence are mapped to feature vectors and used in training. According to cross-validation analysis and a comparison of computational predictions against ChIP-chip data available for the TF Fis, SiteSleuth outperforms three conventional approaches: Match, MATRIX SEARCH, and the method of Berg and von Hippel. SiteSleuth also outperforms QPMEME, a method similar to SiteSleuth in that it involves a learning algorithm. The main advantage of SiteSleuth is a lower false positive rate.

Structural and binding studies of a C-type galactose-binding lectin from Bothrops jararacussu snake venom.

Science.gov (United States)

Sartim, Marco A; Pinheiro, Matheus P; de Pádua, Ricardo A P; Sampaio, Suely V; Nonato, M Cristina

2017-02-01

BJcuL is a snake venom galactoside-binding lectin (SVgalL) isolated from Bothrops jararacussu and is involved in a wide variety of biological activities including triggering of pro-inflammatory response, disruption of microbial biofilm structure and induction of apoptosis. In the present work, we determined the crystallographic structure of BJcuL, the first holo structure of a SVgalL, and introduced the fluorescence-based thermal stability assay (Thermofluor) as a tool for screening and characterization of the binding mechanism of SVgalL ligands. BJcuL structure revealed the existence of a porous and flexible decameric arrangement composed of disulfide-linked dimers related by a five-fold symmetry. Each monomer contains the canonical carbohydrate recognition domain, a calcium ion required for BJcuL lectinic activity and a sodium ion required for protein stabilization. BJcuL thermostability was found to be induced by calcium ion and galactoside sugars which exhibit hyperbolic saturation profiles dependent on ligand concentration. Serendipitously, the gentamicin group of aminoglycoside antibiotics (gAGAs) was also identified as BJcuL ligands. On contrast, gAGAs exhibited a sigmoidal saturation profile compatible with a cooperative mechanism of binding. Thermofluor, hemagglutination inhibition assay and molecular docking strategies were used to identify a distinct binding site in BJcuL localized at the dimeric interface near the fully conserved intermolecular Cys86-Cys86 disulfide bond. The hybrid approach used in the present work provided novel insights into structural behavior and functional diversification of SVgaLs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Multiple DNA binding proteins contribute to timing of chromosome replication in E. coli

DEFF Research Database (Denmark)

Riber, Leise; Frimodt-Møller, Jakob; Charbon, Godefroid

2016-01-01

Chromosome replication in Escherichia coli is initiated from a single origin, oriC. Initiation involves a number of DNA binding proteins, but only DnaA is essential and specific for the initiation process. DnaA is an AAA+ protein that binds both ATP and ADP with similar high affinities. Dna...... replication is initiated, or the time window in which all origins present in a single cell are initiated, i.e. initiation synchrony, or both. Overall, these DNA binding proteins modulate the initiation frequency from oriC by: (i) binding directly to oriC to affect DnaA binding, (ii) altering the DNA topology...... in or around oriC, (iii) altering the nucleotide bound status of DnaA by interacting with non-coding chromosomal sequences, distant from oriC, that are important for DnaA activity. Thus, although DnaA is the key protein for initiation of replication, other DNA-binding proteins act not only on ori...

A peek into tropomyosin binding and unfolding on the actin filament.

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Abhishek Singh

Full Text Available BACKGROUND: Tropomyosin is a prototypical coiled coil along its length with subtle variations in structure that allow interactions with actin and other proteins. Actin binding globally stabilizes tropomyosin. Tropomyosin-actin interaction occurs periodically along the length of tropomyosin. However, it is not well understood how tropomyosin binds actin. PRINCIPAL FINDINGS: Tropomyosin's periodic binding sites make differential contributions to two components of actin binding, cooperativity and affinity, and can be classified as primary or secondary sites. We show through mutagenesis and analysis of recombinant striated muscle alpha-tropomyosins that primary actin binding sites have a destabilizing coiled-coil interface, typically alanine-rich, embedded within a non-interface recognition sequence. Introduction of an Ala cluster in place of the native, more stable interface in period 2 and/or period 3 sites (of seven increased the affinity or cooperativity of actin binding, analysed by cosedimentation and differential scanning calorimetry. Replacement of period 3 with period 5 sequence, an unstable region of known importance for cooperative actin binding, increased the cooperativity of binding. Introduction of the fluorescent probe, pyrene, near the mutation sites in periods 2 and 3 reported local instability, stabilization by actin binding, and local unfolding before or coincident with dissociation from actin (measured using light scattering, and chain dissociation (analyzed using circular dichroism. CONCLUSIONS: This, and previous work, suggests that regions of tropomyosin involved in binding actin have non-interface residues specific for interaction with actin and an unstable interface that is locally stabilized upon binding. The destabilized interface allows residues on the coiled-coil surface to obtain an optimal conformation for interaction with actin by increasing the number of local substates that the side chains can sample. We suggest

Molecular simulations of lactose-bound and unbound forms of the FaeG adhesin reveal critical amino acids involved in sugar binding.

Science.gov (United States)

Baker, Joseph L; Jafri, Heba

2016-11-01

F4 fimbriae are protein filaments found in enterotoxigenic Escherichia coli cells and are implicated in the process of bacterial infection due to their function as bacterial adhesins. These filaments are comprised from several proteins, but the bacterial adhesin FaeG, which is a lactose-binding protein, is the major subunit comprising F4 fimbriae. Crystal structures for three variants of the FaeG protein were recently solved, including the ad variant of FaeG that was crystallized in complex with lactose. However, the dynamics of the FaeG protein bound to lactose have not been explored previously using molecular dynamics simulations. Therefore, in order to study the dynamical interactions between the FaeG ad variant and lactose, we have carried out the first all-atom molecular dynamics simulations of this system. We have also probed the role of crystallographic water molecules on the stability of lactose in the FaeG binding site, and have simulated seven FaeG mutants to probe the influence of amino acid substitutions on the ability of FaeG to bind lactose effectively. Our simulations agree well with experimental results for the influence of mutations on lactose binding, provide dynamical insights into the interactions of FaeG with lactose, and also suggest the possibility of additional regions of the FaeG protein that may act as secondary lactose binding sites. Copyright © 2016 Elsevier Inc. All rights reserved.

N-Acetylgalactosaminyltransferase 14, a novel insulin-like growth factor binding protein-3 binding partner

International Nuclear Information System (INIS)

Wu, Chen; Yao, Guangyin; Zou, Minji; Chen, Guangyu; Wang, Min; Liu, Jingqian; Wang, Jiaxi; Xu, Donggang

2007-01-01

Insulin-like growth factor binding protein-3 (IGFBP-3) is known to inhibit cell proliferation and induce apoptosis in IGF-dependent and IGF-independent manners, but the mechanism underlying IGF-independent effects is not yet clear. In a yeast two-hybrid assay, IGFBP-3 was used as the bait to screen a human fetal liver cDNA library for it interactors that may potentially mediate IGFBP-3-regulated functions. N-Acetylgalactosaminyltransferase 14 (GalNAc-T14), a member of the GalNAc-Tases family, was identified as a novel IGFBP-3 binding partner. This interaction involved the ricin-type beta-trefoil domain of GalNAc-T14. The interaction between IGFBP-3 and GalNAc-T14 was reconfirmed in vitro and in vivo, using GST pull-down, co-immunoprecipitation and mammalian two-hybrid assays. Our findings may provide new clues for further study on the mechanism behind the IGF-independent effects of IGFBP-3 promoting apoptosis. The role of GalNAc-T14 as an intracellular mediator of the effects of IGFBP-3 need to be verified in future studies

Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

International Nuclear Information System (INIS)

Walker, G.; Bourguignon, L.Y.

1990-01-01

Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation

Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

Energy Technology Data Exchange (ETDEWEB)

Walker, G.; Bourguignon, L.Y. (Univ. of Miami Medical School, FL (USA))

1990-08-01

Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation.

Trp[superscript 2313]-His[superscript 2315] of Factor VIII C2 Domain Is Involved in Membrane Binding Structure of a Complex Between the C[subscript 2] Domain and an Inhibitor of Membrane Binding

Energy Technology Data Exchange (ETDEWEB)

Liu, Zhuo; Lin, Lin; Yuan, Cai; Nicolaes, Gerry A.F.; Chen, Liqing; Meehan, Edward J.; Furie, Bruce; Furie, Barbara; Huang, Mingdong (Harvard-Med); (UAH); (Maastricht); (Chinese Aca. Sci.)

2010-11-03

Factor VIII (FVIII) plays a critical role in blood coagulation by forming the tenase complex with factor IXa and calcium ions on a membrane surface containing negatively charged phospholipids. The tenase complex activates factor X during blood coagulation. The carboxyl-terminal C2 domain of FVIII is the main membrane-binding and von Willebrand factor-binding region of the protein. Mutations of FVIII cause hemophilia A, whereas elevation of FVIII activity is a risk factor for thromboembolic diseases. The C2 domain-membrane interaction has been proposed as a target of intervention for regulation of blood coagulation. A number of molecules that interrupt FVIII or factor V (FV) binding to cell membranes have been identified through high throughput screening or structure-based design. We report crystal structures of the FVIII C2 domain under three new crystallization conditions, and a high resolution (1.15 {angstrom}) crystal structure of the FVIII C2 domain bound to a small molecular inhibitor. The latter structure shows that the inhibitor binds to the surface of an exposed {beta}-strand of the C2 domain, Trp{sup 2313}-His{sup 2315}. This result indicates that the Trp{sup 2313}-His{sup 2315} segment is an important constituent of the membrane-binding motif and provides a model to understand the molecular mechanism of the C2 domain membrane interaction.

Six independent fucose-binding sites in the crystal structure of Aspergillus oryzae lectin

Energy Technology Data Exchange (ETDEWEB)

Makyio, Hisayoshi [Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki, 305-0801 (Japan); Shimabukuro, Junpei; Suzuki, Tatsuya [Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193 (Japan); Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501 (Japan); Imamura, Akihiro; Ishida, Hideharu [Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193 (Japan); Kiso, Makoto [Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193 (Japan); Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501 (Japan); Ando, Hiromune, E-mail: hando@gifu-u.ac.jp [Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193 (Japan); Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501 (Japan); Kato, Ryuichi, E-mail: ryuichi.kato@kek.jp [Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki, 305-0801 (Japan)

2016-08-26

The crystal structure of AOL (a fucose-specific lectin of Aspergillus oryzae) has been solved by SAD (single-wavelength anomalous diffraction) and MAD (multi-wavelength anomalous diffraction) phasing of seleno-fucosides. The overall structure is a six-bladed β-propeller similar to that of other fucose-specific lectins. The fucose moieties of the seleno-fucosides are located in six fucose-binding sites. Although the Arg and Glu/Gln residues bound to the fucose moiety are common to all fucose-binding sites, the amino-acid residues involved in fucose binding at each site are not identical. The varying peak heights of the seleniums in the electron density map suggest that each fucose-binding site has a different carbohydrate binding affinity. - Highlights: • The six-bladed β-propeller structure of AOL was solved by seleno-sugar phasing. • The mode of fucose binding is essentially conserved at all six binding sites. • The seleno-fucosides exhibit slightly different interactions and electron densities. • These findings suggest that the affinity for fucose is not identical at each site.

Six independent fucose-binding sites in the crystal structure of Aspergillus oryzae lectin

International Nuclear Information System (INIS)

Makyio, Hisayoshi; Shimabukuro, Junpei; Suzuki, Tatsuya; Imamura, Akihiro; Ishida, Hideharu; Kiso, Makoto; Ando, Hiromune; Kato, Ryuichi

2016-01-01

The crystal structure of AOL (a fucose-specific lectin of Aspergillus oryzae) has been solved by SAD (single-wavelength anomalous diffraction) and MAD (multi-wavelength anomalous diffraction) phasing of seleno-fucosides. The overall structure is a six-bladed β-propeller similar to that of other fucose-specific lectins. The fucose moieties of the seleno-fucosides are located in six fucose-binding sites. Although the Arg and Glu/Gln residues bound to the fucose moiety are common to all fucose-binding sites, the amino-acid residues involved in fucose binding at each site are not identical. The varying peak heights of the seleniums in the electron density map suggest that each fucose-binding site has a different carbohydrate binding affinity. - Highlights: • The six-bladed β-propeller structure of AOL was solved by seleno-sugar phasing. • The mode of fucose binding is essentially conserved at all six binding sites. • The seleno-fucosides exhibit slightly different interactions and electron densities. • These findings suggest that the affinity for fucose is not identical at each site.

Profiling of Parkin-binding partners using tandem affinity purification.

Directory of Open Access Journals (Sweden)

Alessandra Zanon

Full Text Available Parkinson's disease (PD is a progressive neurodegenerative disorder affecting approximately 1-2% of the general population over age 60. It is characterized by a rather selective loss of dopaminergic neurons in the substantia nigra and the presence of α-synuclein-enriched Lewy body inclusions. Mutations in the Parkin gene (PARK2 are the major cause of autosomal recessive early-onset parkinsonism. The Parkin protein is an E3 ubiquitin ligase with various cellular functions, including the induction of mitophagy upon mitochondrial depolarizaton, but the full repertoire of Parkin-binding proteins remains poorly defined. Here we employed tandem affinity purification interaction screens with subsequent mass spectrometry to profile binding partners of Parkin. Using this approach for two different cell types (HEK293T and SH-SY5Y neuronal cells, we identified a total of 203 candidate Parkin-binding proteins. For the candidate proteins and the proteins known to cause heritable forms of parkinsonism, protein-protein interaction data were derived from public databases, and the associated biological processes and pathways were analyzed and compared. Functional similarity between the candidates and the proteins involved in monogenic parkinsonism was investigated, and additional confirmatory evidence was obtained using published genetic interaction data from Drosophila melanogaster. Based on the results of the different analyses, a prioritization score was assigned to each candidate Parkin-binding protein. Two of the top ranking candidates were tested by co-immunoprecipitation, and interaction to Parkin was confirmed for one of them. New candidates for involvement in cell death processes, protein folding, the fission/fusion machinery, and the mitophagy pathway were identified, which provide a resource for further elucidating Parkin function.

Profiling of Parkin-Binding Partners Using Tandem Affinity Purification

Science.gov (United States)

Blankenburg, Hagen; Doncheva, Nadezhda T.; Schwienbacher, Christine; Serafin, Alice; Alexa, Adrian; Weichenberger, Christian X.; Albrecht, Mario; Klein, Christine; Hicks, Andrew A.; Pramstaller, Peter P.

2013-01-01

Parkinson's disease (PD) is a progressive neurodegenerative disorder affecting approximately 1–2% of the general population over age 60. It is characterized by a rather selective loss of dopaminergic neurons in the substantia nigra and the presence of α-synuclein-enriched Lewy body inclusions. Mutations in the Parkin gene (PARK2) are the major cause of autosomal recessive early-onset parkinsonism. The Parkin protein is an E3 ubiquitin ligase with various cellular functions, including the induction of mitophagy upon mitochondrial depolarizaton, but the full repertoire of Parkin-binding proteins remains poorly defined. Here we employed tandem affinity purification interaction screens with subsequent mass spectrometry to profile binding partners of Parkin. Using this approach for two different cell types (HEK293T and SH-SY5Y neuronal cells), we identified a total of 203 candidate Parkin-binding proteins. For the candidate proteins and the proteins known to cause heritable forms of parkinsonism, protein-protein interaction data were derived from public databases, and the associated biological processes and pathways were analyzed and compared. Functional similarity between the candidates and the proteins involved in monogenic parkinsonism was investigated, and additional confirmatory evidence was obtained using published genetic interaction data from Drosophila melanogaster. Based on the results of the different analyses, a prioritization score was assigned to each candidate Parkin-binding protein. Two of the top ranking candidates were tested by co-immunoprecipitation, and interaction to Parkin was confirmed for one of them. New candidates for involvement in cell death processes, protein folding, the fission/fusion machinery, and the mitophagy pathway were identified, which provide a resource for further elucidating Parkin function. PMID:24244333

Cyclic phosphopeptides to rationalize the role of phosphoamino acids in uranyl binding to biological targets

Energy Technology Data Exchange (ETDEWEB)

Starck, Matthieu; Laporte, Fanny A.; Oros, Stephane; Sisommay, Nathalie; Gathu, Vicky; Lebrun, Colette; Delangle, Pascale [INAC/SyMMES, UMR5819, Universite Grenoble Alpes, CEA, CNRS, Grenoble (France); Solari, Pier Lorenzo [Synchrotron SOLEIL, L' orme des Merisiers, Saint-Aubin, Gif-sur-Yvette (France); Creff, Gaelle; Den Auwer, Christophe [Institut de Chimie de Nice, UMR7272, Universite Cote d' Azur, Nice (France); Roques, Jerome [Institut de Physique Nucleaire d' Orsay, CNRS-IN2P3, Univ. Paris-Sud, Universite Paris-Saclay (France)

2017-04-19

The specific molecular interactions responsible for uranium toxicity are not yet understood. The uranyl binding sites in high-affinity target proteins have not been identified yet and the involvement of phosphoamino acids is still an important question. Short cyclic peptide sequences, with three glutamic acids and one phosphoamino acid, are used as simple models to mimic metal binding sites in phosphoproteins and to help understand the mechanisms involved in uranium toxicity. A combination of peptide design and synthesis, analytical chemistry, extended X-ray absorption fine structure (EXAFS) spectroscopy, and DFT calculations demonstrates the involvement of the phosphate group in the uranyl coordination sphere together with the three carboxylates of the glutamate moieties. The affinity constants measured with a reliable analytical competitive approach at physiological pH are significantly enhanced owing to the presence of the phosphorous moiety. These findings corroborate the importance of phosphoamino acids in uranyl binding in proteins and the relevance of considering phosphoproteins as potential uranyl targets in vivo. (copyright 2017 Wiley-VCH Verlag GmbH and Co. KGaA, Weinheim)

Alternative Conformations of the Tau Repeat Domain in Complex with an Engineered Binding Protein*

Science.gov (United States)

Grüning, Clara S. R.; Mirecka, Ewa A.; Klein, Antonia N.; Mandelkow, Eckhard; Willbold, Dieter; Marino, Stephen F.; Stoldt, Matthias; Hoyer, Wolfgang

2014-01-01

The aggregation of Tau into paired helical filaments is involved in the pathogenesis of several neurodegenerative diseases, including Alzheimer disease. The aggregation reaction is characterized by conformational conversion of the repeat domain, which partially adopts a cross-β-structure in the resulting amyloid-like fibrils. Here, we report the selection and characterization of an engineered binding protein, β-wrapin TP4, targeting the Tau repeat domain. TP4 was obtained by phage display using the four-repeat Tau construct K18ΔK280 as a target. TP4 binds K18ΔK280 as well as the longest isoform of human Tau, hTau40, with nanomolar affinity. NMR spectroscopy identified two alternative TP4-binding sites in the four-repeat domain, with each including two hexapeptide motifs with high β-sheet propensity. Both binding sites contain the aggregation-determining PHF6 hexapeptide within repeat 3. In addition, one binding site includes the PHF6* hexapeptide within repeat 2, whereas the other includes the corresponding hexapeptide Tau(337–342) within repeat 4, denoted PHF6**. Comparison of TP4-binding with Tau aggregation reveals that the same regions of Tau are involved in both processes. TP4 inhibits Tau aggregation at substoichiometric concentration, demonstrating that it interferes with aggregation nucleation. This study provides residue-level insight into the interaction of Tau with an aggregation inhibitor and highlights the structural flexibility of Tau. PMID:24966331

Radiometric immunosorbent assay for the detection of anti-hormone-binding protein antibodies

International Nuclear Information System (INIS)

Pierce, E.A.; Dame, M.C.; DeLuca, H.F.

1986-01-01

A radiometric immunosorbent assay (RISA) for the detection of monoclonal antibodies to hormone-binding proteins has been developed. The assay involves incubating hybridoma supernatants in microtiter wells that have been coated with goat anti-mouse IgG antibodies. Any mouse IgG in the test supernatant is thus specifically retained in the wells. Radioactive ligand-binding protein complexes are then incubated in the wells. The presence of anti-binding protein antibodies in the supernatant is indicated by specific retention of radioactive ligand-binding protein complexes in the wells. Crude antigen preparations, such as tissue homogenates, can be used to detect antibodies. The assay is capable of detecting antibody at concentrations 20 ng/ml (approx. 100 pM IgG). The RISA has been used successfully to screen for monoclonal antibodies to the intracellular receptor for 1,25-dihydroxyvitamin D 3 and should be useful for the detection of antibodies to ligand-binding proteins in general

Substrate-Triggered Exosite Binding: Synergistic Dendrimer/Folic Acid Action for Achieving Specific, Tight-Binding to Folate Binding Protein.

Science.gov (United States)

Chen, Junjie; van Dongen, Mallory A; Merzel, Rachel L; Dougherty, Casey A; Orr, Bradford G; Kanduluru, Ananda Kumar; Low, Philip S; Marsh, E Neil G; Banaszak Holl, Mark M

2016-03-14

Polymer-ligand conjugates are designed to bind proteins for applications as drugs, imaging agents, and transport scaffolds. In this work, we demonstrate a folic acid (FA)-triggered exosite binding of a generation five poly(amidoamine) (G5 PAMAM) dendrimer scaffold to bovine folate binding protein (bFBP). The protein exosite is a secondary binding site on the protein surface, separate from the FA binding pocket, to which the dendrimer binds. Exosite binding is required to achieve the greatly enhanced binding constants and protein structural change observed in this study. The G5Ac-COG-FA1.0 conjugate bound tightly to bFBP, was not displaced by a 28-fold excess of FA, and quenched roughly 80% of the initial fluorescence. Two-step binding kinetics were measured using the intrinsic fluorescence of the FBP tryptophan residues to give a KD in the low nanomolar range for formation of the initial G5Ac-COG-FA1.0/FBP* complex, and a slow conversion to the tight complex formed between the dendrimer and the FBP exosite. The extent of quenching was sensitive to the choice of FA-dendrimer linker chemistry. Direct amide conjugation of FA to G5-PAMAM resulted in roughly 50% fluorescence quenching of the FBP. The G5Ac-COG-FA, which has a longer linker containing a 1,2,3-triazole ring, exhibited an ∼80% fluorescence quenching. The binding of the G5Ac-COG-FA1.0 conjugate was compared to poly(ethylene glycol) (PEG) conjugates of FA (PEGn-FA). PEG2k-FA had a binding strength similar to that of FA, whereas other PEG conjugates with higher molecular weight showed weaker binding. However, no PEG conjugates gave an increased degree of total fluorescence quenching.

Nucleic acid-binding properties of the RRM-containing protein RDM1

International Nuclear Information System (INIS)

Hamimes, Samia; Bourgeon, Dominique; Stasiak, Alicja Z.; Stasiak, Andrzej; Van Dyck, Eric

2006-01-01

RDM1 (RAD52 Motif 1) is a vertebrate protein involved in the cellular response to the anti-cancer drug cisplatin. In addition to an RNA recognition motif, RDM1 contains a small amino acid motif, named RD motif, which it shares with the recombination and repair protein, RAD52. RDM1 binds to single- and double-stranded DNA, and recognizes DNA distortions induced by cisplatin adducts in vitro. Here, we have performed an in-depth analysis of the nucleic acid-binding properties of RDM1 using gel-shift assays and electron microscopy. We show that RDM1 possesses acidic pH-dependent DNA-binding activity and that it binds RNA as well as DNA, and we present evidence from competition gel-shift experiments that RDM1 may be capable of discrimination between the two nucleic acids. Based on reported studies of RAD52, we have generated an RDM1 variant mutated in its RD motif. We find that the L 119 GF → AAA mutation affects the mode of RDM1 binding to single-stranded DNA

Cloning of a novel phosphotyrosine binding domain containing molecule, Odin, involved in signaling by receptor tyrosine kinases

DEFF Research Database (Denmark)

Pandey, A.; Blagoev, B.; Kratchmarova, I.

2002-01-01

. Deletion analysis showed that the phosphotyrosine binding domain of Odin is not required for its tyrosine phosphorylation. Overexpression of Odin, but not an unrelated adapter protein, Grb2, inhibited EGF-induced activation of c-Fos promoter. Microinjection of wild-type or a mutant version lacking the PTB...

Expression of melatonin receptors in arteries involved in thermoregulation

International Nuclear Information System (INIS)

Viswanathan, M.; Laitinen, J.T.; Saavedra, J.M.

1990-01-01

Melatonin binding sites were localized and characterized in the vasculature of the rat by using the melatonin analogue 2-[125I]iodomelatonin (125I-melatonin) and quantitative in vitro autoradiography. The expression of these sites was restricted to the caudal artery and to the arteries that form the circle of Willis at the base of the brain. The arterial 125I-melatonin binding was stable, saturable, and reversible. Saturation studies revealed that the binding represented a single class of high-affinity binding sites with a dissociation constant (Kd) of 3.4 x 10(-11) M in the anterior cerebral artery and 1.05 x 10(-10) M in the caudal artery. The binding capacities (Bmax) in these arteries were 19 and 15 fmol/mg of protein, respectively. The relative order of potency of indoles for inhibition of 125I-melatonin binding at these sites was typical of a melatonin receptor: 2-iodomelatonin greater than melatonin greater than N-acetylserotonin much much greater than 5-hydroxytryptamine. Norepinephrine-induced contraction of the caudal artery in vitro was significantly prolonged and potentiated by melatonin in a concentration-dependent manner, suggesting that these arterial binding sites are functional melatonin receptors. Neither primary steps in smooth muscle contraction (inositol phospholipid hydrolysis) nor relaxation (adenylate cyclase activation) were affected by melatonin. Melatonin, through its action on the tone of these arteries, may cause circulatory adjustments in these arteries, which are believed to be involved in thermoregulation

Strong Ligand-Protein Interactions Derived from Diffuse Ligand Interactions with Loose Binding Sites.

Science.gov (United States)

Marsh, Lorraine

2015-01-01

Many systems in biology rely on binding of ligands to target proteins in a single high-affinity conformation with a favorable ΔG. Alternatively, interactions of ligands with protein regions that allow diffuse binding, distributed over multiple sites and conformations, can exhibit favorable ΔG because of their higher entropy. Diffuse binding may be biologically important for multidrug transporters and carrier proteins. A fine-grained computational method for numerical integration of total binding ΔG arising from diffuse regional interaction of a ligand in multiple conformations using a Markov Chain Monte Carlo (MCMC) approach is presented. This method yields a metric that quantifies the influence on overall ligand affinity of ligand binding to multiple, distinct sites within a protein binding region. This metric is essentially a measure of dispersion in equilibrium ligand binding and depends on both the number of potential sites of interaction and the distribution of their individual predicted affinities. Analysis of test cases indicates that, for some ligand/protein pairs involving transporters and carrier proteins, diffuse binding contributes greatly to total affinity, whereas in other cases the influence is modest. This approach may be useful for studying situations where "nonspecific" interactions contribute to biological function.

Discrete persistent-chain model for protein binding on DNA.

Science.gov (United States)

Lam, Pui-Man; Zhen, Yi

2011-04-01

We describe and solve a discrete persistent-chain model of protein binding on DNA, involving an extra σ(i) at a site i of the DNA. This variable takes the value 1 or 0, depending on whether or not the site is occupied by a protein. In addition, if the site is occupied by a protein, there is an extra energy cost ɛ. For a small force, we obtain analytic expressions for the force-extension curve and the fraction of bound protein on the DNA. For higher forces, the model can be solved numerically to obtain force-extension curves and the average fraction of bound proteins as a function of applied force. Our model can be used to analyze experimental force-extension curves of protein binding on DNA, and hence deduce the number of bound proteins in the case of nonspecific binding. ©2011 American Physical Society

EWS and FUS bind a subset of transcribed genes encoding proteins enriched in RNA regulatory functions.

Science.gov (United States)

Luo, Yonglun; Blechingberg, Jenny; Fernandes, Ana Miguel; Li, Shengting; Fryland, Tue; Børglum, Anders D; Bolund, Lars; Nielsen, Anders Lade

2015-11-14

FUS (TLS) and EWS (EWSR1) belong to the FET-protein family of RNA and DNA binding proteins. FUS and EWS are structurally and functionally related and participate in transcriptional regulation and RNA processing. FUS and EWS are identified in translocation generated cancer fusion proteins and involved in the human neurological diseases amyotrophic lateral sclerosis and fronto-temporal lobar degeneration. To determine the gene regulatory functions of FUS and EWS at the level of chromatin, we have performed chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Our results show that FUS and EWS bind to a subset of actively transcribed genes, that binding often is downstream the poly(A)-signal, and that binding overlaps with RNA polymerase II. Functional examinations of selected target genes identified that FUS and EWS can regulate gene expression at different levels. Gene Ontology analyses showed that FUS and EWS target genes preferentially encode proteins involved in regulatory processes at the RNA level. The presented results yield new insights into gene interactions of EWS and FUS and have identified a set of FUS and EWS target genes involved in pathways at the RNA regulatory level with potential to mediate normal and disease-associated functions of the FUS and EWS proteins.

Novel interactions of ankyrins-G at the costameres: The muscle-specific Obscurin/Titin-Binding-related Domain (OTBD) binds plectin and filamin C

International Nuclear Information System (INIS)

Maiweilidan, Yimingjiang; Klauza, Izabela; Kordeli, Ekaterini

2011-01-01

Ankyrins, the adapters of the spectrin skeleton, are involved in local accumulation and stabilization of integral proteins to the appropriate membrane domains. In striated muscle, tissue-dependent alternative splicing generates unique Ank3 gene products (ankyrins-G); they share the Obscurin/Titin-Binding-related Domain (OTBD), a muscle-specific insert of the C-terminal domain which is highly conserved among ankyrin genes, and binds obscurin and titin to Ank1 gene products. We previously proposed that OTBD sequences constitute a novel domain of protein-protein interactions which confers ankyrins with specific cellular functions in muscle. Here we searched for muscle proteins binding to ankyrin-G OTBD by yeast two hybrid assay, and we found plectin and filamin C, two organizing elements of the cytoskeleton with essential roles in myogenesis, muscle cell cytoarchitecture, and muscle disease. The three proteins coimmunoprecipitate from skeletal muscle extracts and colocalize at costameres in adult muscle fibers. During in vitro myogenesis, muscle ankyrins-G are first expressed in postmitotic myocytes undergoing fusion to myotubes. In western blots of subcellular fractions from C2C12 cells, the majority of muscle ankyrins-G appear associated with membrane compartments. Occasional but not extensive co-localization at nascent costameres suggested that ankyrin-G interactions with plectin and filamin C are not involved in costamere assembly; they would rather reinforce stability and/or modulate molecular interactions in sarcolemma microdomains by establishing novel links between muscle-specific ankyrins-G and the two costameric dystrophin-associated glycoprotein and integrin-based protein complexes. These results report the first protein-protein interactions involving the ankyrin-G OTBD domain and support the hypothesis that OTBD sequences confer ankyrins with a gain of function in vertebrates, bringing further consolidation and resilience of the linkage between sarcomeres

Identification of DNA-binding sites for the activator involved in late transcription of the temperate lactococcal phage TP901-1

DEFF Research Database (Denmark)

Pedersen, Margit; Kilstrup, Mogens; Hammer, Karin

2006-01-01

Alt, encoded by the lactococcal phage TP901-1, is needed for late transcription. We identify Alt as a DNA-binding protein, and footprint analysis shows that Alt binds to a region containing four imperfect direct repeats (ALT boxes) located -76 to -32 relative to the P-late transcriptional start...... site. The importance of the ALT boxes was confirmed by deletion of one or two ALT boxes and by introducing mutations in ALT boxes 1 and 4. Alt is proposed to act as a tetramer or higher multimer activating transcription of TP901-1 late genes by binding to the four ALT boxes, and bending of the DNA may...... be important for transcriptional activation of P-late. Furthermore, our results suggest that DNA replication may be required for late transcription in TP901-1. Additionally, we identify gp28 of the related lactococcal phage Tuc2009 as an activator and show that the activators required for late transcription...

In silico docking of forchlorfenuron (FCF to septins suggests that FCF interferes with GTP binding.

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Dimitrios Angelis

Full Text Available Septins are GTP-binding proteins that form cytoskeleton-like filaments, which are essential for many functions in eukaryotic organisms. Small molecule compounds that disrupt septin filament assembly are valuable tools for dissecting septin functions with high temporal control. To date, forchlorfenuron (FCF is the only compound known to affect septin assembly and functions. FCF dampens the dynamics of septin assembly inducing the formation of enlarged stable polymers, but the underlying mechanism of action is unknown. To investigate how FCF binds and affects septins, we performed in silico simulations of FCF docking to all available crystal structures of septins. Docking of FCF with SEPT2 and SEPT3 indicated that FCF interacts preferentially with the nucleotide-binding pockets of septins. Strikingly, FCF is predicted to form hydrogen bonds with residues involved in GDP-binding, mimicking nucleotide binding. FCF docking with the structure of SEPT2-GppNHp, a nonhydrolyzable GTP analog, and SEPT7 showed that FCF may assume two alternative non-overlapping conformations deeply into and on the outer side of the nucleotide-binding pocket. Surprisingly, FCF was predicted to interact with the P-loop Walker A motif GxxxxGKS/T, which binds the phosphates of GTP, and the GTP specificity motif AKAD, which interacts with the guanine base of GTP, and highly conserved amino acids including a threonine, which is critical for GTP hydrolysis. Thus, in silico FCF exhibits a conserved mechanism of binding, interacting with septin signature motifs and residues involved in GTP binding and hydrolysis. Taken together, our results suggest that FCF stabilizes septins by locking them into a conformation that mimics a nucleotide-bound state, preventing further GTP binding and hydrolysis. Overall, this study provides the first insight into how FCF may bind and stabilize septins, and offers a blueprint for the rational design of FCF derivatives that could target septins with

Steric hindrance mutagenesis in the conserved extracellular vestibule impedes allosteric binding of antidepressants to the serotonin transporter

DEFF Research Database (Denmark)

Plenge, Per; Shi, Lei; Beuming, Thijs

2012-01-01

be involved in the allosteric binding in the extracellular vestibule located above the central substrate binding (S1) site. Indeed, mutagenesis of selected residues in the vestibule reduces the allosteric potency of (S)-citalopram and clomipramine. The identified site is further supported by the inhibitory...

[3]tetrahydrotrazodone binding. Association with serotonin binding sites

International Nuclear Information System (INIS)

Kendall, D.A.; Taylor, D.P.; Enna, S.J.

1983-01-01

High (17 nM) and low (603 nM) affinity binding sites for [ 3 ]tetrahydrotrazodone ([ 3 ] THT), a biologically active analogue of trazodone, have been identified in rat brain membranes. The substrate specificity, concentration, and subcellular and regional distributions of these sites suggest that they may represent a component of the serotonin transmitter system. Pharmacological analysis of [ 3 ]THT binding, coupled with brain lesion and drug treatment experiments, revealed that, unlike other antidepressants, [ 3 ] THT does not attach to either a biogenic amine transporter or serotonin binding sites. Rather, it would appear that [ 3 ]THT may be an antagonist ligand for the serotonin binding site. This probe may prove of value in defining the mechanism of action of trazodone and in further characterizing serotonin receptors

RNA binding efficacy of theophylline, theobromine and caffeine.

Science.gov (United States)

Johnson, I Maria; Kumar, S G Bhuvan; Malathi, R

2003-04-01

The binding of naturally occurring methylxanthines such as theophylline, theobromine and caffeine to nucleic acids are reckoned to be pivotal as they are able to modulate the cellular activities. We explore the interaction of yeast RNA binding efficacy of the above xanthine derivatives by using UV absorption differential spectroscopy and Fourier Transform Infrared (FTIR) spectroscopy. Both the analyses show discrimination in their binding affinity to RNA. The differential UV-spectrum at P/D 3.3 reveals the greater RNA binding activity for theophylline (85 +/- 5%), whereas moderate and comparatively less binding activity for theobromine (45 +/- 5%) and caffeine (30 +/- 5%) and the binding activity was found to depend on concentration of the drugs. In FTIR analysis we observed changes in the amino group (NH) of RNA complexed by drugs, where the NH band is found to become very broad, indicating hydrogen bonding (H-bonding) with theophylline (3343.4 cm(-1)), theobromine (3379.8 cm(-1)) and caffeine (3343 cm(-1)) as compared to the free RNA (3341.6 cm(-1)). Furthermore in RNA-theophylline complex, it is observed that the carbonyl (C=O) vibration frequency (nu(C=O)) of both drug (nu(C=O)=1718, 1666 cm(-1)) as well as RNA (nu(C=O)=1699, 1658 cm(-1)) disappeared and a new vibration band appeared around 1703 cm(-1), indicating that the C=O and NH groups of drug and RNA are effectively involved in H-bonding. Whereas in RNA-theobromine and RNA-caffeine complexes, we found very little changes in C=O frequency and only broadening of the NH band of RNA due to complexation is observed in these groups. The changes in the vibrations of G-C/A-U bands and other bending frequencies are discussed. Thus the discrimination in the binding affinity of methylxanthines with RNA molecule shows that strong RNA binding drugs like theophylline can selectively be delivered to RNA targets of microbial pathogens having the mechanism of RNA catalysis.

Spectroscopic study of drug-binding characteristics of unmodified and pNPA-based acetylated human serum albumin: Does esterase activity affect microenvironment of drug binding sites on the protein?

Energy Technology Data Exchange (ETDEWEB)

Moradi, Nastaran [Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Faculty of Pharmaceutical Sciences, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Ashrafi-Kooshk, Mohammad Reza [Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Ghobadi, Sirous [Department of Biology, Faculty of Sciences, Razi University, Kermanshah (Iran, Islamic Republic of); Shahlaei, Mohsen [Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Faculty of Pharmaceutical Sciences, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Khodarahmi, Reza, E-mail: rkhodarahmi@mbrc.ac.ir [Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Faculty of Pharmaceutical Sciences, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of)

2015-04-15

Human serum albumin (HSA) is the most prominent extracellular protein in blood plasma. There are several binding sites on the protein which provide accommodation for structurally-unrelated endogenous and exogenous ligands and a wide variety of drugs. “Esterase-like” activity (hydrolysis of p-nitrophenyl esters) by the protein has been also reported. In the current study, we set out to investigate the interaction of indomethacin and ibuprofen with the unmodified and modified HSA (pNPA-modified HSA) using various spectroscopic techniques. Fluorescence data showed that 1:1 binding of drug to HSA is associated with quenching of the protein intrinsic fluorescence. Decrease of protein surface hydrophobicity (PSH), alteration in drug binding affinity and change of the protein stability, after esterase-like activity and permanent acetylation of HSA, were also documented. Analysis of the quenching and thermodynamic parameters indicated that forces involved in drug–HSA interactions change upon the protein modification. - Highlights: • Binding propensity of indomethacin extremely decreased upon the protein acetylation. • There is no ibuprofen binding after protein acetylation. • Protein stability changes upon drug binding as well as protein acetylation. • Drug pharmacokinetics may be influenced under co-administration of HSA-modifier drugs.

Spectroscopic study of drug-binding characteristics of unmodified and pNPA-based acetylated human serum albumin: Does esterase activity affect microenvironment of drug binding sites on the protein?

International Nuclear Information System (INIS)

Moradi, Nastaran; Ashrafi-Kooshk, Mohammad Reza; Ghobadi, Sirous; Shahlaei, Mohsen; Khodarahmi, Reza

2015-01-01

Human serum albumin (HSA) is the most prominent extracellular protein in blood plasma. There are several binding sites on the protein which provide accommodation for structurally-unrelated endogenous and exogenous ligands and a wide variety of drugs. “Esterase-like” activity (hydrolysis of p-nitrophenyl esters) by the protein has been also reported. In the current study, we set out to investigate the interaction of indomethacin and ibuprofen with the unmodified and modified HSA (pNPA-modified HSA) using various spectroscopic techniques. Fluorescence data showed that 1:1 binding of drug to HSA is associated with quenching of the protein intrinsic fluorescence. Decrease of protein surface hydrophobicity (PSH), alteration in drug binding affinity and change of the protein stability, after esterase-like activity and permanent acetylation of HSA, were also documented. Analysis of the quenching and thermodynamic parameters indicated that forces involved in drug–HSA interactions change upon the protein modification. - Highlights: • Binding propensity of indomethacin extremely decreased upon the protein acetylation. • There is no ibuprofen binding after protein acetylation. • Protein stability changes upon drug binding as well as protein acetylation. • Drug pharmacokinetics may be influenced under co-administration of HSA-modifier drugs

Variation in 12 porcine genes involved in the carbohydrate moiety assembly of glycosphingolipids does not account for differential binding of F4 Escherichia coli and their fimbriae.

Science.gov (United States)

Goetstouwers, Tiphanie; Van Poucke, Mario; Coddens, Annelies; Nguyen, Van Ut; Melkebeek, Vesna; Deforce, Dieter; Cox, Eric; Peelman, Luc J

2014-10-03

Glycosphingolipids (GSLs) are important membrane components composed of a carbohydrate structure attached to a hydrophobic ceramide. They can serve as specific membrane receptors for microbes and microbial products, such as F4 Escherichia coli (F4 ETEC) and isolated F4 fimbriae. The aim of this study was to investigate the hypothesis that variation in genes involved in the assembly of the F4 binding carbohydrate moiety of GSLs (i.e. ARSA, B4GALT6, GAL3ST1, GALC, GBA, GLA, GLB1, GLB1L, NEU1, NEU2, UGCG, UGT8) could account for differential binding of F4 ETEC and their fimbriae. RT-PCR could not reveal any differential expression of the 12 genes in the jejunum of F4 receptor-positive (F4R(+)) and F4 receptor-negative (F4R(-)) pigs. Sequencing the complete open reading frame of the 11 expressed genes (NEU2 was not expressed) identified 72 mutations. Although some of them might have a structural effect, none of them could be associated with a F4R phenotype. We conclude that no regulatory or structural variation in any of the investigated genes is responsible for the genetic susceptibility of pigs towards F4 ETEC.

Monomeric Yeast Frataxin is an Iron-Binding Protein

International Nuclear Information System (INIS)

Cook, J.; Bencze, K.; Jankovic, A.; Crater, A.; Busch, C.; Bradley, P.; Stemmler, A.; Spaller, M.; Stemmler, T.

2006-01-01

Friedreich's ataxia, an autosomal cardio- and neurodegenerative disorder that affects 1 in 50 000 humans, is caused by decreased levels of the protein frataxin. Although frataxin is nuclear-encoded, it is targeted to the mitochondrial matrix and necessary for proper regulation of cellular iron homeostasis. Frataxin is required for the cellular production of both heme and iron-sulfur (Fe-S) clusters. Monomeric frataxin binds with high affinity to ferrochelatase, the enzyme involved in iron insertion into porphyrin during heme production. Monomeric frataxin also binds to Isu, the scaffold protein required for assembly of Fe-S cluster intermediates. These processes (heme and Fe-S cluster assembly) share requirements for iron, suggesting that monomeric frataxin might function as the common iron donor. To provide a molecular basis to better understand frataxin's function, we have characterized the binding properties and metal-site structure of ferrous iron bound to monomeric yeast frataxin. Yeast frataxin is stable as an iron-loaded monomer, and the protein can bind two ferrous iron atoms with micromolar binding affinity. Frataxin amino acids affected by the presence of iron are localized within conserved acidic patches located on the surfaces of both helix-1 and strand-1. Under anaerobic conditions, bound metal is stable in the high-spin ferrous state. The metal-ligand coordination geometry of both metal-binding sites is consistent with a six-coordinate iron-(oxygen/nitrogen) based ligand geometry, surely constructed in part from carboxylate and possibly imidazole side chains coming from residues within these conserved acidic patches on the protein. On the basis of our results, we have developed a model for how we believe yeast frataxin interacts with iron

Rational redesign of neutral endopeptidase binding to merlin and moesin proteins

Science.gov (United States)

Niv, Masha Y; Iida, Katsuyuki; Zheng, Rong; Horiguchi, Akio; Shen, Ruoqian; Nanus, David M

2009-01-01

Neutral endopeptidase (NEP) is a 90- to 110-kDa cell-surface peptidase that is normally expressed by numerous tissues but whose expression is lost or reduced in a variety of malignancies. The anti-tumorigenic function of NEP is mediated not only by its catalytic activity but also through direct protein–protein interactions of its cytosolic region with several binding partners, including Lyn kinase, PTEN, and ezrin/radixin/moesin (ERM) proteins. We have previously shown that mutation of the K19K20K21 basic cluster in NEPs' cytosolic region to residues QNI disrupts binding to the ERM proteins. Here we show that the ERM-related protein merlin (NF2) does not bind NEP or its cytosolic region. Using experimental data, threading, and sequence analysis, we predicted the involvement of moesin residues E159Q160 in binding to the NEP cytosolic domain. Mutation of these residues to NL (to mimic the corresponding N159L160 residues in the nonbinder merlin) disrupted moesin binding to NEP. Mutation of residues N159L160Y161K162M163 in merlin to the corresponding moesin residues resulted in NEP binding to merlin. This engineered NEP peptide–merlin interaction was diminished by the QNI mutation in NEP, supporting the role of the NEP basic cluster in binding. We thus identified the region of interaction between NEP and moesin, and engineered merlin into a NEP-binding protein. These data form the basis for further exploration of the details of NEP-ERM binding and function. PMID:19388049

Identification of the site of human mannan-binding lectin involved in the interaction with its partner serine proteases: the essential role of Lys55

DEFF Research Database (Denmark)

Teillet, F; Lacroix, M; Thiel, Steffen

2007-01-01

Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2. To ident......Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2....... To identify the MASP binding site(s) of human MBL, point mutants targeting residues C-terminal to the hinge region were produced and tested for their interaction with the MASPs and MAp19 using surface plasmon resonance and functional assays. Mutation Lys(55)Ala abolished interaction with the MASPs and MAp19...... and prevented formation of functional MBL-MASP-2 complexes. Mutations Lys(55)Gln and Lys(55)Glu abolished binding to MASP-1 and -3 and strongly inhibited interaction with MAp19. Conversely, mutation Lys(55)Arg abolished interaction with MASP-2 and MAp19, but only weakened interaction with MASP-1 and -3...

Cyclophilin B binding to platelets supports calcium-dependent adhesion to collagen.

Science.gov (United States)

Allain, F; Durieux, S; Denys, A; Carpentier, M; Spik, G

1999-08-01

We have recently reported that cyclophilin B (CyPB), a secreted cyclosporine-binding protein, could bind to T lymphocytes through interactions with two types of binding sites. The first ones, referred to as type I, involve interactions with the conserved domain of CyPB and promote the endocytosis of surface-bound ligand, while the second type of binding sites, termed type II, are represented by glycosaminoglycans (GAG). Here, we further investigated the interactions of CyPB with blood cell populations. In addition to lymphocytes, CyPB was found to interact mainly with platelets. The binding is specific, with a dissociation constant (kd) of 9 +/- 3 nmol/L and the number of sites estimated at 960 +/- 60 per cell. Platelet glycosaminoglycans are not required for the interactions, but the binding is dramatically reduced by active cyclosporine derivatives. We then analyzed the biologic effects of CyPB and found a significant increase in platelet adhesion to collagen. Concurrently, CyPB initiates a transmembranous influx of Ca(2+) and induces the phosphorylation of the P-20 light chains of myosin. Taken together, the present results demonstrate for the first time that extracellular CyPB specifically interacts with platelets through a functional receptor related to the lymphocyte type I binding sites and might act by regulating the activity of a receptor-operated membrane Ca(2+) channel.

Binding of tissue plasminogen activator to human umbilical vein endothelial cells

International Nuclear Information System (INIS)

Beebe, D.P.

1987-01-01

The binding of purified, recombinant tissue plasminogen activator (tPA) to human umbilical vein endothelial cells (HUVEC) was studied in vitro using immunofluorescence as well as radiolabeled tPA. Immunofluorescence was performed on HUVEC grown on round glass coverslips using rabbit anti-human tPA and fluorescein-conjugated anti-rabbit immunoglobulin. Positive fluorescence was observed only after incubation of HUVEC with tPA. HUVEC were grown to confluence in 24-well tissue culture plates, washed, and incubated with a constant amount of 125 I-tPA and various concentrations of unlabeled tPA. The binding of tPA to HUVEC was found to be specific, saturable, and reversible. Scatchard analysis yielded as equilibrium constant (K/sub eq/) of 4.2 x 10 6 M -1 and 1.2 x 10 7 binding sites per cell. Binding was inhibited by positively charged amino acids and by D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone but not by carbohydrates including mannose, galactose, N-acetyl glucosamine and N-acetyl galactosamine. Neat human plasma abrogates but does not totally inhibit binding of tPA to HUVEC. Binding was neither enhanced nor inhibited by fibronectin. Although the affinity of binding of tPA to HUVEC is low, the endothelial cell may be involved in regulating plasma levels of tPA in vivo which may have therapeutic significance

Cavity Versus Ligand Shape Descriptors: Application to Urokinase Binding Pockets.

Science.gov (United States)

Cerisier, Natacha; Regad, Leslie; Triki, Dhoha; Camproux, Anne-Claude; Petitjean, Michel

2017-11-01

We analyzed 78 binding pockets of the human urokinase plasminogen activator (uPA) catalytic domain extracted from a data set of crystallized uPA-ligand complexes. These binding pockets were computed with an original geometric method that does NOT involve any arbitrary parameter, such as cutoff distances, angles, and so on. We measured the deviation from convexity of each pocket shape with the pocket convexity index (PCI). We defined a new pocket descriptor called distributional sphericity coefficient (DISC), which indicates to which extent the protein atoms of a given pocket lie on the surface of a sphere. The DISC values were computed with the freeware PCI. The pocket descriptors and their high correspondences with ligand descriptors are crucial for polypharmacology prediction. We found that the protein heavy atoms lining the urokinases binding pockets are either located on the surface of their convex hull or lie close to this surface. We also found that the radii of the urokinases binding pockets and the radii of their ligands are highly correlated (r = 0.9).

Streptococcus mutans SpaP binds to RadD of Fusobacterium nucleatum ssp. polymorphum.

Science.gov (United States)

Guo, Lihong; Shokeen, Bhumika; He, Xuesong; Shi, Wenyuan; Lux, Renate

2017-10-01

Adhesin-mediated bacterial interspecies interactions are important elements in oral biofilm formation. They often occur on a species-specific level, which could determine health or disease association of a biofilm community. Among the key players involved in these processes are the ubiquitous fusobacteria that have been recognized for their ability to interact with numerous different binding partners. Fusobacterial interactions with Streptococcus mutans, an important oral cariogenic pathogen, have previously been described but most studies focused on binding to non-mutans streptococci and specific cognate adhesin pairs remain to be identified. Here, we demonstrated differential binding of oral fusobacteria to S. mutans. Screening of existing mutant derivatives indicated SpaP as the major S. mutans adhesin specific for binding to Fusobacterium nucleatum ssp. polymorphum but none of the other oral fusobacteria tested. We inactivated RadD, a known adhesin of F. nucleatum ssp. nucleatum for interaction with a number of gram-positive species, in F. nucleatum ssp. polymorphum and used a Lactococcus lactis heterologous SpaP expression system to demonstrate SpaP interaction with RadD of F. nucleatum ssp. polymorphum. This is a novel function for SpaP, which has mainly been characterized as an adhesin for binding to host proteins including salivary glycoproteins. In conclusion, we describe an additional role for SpaP as adhesin in interspecies adherence with RadD-SpaP as the interacting adhesin pair for binding between S. mutans and F. nucleatum ssp. polymorphum. Furthermore, S. mutans attachment to oral fusobacteria appears to involve species- and subspecies-dependent adhesin interactions. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Purification and characterization of sheep platelet cyclo-oxygenase. Acetylation by aspirin prevents haemin binding to the enzyme.

Science.gov (United States)

Boopathy, R; Balasubramanian, A S

1986-01-01

Arachidonate cyclo-oxygenase (prostaglandin synthetase; prostaglandin endoperoxide synthetase; EC 1.14.99.1) was purified from sheep platelets. The purification procedure involved hydrophobic column chromatography using either Ibuprofen-Sepharose, phenyl-Sepharose or arachidic acid-Sepharose as the first step followed by metal-chelate Sepharose and haemin-Sepharose affinity chromatography. The purified enzyme (Mr approximately 65,000) was homogeneous as observed by SDS/polyacrylamide-gel electrophoresis and silver staining. The enzyme was a glycoprotein with mannose as the neutral sugar. Haemin or haemoglobin was essential for activity. The purified enzyme could bind haemin exhibiting a characteristic absorption maximum at 410 nm. The enzyme after metal-chelate column chromatography could undergo acetylation by [acetyl-3H]aspirin. The labelled acetylated enzyme could not bind to haemin-Sepharose, presumably due to acetylation of a serine residue involved in the binding to haemin. The acetylated enzyme also failed to show its characteristic absorption maximum at 410 nm when allowed to bind haemin. Images Fig. 1. Fig. 4. PMID:3101664

Artemin Crystal Structure Reveals Insights into Heparan Sulfate Binding

Energy Technology Data Exchange (ETDEWEB)

Silvian,L.; Jin, P.; Carmillo, P.; Boriack-Sjodin, P.; Pelletier, C.; Rushe, M.; Gong, B.; Sah, D.; Pepinsky, B.; Rossomando, A.

2006-01-01

Artemin (ART) promotes the growth of developing peripheral neurons by signaling through a multicomponent receptor complex comprised of a transmembrane tyrosine kinase receptor (cRET) and a specific glycosylphosphatidylinositol-linked co-receptor (GFR{alpha}3). Glial cell line-derived neurotrophic factor (GDNF) signals through a similar ternary complex but requires heparan sulfate proteoglycans (HSPGs) for full activity. HSPG has not been demonstrated as a requirement for ART signaling. We crystallized ART in the presence of sulfate and solved its structure by isomorphous replacement. The structure reveals ordered sulfate anions bound to arginine residues in the pre-helix and amino-terminal regions that were organized in a triad arrangement characteristic of heparan sulfate. Three residues in the pre-helix were singly or triply substituted with glutamic acid, and the resulting proteins were shown to have reduced heparin-binding affinity that is partly reflected in their ability to activate cRET. This study suggests that ART binds HSPGs and identifies residues that may be involved in HSPG binding.

RNA-binding IMPs promote cell adhesion and invadopodia formation

DEFF Research Database (Denmark)

Vikesaa, Jonas; Hansen, Thomas V O; Jønson, Lars

2006-01-01

Oncofetal RNA-binding IMPs have been implicated in mRNA localization, nuclear export, turnover and translational control. To depict the cellular actions of IMPs, we performed a loss-of-function analysis, which showed that IMPs are necessary for proper cell adhesion, cytoplasmic spreading and inva......Oncofetal RNA-binding IMPs have been implicated in mRNA localization, nuclear export, turnover and translational control. To depict the cellular actions of IMPs, we performed a loss-of-function analysis, which showed that IMPs are necessary for proper cell adhesion, cytoplasmic spreading...... and invadopodia formation. Loss of IMPs was associated with a coordinate downregulation of mRNAs encoding extracellular matrix and adhesion proteins. The transcripts were present in IMP RNP granules, implying that IMPs were directly involved in the post-transcriptional control of the transcripts. In particular......-mediated invadopodia formation. Taken together, our results indicate that RNA-binding proteins exert profound effects on cellular adhesion and invasion during development and cancer formation....

Maize DRE-binding proteins DBF1 and DBF2 are involved in rab17 regulation through the drought-responsive element in an ABA-dependent pathway.

Science.gov (United States)

Kizis, Dimosthenis; Pagès, Montserrat

2002-06-01

The abscisic acid-responsive gene rab17 of maize is expressed during late embryogenesis, and is induced by ABA and desiccation in embryo and vegetative tissues. ABRE and DRE cis-elements are involved in regulation of the gene by ABA and drought. Using yeast one-hybrid screening, we isolated two cDNAs encoding two new DRE-binding proteins, designated DBF1 and DBF2, that are members of the AP2/EREBP transcription factor family. Analysis of mRNA accumulation profiles showed that DBF1 is induced during maize embryogenesis and after desiccation, NaCl and ABA treatments in plant seedlings, whereas the DBF2 mRNA is not induced. DNA-binding preferences of DBFs were analysed by electrophoretic mobility shift assays, and showed that both DBF1 and DBF2 bound to the wild-type DRE2 element, but not to the DRE2 mutant or to the DRE1 element which differs only in a single nucleotide. Transactivation activity using particle bombardment showed that DBF1 functioned as activator of DRE2-dependent transcription of rab17 promoter by ABA, whereas DBF2 overexpression had a repression action downregulating not only the basal promoter activity, but also the ABA effect. These results show that ABA plays a role in the regulation of DBF activity, and suggests the existence of an ABA-dependent pathway for the regulation of genes through the C-repeat/DRE element.

Structural and Histone Binding Ability Characterizations of Human PWWP Domains

Energy Technology Data Exchange (ETDEWEB)

Wu, Hong; Zeng, Hong; Lam, Robert; Tempel, Wolfram; Amaya, Maria F.; Xu, Chao; Dombrovski, Ludmila; Qiu, Wei; Wang, Yanming; Min, Jinrong (Toronto); (Penn)

2013-09-25

The PWWP domain was first identified as a structural motif of 100-130 amino acids in the WHSC1 protein and predicted to be a protein-protein interaction domain. It belongs to the Tudor domain 'Royal Family', which consists of Tudor, chromodomain, MBT and PWWP domains. While Tudor, chromodomain and MBT domains have long been known to bind methylated histones, PWWP was shown to exhibit histone binding ability only until recently. The PWWP domain has been shown to be a DNA binding domain, but sequence analysis and previous structural studies show that the PWWP domain exhibits significant similarity to other 'Royal Family' members, implying that the PWWP domain has the potential to bind histones. In order to further explore the function of the PWWP domain, we used the protein family approach to determine the crystal structures of the PWWP domains from seven different human proteins. Our fluorescence polarization binding studies show that PWWP domains have weak histone binding ability, which is also confirmed by our NMR titration experiments. Furthermore, we determined the crystal structures of the BRPF1 PWWP domain in complex with H3K36me3, and HDGF2 PWWP domain in complex with H3K79me3 and H4K20me3. PWWP proteins constitute a new family of methyl lysine histone binders. The PWWP domain consists of three motifs: a canonical {beta}-barrel core, an insertion motif between the second and third {beta}-strands and a C-terminal {alpha}-helix bundle. Both the canonical {beta}-barrel core and the insertion motif are directly involved in histone binding. The PWWP domain has been previously shown to be a DNA binding domain. Therefore, the PWWP domain exhibits dual functions: binding both DNA and methyllysine histones.

Structural determination of functional units of the nucleotide binding domain (NBD94 of the reticulocyte binding protein Py235 of Plasmodium yoelii.

Directory of Open Access Journals (Sweden)

Ardina Grüber

2010-02-01

Full Text Available Invasion of the red blood cells (RBC by the merozoite of malaria parasites involves a large number of receptor ligand interactions. The reticulocyte binding protein homologue family (RH plays an important role in erythrocyte recognition as well as virulence. Recently, it has been shown that members of RH in addition to receptor binding may also have a role as ATP/ADP sensor. A 94 kDa region named Nucleotide-Binding Domain 94 (NBD94 of Plasmodium yoelii YM, representative of the putative nucleotide binding region of RH, has been demonstrated to bind ATP and ADP selectively. Binding of ATP or ADP induced nucleotide-dependent structural changes in the C-terminal hinge-region of NBD94, and directly impacted on the RBC binding ability of RH.In order to find the smallest structural unit, able to bind nucleotides, and its coupling module, the hinge region, three truncated domains of NBD94 have been generated, termed NBD94(444-547, NBD94(566-663 and NBD94(674-793, respectively. Using fluorescence correlation spectroscopy NBD94(444-547 has been identified to form the smallest nucleotide binding segment, sensitive for ATP and ADP, which became inhibited by 4-Chloro-7-nitrobenzofurazan. The shape of NBD94(444-547 in solution was calculated from small-angle X-ray scattering data, revealing an elongated molecule, comprised of two globular domains, connected by a spiral segment of about 73.1 A in length. The high quality of the constructs, forming the hinge-region, NBD94(566-663 and NBD94(674-793 enabled to determine the first crystallographic and solution structure, respectively. The crystal structure of NBD94(566-663 consists of two helices with 97.8 A and 48.6 A in length, linked by a loop. By comparison, the low resolution structure of NBD94(674-793 in solution represents a chair-like shape with three architectural segments.These structures give the first insight into how nucleotide binding impacts on the overall structure of RH and demonstrates the

Discriminating binding mechanisms of an intrinsically disordered protein via a multi-state coarse-grained model

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Knott, Michael [Department of Chemistry, Cambridge University, Lensfield Road, Cambridge CB2 1EW (United Kingdom); Best, Robert B., E-mail: robertbe@helix.nih.gov [Department of Chemistry, Cambridge University, Lensfield Road, Cambridge CB2 1EW (United Kingdom); Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520 (United States)

2014-05-07

Many proteins undergo a conformational transition upon binding to their cognate binding partner, with intrinsically disordered proteins (IDPs) providing an extreme example in which a folding transition occurs. However, it is often not clear whether this occurs via an “induced fit” or “conformational selection” mechanism, or via some intermediate scenario. In the first case, transient encounters with the binding partner favour transitions to the bound structure before the two proteins dissociate, while in the second the bound structure must be selected from a subset of unbound structures which are in the correct state for binding, because transient encounters of the incorrect conformation with the binding partner are most likely to result in dissociation. A particularly interesting situation involves those intrinsically disordered proteins which can bind to different binding partners in different conformations. We have devised a multi-state coarse-grained simulation model which is able to capture the binding of IDPs in alternate conformations, and by applying it to the binding of nuclear coactivator binding domain (NCBD) to either ACTR or IRF-3 we are able to determine the binding mechanism. By all measures, the binding of NCBD to either binding partner appears to occur via an induced fit mechanism. Nonetheless, we also show how a scenario closer to conformational selection could arise by choosing an alternative non-binding structure for NCBD.

Discriminating binding mechanisms of an intrinsically disordered protein via a multi-state coarse-grained model

International Nuclear Information System (INIS)

Knott, Michael; Best, Robert B.

2014-01-01

Many proteins undergo a conformational transition upon binding to their cognate binding partner, with intrinsically disordered proteins (IDPs) providing an extreme example in which a folding transition occurs. However, it is often not clear whether this occurs via an “induced fit” or “conformational selection” mechanism, or via some intermediate scenario. In the first case, transient encounters with the binding partner favour transitions to the bound structure before the two proteins dissociate, while in the second the bound structure must be selected from a subset of unbound structures which are in the correct state for binding, because transient encounters of the incorrect conformation with the binding partner are most likely to result in dissociation. A particularly interesting situation involves those intrinsically disordered proteins which can bind to different binding partners in different conformations. We have devised a multi-state coarse-grained simulation model which is able to capture the binding of IDPs in alternate conformations, and by applying it to the binding of nuclear coactivator binding domain (NCBD) to either ACTR or IRF-3 we are able to determine the binding mechanism. By all measures, the binding of NCBD to either binding partner appears to occur via an induced fit mechanism. Nonetheless, we also show how a scenario closer to conformational selection could arise by choosing an alternative non-binding structure for NCBD

α-Synuclein and huntingtin exon 1 amyloid fibrils bind laterally to the cellular membrane.

Science.gov (United States)

Monsellier, Elodie; Bousset, Luc; Melki, Ronald

2016-01-13

Fibrillar aggregates involved in neurodegenerative diseases have the ability to spread from one cell to another in a prion-like manner. The underlying molecular mechanisms, in particular the binding mode of the fibrils to cell membranes, are poorly understood. In this work we decipher the modality by which aggregates bind to the cellular membrane, one of the obligatory steps of the propagation cycle. By characterizing the binding properties of aggregates made of α-synuclein or huntingtin exon 1 protein displaying similar composition and structure but different lengths to mammalian cells we demonstrate that in both cases aggregates bind laterally to the cellular membrane, with aggregates extremities displaying little or no role in membrane binding. Lateral binding to artificial liposomes was also observed by transmission electron microscopy. In addition we show that although α-synuclein and huntingtin exon 1 fibrils bind both laterally to the cellular membrane, their mechanisms of interaction differ. Our findings have important implications for the development of future therapeutic tools that aim to block protein aggregates propagation in the brain.

An HIV-1 encoded peptide mimics the DNA binding loop of NF-κB and binds thioredoxin with high affinity

International Nuclear Information System (INIS)

Su Guoping; Wang Min; Taylor, Ethan Will

2005-01-01

Pro-fs is a human immunodeficiency virus type 1 (HIV-l)-encoded putative selenoprotein, predicted by a theoretical analysis of the viral genome; it is potentially expressed by a -1 frameshift from the protease coding region. Pro-fs has significant sequence similarity to the DNA binding loop of nuclear factor kappa B (NF-κB), which is known to bind thioredoxin (Trx). We hypothesize that the putative HIV-1 pro-fs gene product functions by mimicry of NF-κB via binding to Trx. The hypothesis was tested in vitro by co-immunoprecipitation and GST-pull down assays, using a purified mutant pro-fs protein, in which the two potential selenocysteine residues were mutated to cysteines, in order to permit expression in bacteria. Both experiments showed that pro-fs binds to human wild type Trx (Trx-wt) with high affinity. Mutation of the two conserved cysteine residues in the Trx active site redox center to serine (Ser) (Trx-CS) weakened but failed to abolish the interaction. In pro-fs-transfected 293T cells, using confocal microscopy and fluorescence resonance energy transfer (FRET), we have observed that pro-fs localizes in cell nuclei and forms oligomers. Upon stimulation by phorbol 12-myristate 13-acetate (PMA), Trx translocates into cell nuclei. Significant FRET efficiency was detected in the nuclei of PMA-stimulated 293T cells co-expressing fluorescence-tagged pro-fs and Trx-wt or Trx-CS. These results indicate that in living cells the double cysteine mutant of pro-fs binds to both Trx and Trx-CS with high affinity, suggesting that Trx-pro-fs binding is a structurally-specific interaction, involving more of the Trx molecule than just its active site cysteine residues. These results establish the capacity for functional mimicry of the Trx binding ability of the NF-κB/Rel family of transcription factors by the putative HIV-1 pro-fs protein

DNA-binding proteins essential for protein-primed bacteriophage ø29 DNA replication

Directory of Open Access Journals (Sweden)

Margarita Salas

2016-08-01

Full Text Available Bacillus subtilis phage Φ29 has a linear, double-stranded DNA 19 kb long with an inverted terminal repeat of 6 nucleotides and a protein covalently linked to the 5’ ends of the DNA. This protein, called terminal protein (TP, is the primer for the initiation of replication, a reaction catalyzed by the viral DNA polymerase at the two DNA ends. The DNA polymerase further elongates the nascent DNA chain in a processive manner, coupling strand displacement with elongation. The viral protein p5 is a single-stranded DNA binding protein (SSB that binds to the single strands generated by strand displacement during the elongation process. Viral protein p6 is a double-stranded DNA binding protein (DBP that preferentially binds to the origins of replication at the Φ29 DNA ends and is required for the initiation of replication. Both SSB and DBP are essential for Φ29 DNA amplification. This review focuses on the role of these phage DNA-binding proteins in Φ29 DNA replication both in vitro and in vivo, as well as on the implication of several B. subtilis DNA-binding proteins in different processes of the viral cycle. We will revise the enzymatic activities of the Φ29 DNA polymerase: TP-deoxynucleotidylation, processive DNA polymerization coupled to strand displacement, 3’-5’ exonucleolysis and pyrophosphorolysis. The resolution of the Φ29 DNA polymerase structure has shed light on the translocation mechanism and the determinants responsible for processivity and strand displacement. These two properties have made Φ29 DNA polymerase one of the main enzymes used in the current DNA amplification technologies. The determination of the structure of Φ29 TP revealed the existence of three domains: the priming domain, where the primer residue Ser232, as well as Phe230, involved in the determination of the initiating nucleotide, are located, the intermediate domain, involved in DNA polymerase binding, and the N-terminal domain, responsible for DNA binding

Analysis of the PDZ binding specificities of Influenza A Virus NS1 proteins

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Nagasaka Kazunori

2011-01-01

Full Text Available Abstract The Influenza A virus non-structural protein 1 (NS1 is a multifunctional virulence factor with several protein-protein interaction domains, involved in preventing apoptosis of the infected cell and in evading the interferon response. In addition, the majority of influenza A virus NS1 proteins have a class I PDZ-binding motif at the C-terminus, and this itself has been shown to be a virulence determinant. In the majority of human influenza NS1 proteins the consensus motif is RSxV: in avian NS1 it is ESxV. Of the few human strains that have the avian motif, all were from very high mortality outbreaks of the disease. Previous work has shown that minor differences in PDZ-binding motifs can have major effects on the spectrum of cellular proteins targeted. In this study we analyse the effect of these differences upon the binding of Influenza A virus NS1 protein to a range of cellular proteins involved in polarity and signal transduction.

Binding of ReO[subscript 4];#8722; with an engineered MoO[subscript 4 superscript 2];#8722;-binding protein: towards a new approach in radiopharmaceutical applications

Energy Technology Data Exchange (ETDEWEB)

Aryal, Baikuntha P.; Brugarolas, Pedro; He, Chuan (UC)

2012-05-25

Radiolabeled biomolecules are routinely used for clinical diagnostics. {sup 99m}Tc is the most commonly used radioactive tracer in radiopharmaceuticals. {sup 188}Re and {sup 186}Re are also commonly used as radioactive tracers in medicine. However, currently available methods for radiolabeling are lengthy and involve several steps in bioconjugation processes. In this work we present a strategy to engineer proteins that may selectively recognize the perrhenate (ReO{sub 4}{sup -}) ion as a new way to label proteins. We found that a molybdate (MoO{sub 4}{sup 2-})-binding protein (ModA) from Escherichia coli can bind perrhenate with high affinity. Using fluorescence and isothermal titration calorimetry measurements, we determined the dissociation constant of ModA for ReO{sub 4}{sup -} to be 541 nM and we solved a crystal structure of ModA with a bound ReO{sub 4}{sup -}. On the basis of the structure we created a mutant protein containing a disulfide linkage, which exhibited increased affinity for perrhenate (K{sub d} = 104 nM). High-resolution crystal structures of ModA (1.7 {angstrom}) and A11C/R153C mutant (2.0 {angstrom}) were solved with bound perrhenate. Both structures show that a perrhenate ion occupies the molybdate binding site using the same amino acid residues that are involved in molybdate binding. The overall structure of the perrhenate-bound ModA is unchanged compared with that of the molybdate-bound form. In the mutant protein, the bound perrhenate is further stabilized by the engineered disulfide bond.

Relaxed selection against accidental binding of transcription factors with conserved chromatin contexts.

Science.gov (United States)

Babbitt, G A

2010-10-15

The spurious (or nonfunctional) binding of transcription factors (TF) to the wrong locations on DNA presents a formidable challenge to genomes given the relatively low ceiling for sequence complexity within the short lengths of most binding motifs. The high potential for the occurrence of random motifs and subsequent nonfunctional binding of many transcription factors should theoretically lead to natural selection against the occurrence of spurious motif throughout the genome. However, because of the active role that chromatin can influence over eukaryotic gene regulation, it may also be expected that many supposed spurious binding sites could escape purifying selection if (A) they simply occur in regions of high nucleosome occupancy or (B) their surrounding chromatin was dynamically involved in their identity and function. We compared nucleosome occupancy and the presence/absence of functionally conserved chromatin context to the strength of selection against spurious binding of various TF binding motifs in Saccharomyces yeast. While we find no direct relationship with nucleosome occupancy, we find strong evidence that transcription factors spatially associated with evolutionarily conserved chromatin states are under relaxed selection against accidental binding. Transcription factors (with/without) a conserved chromatin context were found to occur on average, (87.7%/49.3%) of their expected frequencies. Functional binding motifs with conserved chromatin contexts were also significantly shorter in length and more often clustered. These results indicate a role of chromatin context dependency in relaxing selection against spurious binding in nearly half of all TF binding motifs throughout the yeast genome. 2010 Elsevier B.V. All rights reserved.

The role of CH/π interactions in the high affinity binding of streptavidin and biotin.

Science.gov (United States)

Ozawa, Motoyasu; Ozawa, Tomonaga; Nishio, Motohiro; Ueda, Kazuyoshi

2017-08-01

The streptavidin-biotin complex has an extraordinarily high affinity (Ka: 10 15 mol -1 ) and contains one of the strongest non-covalent interactions known. This strong interaction is widely used in biological tools, including for affinity tags, detection, and immobilization of proteins. Although hydrogen bond networks and hydrophobic interactions have been proposed to explain this high affinity, the reasons for it remain poorly understood. Inspired by the deceased affinity of biotin observed for point mutations of streptavidin at tryptophan residues, we hypothesized that a CH/π interaction may also contribute to the strong interaction between streptavidin and biotin. CH/π interactions were explored and analyzed at the biotin-binding site and at the interface of the subunits by the fragment molecular orbital method (FMO) and extended applications: PIEDA and FMO4. The results show that CH/π interactions are involved in the high affinity for biotin at the binding site of streptavidin. We further suggest that the involvement of CH/π interactions at the subunit interfaces and an extended CH/π network play more critical roles in determining the high affinity, rather than involvement at the binding site. Copyright © 2017 Elsevier Inc. All rights reserved.

TIA-1 RRM23 binding and recognition of target oligonucleotides.

Science.gov (United States)

Waris, Saboora; García-Mauriño, Sofía M; Sivakumaran, Andrew; Beckham, Simone A; Loughlin, Fionna E; Gorospe, Myriam; Díaz-Moreno, Irene; Wilce, Matthew C J; Wilce, Jacqueline A

2017-05-05

TIA-1 (T-cell restricted intracellular antigen-1) is an RNA-binding protein involved in splicing and translational repression. It mainly interacts with RNA via its second and third RNA recognition motifs (RRMs), with specificity for U-rich sequences directed by RRM2. It has recently been shown that RRM3 also contributes to binding, with preferential binding for C-rich sequences. Here we designed UC-rich and CU-rich 10-nt sequences for engagement of both RRM2 and RRM3 and demonstrated that the TIA-1 RRM23 construct preferentially binds the UC-rich RNA ligand (5΄-UUUUUACUCC-3΄). Interestingly, this binding depends on the presence of Lys274 that is C-terminal to RRM3 and binding to equivalent DNA sequences occurs with similar affinity. Small-angle X-ray scattering was used to demonstrate that, upon complex formation with target RNA or DNA, TIA-1 RRM23 adopts a compact structure, showing that both RRMs engage with the target 10-nt sequences to form the complex. We also report the crystal structure of TIA-1 RRM2 in complex with DNA to 2.3 Å resolution providing the first atomic resolution structure of any TIA protein RRM in complex with oligonucleotide. Together our data support a specific mode of TIA-1 RRM23 interaction with target oligonucleotides consistent with the role of TIA-1 in binding RNA to regulate gene expression. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Radiochemical and immunohistochemical detection of low density lipoprotein surface binding by lymphocytes

International Nuclear Information System (INIS)

Melzner, I.; Hambitzer, R.; Haferkamp, O.

1983-01-01

Human peripheral blood lymphocytes bind and take up low density lipoprotein (LDL) by receptor-mediated endocytosis. The binding of LDL was determiend by incubation with 125 I-LDL and an immunohistochemical assay. By both techniques a diminished rate of binding was found when cells were freshly isolated from the blood, but increased 5 to 10 fold when lymphocytes were incubated in lipoprotein-deficient medium for 72 hours. In addition, it was shown immunohistochemically that only few ceels showed an LDL-dependent fluorescent labelling: approximately 5 to 10 % of the freshly isolated lymphocytes and 40 to 50 % of the cells incubated for 72 hours under lipoprotein-free conditions. The present data indicate that not only the high affinity LDL receptor described by Goldstein and Braun may be involved in the uptake of cholesterol by lymphocytes, but also other binding sites, which may have immunological function in some lymphocyte subpopulations. (author)

Proteomics shows Hsp70 does not bind peptide sequences indiscriminately in vivo

International Nuclear Information System (INIS)

Grossmann, Michael E.; Madden, Benjamin J.; Gao, Fan; Pang, Yuan-Ping; Carpenter, John E.; McCormick, Daniel; Young, Charles Y.F.

2004-01-01

Heat shock protein 70 (Hsp70) binds peptide and has several functions that include protein folding, protein trafficking, and involvement with immune function. However, endogenous Hsp70-binding peptides had not previously been identified. Therefore, we eluted and identified several hundred endogenously bound peptides from Hsp70 using liquid chromatography ion trap mass spectrophotometry (LC-ITMS). Our work shows that the peptides are capable of binding Hsp70 as previously described. They are generally 8-26 amino acids in length and correspond to specific regions of many proteins. Through computationally assisted analysis of peptides eluted from Hsp70 we determined variable amino acid sequences, including a 5 amino acid core sequence that Hsp70 favorably binds. We also developed a computer algorithm that predicts Hsp70 binding within proteins. This work helps to define what peptides are bound by Hsp70 in vivo and suggests that Hsp70 facilitates peptide selection by aiding a funneling mechanism that is flexible but allows only a limited number of peptides to be processed

Beyond Antibodies as Binding Partners: The Role of Antibody Mimetics in Bioanalysis.

Science.gov (United States)

Yu, Xiaowen; Yang, Yu-Ping; Dikici, Emre; Deo, Sapna K; Daunert, Sylvia

2017-06-12

The emergence of novel binding proteins or antibody mimetics capable of binding to ligand analytes in a manner analogous to that of the antigen-antibody interaction has spurred increased interest in the biotechnology and bioanalytical communities. The goal is to produce antibody mimetics designed to outperform antibodies with regard to binding affinities, cellular and tumor penetration, large-scale production, and temperature and pH stability. The generation of antibody mimetics with tailored characteristics involves the identification of a naturally occurring protein scaffold as a template that binds to a desired ligand. This scaffold is then engineered to create a superior binder by first creating a library that is then subjected to a series of selection steps. Antibody mimetics have been successfully used in the development of binding assays for the detection of analytes in biological samples, as well as in separation methods, cancer therapy, targeted drug delivery, and in vivo imaging. This review describes recent advances in the field of antibody mimetics and their applications in bioanalytical chemistry, specifically in diagnostics and other analytical methods.

The SPOR Domain, a Widely Conserved Peptidoglycan Binding Domain That Targets Proteins to the Site of Cell Division.

Science.gov (United States)

Yahashiri, Atsushi; Jorgenson, Matthew A; Weiss, David S

2017-07-15

Sporulation-related repeat (SPOR) domains are small peptidoglycan (PG) binding domains found in thousands of bacterial proteins. The name "SPOR domain" stems from the fact that several early examples came from proteins involved in sporulation, but SPOR domain proteins are quite diverse and contribute to a variety of processes that involve remodeling of the PG sacculus, especially with respect to cell division. SPOR domains target proteins to the division site by binding to regions of PG devoid of stem peptides ("denuded" glycans), which in turn are enriched in septal PG by the intense, localized activity of cell wall amidases involved in daughter cell separation. This targeting mechanism sets SPOR domain proteins apart from most other septal ring proteins, which localize via protein-protein interactions. In addition to SPOR domains, bacteria contain several other PG-binding domains that can exploit features of the cell wall to target proteins to specific subcellular sites. Copyright © 2017 American Society for Microbiology.

New Insights into Functional Roles of the Polypyrimidine Tract-Binding Protein

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Maria Grazia Romanelli

2013-11-01

Full Text Available Polypyrimidine Tract Binding Protein (PTB is an intensely studied RNA binding protein involved in several post-transcriptional regulatory events of gene expression. Initially described as a pre-mRNA splicing regulator, PTB is now widely accepted as a multifunctional protein shuttling between nucleus and cytoplasm. Accordingly, PTB can interact with selected RNA targets, structural elements and proteins. There is increasing evidence that PTB and its paralog PTBP2 play a major role as repressors of alternatively spliced exons, whose transcription is tissue-regulated. In addition to alternative splicing, PTB is involved in almost all steps of mRNA metabolism, including polyadenylation, mRNA stability and initiation of protein translation. Furthermore, it is well established that PTB recruitment in internal ribosome entry site (IRES activates the translation of picornaviral and cellular proteins. Detailed studies of the structural properties of PTB have contributed to our understanding of the mechanism of RNA binding by RNA Recognition Motif (RRM domains. In the present review, we will describe the structural properties of PTB, its paralogs and co-factors, the role in post-transcriptional regulation and actions in cell differentiation and pathogenesis. Defining the multifunctional roles of PTB will contribute to the understanding of key regulatory events in gene expression.

CINPA1 binds directly to constitutive androstane receptor and inhibits its activity.

Science.gov (United States)

Cherian, Milu T; Chai, Sergio C; Wright, William C; Singh, Aman; Alexandra Casal, Morgan; Zheng, Jie; Wu, Jing; Lee, Richard E; Griffin, Patrick R; Chen, Taosheng

2018-03-31

The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic sensors that regulate the expression of drug-metabolizing enzymes and efflux transporters. CAR activation promotes drug elimination, thereby reducing therapeutic effectiveness, or causes adverse drug effects via toxic metabolites. CAR inhibitors could be used to attenuate these adverse drug effects. CAR and PXR share ligands and target genes, confounding the understanding of the regulation of receptor-specific activity. We previously identified a small-molecule inhibitor, CINPA1, that inhibits CAR (without activating PXR at lower concentrations) by altering CAR-coregulator interactions and reducing CAR recruitment to DNA response elements of regulated genes. However, solid evidence was not presented for the direct binding of CINPA1 to CAR. In this study, we demonstrate direct interaction of CINPA1 with the CAR ligand-binding domain (CAR-LBD) and identify key residues involved in such interactions through a combination of biophysical and computational methods. We found that CINPA1 resides in the ligand-binding pocket to stabilize the CAR-LBD in a more rigid, less fluid state. Molecular dynamics simulations, together with our previously reported docking model, enabled us to predict which CAR residues were critical for interactions with CINPA1. The importance of these residues for CINPA1 binding were then validated by directed mutations and testing the mutant CAR proteins in transcription reporter and coregulatory interaction assays. We demonstrated strong hydrogen bonding of CINPA1 with N165 and H203 and identified other residues involved in hydrophobic contacts with CINPA1. Overall, our data confirm that CINPA1 directly binds to CAR. Copyright © 2018 Elsevier Inc. All rights reserved.

Functional analysis of the citrate activator CitO from Enterococcus faecalis implicates a divalent metal in ligand binding

Directory of Open Access Journals (Sweden)

Victor S. Blancato

2016-02-01

Full Text Available The regulator of citrate metabolism, CitO, from Enterococcus faecalis belongs to the FCD family within the GntR superfamily. In the presence of citrate, CitO binds to cis-acting sequences located upstream of the cit promoters inducing the expression of genes involved in citrate utilization. The quantification of the molecular binding affinities, performed by isothermal titration calorimetry (ITC, indicated that CitO has a high affinity for citrate (KD= 1.2±0.2 µM, while it did not recognize other metabolic intermediates. Based on a structural model of CitO where a putative small molecule and a metal binding site were identified, it was hypothesized that the metal ion is required for citrate binding. In agreement with this model, citrate binding to CitO sharply decreased when the protein was incubated with EDTA. This effect was reverted by the addition of Ni2+, and Zn2+ to a lesser extent. Structure-based site-directed mutagenesis was conducted and it was found that changes to alanine in residues Arg97 and His191 resulted in decreased binding affinities for citrate, as determined by EMSA and ITC. Further assays using lacZ fusions confirmed that these residues in CitO are involved in sensing citrate in vivo. These results indicate that the molecular modifications induced by a ligand and a metal binding in the C-terminal domain of CitO are required for optimal DNA binding activity, and consequently, transcriptional activation.

The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange*

Science.gov (United States)

Fenyk, Stepan; Dixon, Christopher H.; Gittens, William H.; Townsend, Philip D.; Sharples, Gary J.; Pålsson, Lars-Olof; Takken, Frank L. W.; Cann, Martin J.

2016-01-01

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. PMID:26601946

The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.

Science.gov (United States)

Fenyk, Stepan; Dixon, Christopher H; Gittens, William H; Townsend, Philip D; Sharples, Gary J; Pålsson, Lars-Olof; Takken, Frank L W; Cann, Martin J

2016-01-15

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of glucagon-like peptide-1 receptor-binding determinants.

Science.gov (United States)

Xiao, Q; Jeng, W; Wheeler, M B

2000-12-01

Glucagon-like peptide 1 (GLP-1) is a potent insulinotropic hormone currently under study as a therapeutic agent for type 2 diabetes. Since an understanding of the molecular mechanisms leading to high-affinity receptor (R) binding and activation may facilitate the development of more potent GLP-1R agonists, we have localized specific regions of GLP-1R required for binding. The purified N-terminal fragment (hereafter referred to as NT) of the GLP-1R produced in either insect (Sf9) or mammalian (COS-7) cells was shown to bind GLP-1. The physical interaction of NT with GLP-1 was first demonstrated by cross-linking ((125)I-GLP-1/NT complex band at approximately 28 kDa) and secondly by attachment to Ni(2+)-NTA beads. The GLP-1R NT protein attached to beads bound GLP-1, but with lower affinity (inhibitory concentration (IC(50)): 4.5 x 10(-7) M) than wild-type (WT) GLP-1R (IC(50): 5.2 x 10(-9)M). The low affinity of GLP-1R NT suggested that other receptor domains may contribute to GLP-1 binding. This was supported by studies using chimeric glucose-dependent insulinotropic polypeptide (GIP)/GLP-1 receptors. GIP(1-151)/GLP-1R, but not GIP(1-222)/GLP-1R, exhibited specific GLP-1 binding and GLP-1-induced cAMP production, suggesting that the region encompassing transmembrane (TM) domain 1 through to TM3 was required for binding. Since it was hypothesized that certain charged or polar amino acids in this region might be involved in binding, these residues (TM2-TM3) were analyzed by substitution mutagenesis. Five mutants (K197A, D198A, K202A, D215A, R227A) displayed remarkably reduced binding affinity. These studies indicate that the NT domain of the GLP-1R is able to bind GLP-1, but charged residues concentrated at the distal TM2/extracellular loop-1 (EC1) interface (K197, D198, K202) and in EC1 (D215 and R227) probably contribute to the binding determinants of the GLP-1R.

Synthesis of Heparan Sulfate with Cyclophilin B-binding Properties Is Determined by Cell Type-specific Expression of Sulfotransferases*

Science.gov (United States)

Deligny, Audrey; Denys, Agnès; Marcant, Adeline; Melchior, Aurélie; Mazurier, Joël; van Kuppevelt, Toin H.; Allain, Fabrice

2010-01-01

Cyclophilin B (CyPB) induces migration and adhesion of T lymphocytes via a mechanism that requires interaction with 3-O-sulfated heparan sulfate (HS). HS biosynthesis is a complex process with many sulfotransferases involved. N-Deacetylases/N-sulfotransferases are responsible for N-sulfation, which is essential for subsequent modification steps, whereas 3-O-sulfotransferases (3-OSTs) catalyze the least abundant modification. These enzymes are represented by several isoforms, which differ in term of distribution pattern, suggesting their involvement in making tissue-specific HS. To elucidate how the specificity of CyPB binding is determined, we explored the relationships between the expression of these sulfotransferases and the generation of HS motifs with CyPB-binding properties. We demonstrated that high N-sulfate density and the presence of 2-O- and 3-O-sulfates determine binding of CyPB, as evidenced by competitive experiments with heparin derivatives, soluble HS, and anti-HS antibodies. We then showed that target cells, i.e. CD4+ lymphocyte subsets, monocytes/macrophages, and related cell lines, specifically expressed high levels of NDST2 and 3-OST3 isoforms. Silencing the expression of NDST1, NDST2, 2-OST, and 3-OST3 by RNA interference efficiently decreased binding and activity of CyPB, thus confirming their involvement in the biosynthesis of binding sequences for CyPB. Moreover, we demonstrated that NDST1 was able to partially sulfate exogenous substrate in the absence of NDST2 but not vice versa, suggesting that both isoenzymes do not have redundant activities but do have rather complementary activities in making N-sulfated sequences with CyPB-binding properties. Altogether, these results suggest a regulatory mechanism in which cell type-specific expression of certain HS sulfotransferases determines the specific binding of CyPB to target cells. PMID:19940140

Synthesis of heparan sulfate with cyclophilin B-binding properties is determined by cell type-specific expression of sulfotransferases.

Science.gov (United States)

Deligny, Audrey; Denys, Agnès; Marcant, Adeline; Melchior, Aurélie; Mazurier, Joël; van Kuppevelt, Toin H; Allain, Fabrice

2010-01-15

Cyclophilin B (CyPB) induces migration and adhesion of T lymphocytes via a mechanism that requires interaction with 3-O-sulfated heparan sulfate (HS). HS biosynthesis is a complex process with many sulfotransferases involved. N-Deacetylases/N-sulfotransferases are responsible for N-sulfation, which is essential for subsequent modification steps, whereas 3-O-sulfotransferases (3-OSTs) catalyze the least abundant modification. These enzymes are represented by several isoforms, which differ in term of distribution pattern, suggesting their involvement in making tissue-specific HS. To elucidate how the specificity of CyPB binding is determined, we explored the relationships between the expression of these sulfotransferases and the generation of HS motifs with CyPB-binding properties. We demonstrated that high N-sulfate density and the presence of 2-O- and 3-O-sulfates determine binding of CyPB, as evidenced by competitive experiments with heparin derivatives, soluble HS, and anti-HS antibodies. We then showed that target cells, i.e. CD4+ lymphocyte subsets, monocytes/macrophages, and related cell lines, specifically expressed high levels of NDST2 and 3-OST3 isoforms. Silencing the expression of NDST1, NDST2, 2-OST, and 3-OST3 by RNA interference efficiently decreased binding and activity of CyPB, thus confirming their involvement in the biosynthesis of binding sequences for CyPB. Moreover, we demonstrated that NDST1 was able to partially sulfate exogenous substrate in the absence of NDST2 but not vice versa, suggesting that both isoenzymes do not have redundant activities but do have rather complementary activities in making N-sulfated sequences with CyPB-binding properties. Altogether, these results suggest a regulatory mechanism in which cell type-specific expression of certain HS sulfotransferases determines the specific binding of CyPB to target cells.

Molecular Events Involved in a Single Cycle of Ligand Transfer from an ATP Binding Cassette Transporter, LolCDE, to a Molecular Chaperone, LolA*

OpenAIRE

Taniguchi, Naohiro; Tokuda, Hajime

2008-01-01

An ATP binding cassette transporter LolCDE complex releases lipoproteins from the inner membrane of Escherichia coli in an ATP-dependent manner, leading to the formation of a complex between a lipoprotein and a periplasmic chaperone, LolA. LolA is proposed to undergo a conformational change upon the lipoprotein binding. The lipoprotein is then transferred from the LolA-lipoprotein complex to the outer membrane via LolB. Unlike most ATP binding cassette transporters med...

ACYLTRANSFERASE ACTIVITIES OF THE HIGH-MOLECULAR-MASS ESSENTIAL PENICILLIN-BINDING PROTEINS

NARCIS (Netherlands)

ADAM, M; DAMBLON, C; JAMIN, M; ZORZI, W; DUSART, [No Value; GALLENI, M; ELKHARROUBI, A; PIRAS, G; SPRATT, BG; KECK, W; COYETTE, J; GHUYSEN, JM; NGUYENDISTECHE, M; FRERE, JM

1991-01-01

The high-molecular-mass penicillin-binding proteins (HMM-PBPs), present in the cytoplasmic membranes of all eubacteria, are involved in important physiological events such as cell elongation, septation or shape determination. Up to now it has, however, been very difficult or impossible to study the

Crystal structure of CbpF, a bifunctional choline-binding protein and autolysis regulator from Streptococcus pneumoniae.

Science.gov (United States)

Molina, Rafael; González, Ana; Stelter, Meike; Pérez-Dorado, Inmaculada; Kahn, Richard; Morales, María; Moscoso, Miriam; Campuzano, Susana; Campillo, Nuria E; Mobashery, Shahriar; García, José L; García, Pedro; Hermoso, Juan A

2009-03-01

Phosphorylcholine, a crucial component of the pneumococcal cell wall, is essential in bacterial physiology and in human pathogenesis because it binds to serum components of the immune system and acts as a docking station for the family of surface choline-binding proteins. The three-dimensional structure of choline-binding protein F (CbpF), one of the most abundant proteins in the pneumococcal cell wall, has been solved in complex with choline. CbpF shows a new modular structure composed both of consensus and non-consensus choline-binding repeats, distributed along its length, which markedly alter its shape, charge distribution and binding ability, and organizing the protein into two well-defined modules. The carboxy-terminal module is involved in cell wall binding and the amino-terminal module is crucial for inhibition of the autolytic LytC muramidase, providing a regulatory function for pneumococcal autolysis.

Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: implications for viral genomic RNA packaging.

Science.gov (United States)

Webb, Joseph A; Jones, Christopher P; Parent, Leslie J; Rouzina, Ioulia; Musier-Forsyth, Karin

2013-08-01

Despite the vast excess of cellular RNAs, precisely two copies of viral genomic RNA (gRNA) are selectively packaged into new human immunodeficiency type 1 (HIV-1) particles via specific interactions between the HIV-1 Gag and the gRNA psi (ψ) packaging signal. Gag consists of the matrix (MA), capsid, nucleocapsid (NC), and p6 domains. Binding of the Gag NC domain to ψ is necessary for gRNA packaging, but the mechanism by which Gag selectively interacts with ψ is unclear. Here, we investigate the binding of NC and Gag variants to an RNA derived from ψ (Psi RNA), as well as to a non-ψ region (TARPolyA). Binding was measured as a function of salt to obtain the effective charge (Zeff) and nonelectrostatic (i.e., specific) component of binding, Kd(1M). Gag binds to Psi RNA with a dramatically reduced Kd(1M) and lower Zeff relative to TARPolyA. NC, GagΔMA, and a dimerization mutant of Gag bind TARPolyA with reduced Zeff relative to WT Gag. Mutations involving the NC zinc finger motifs of Gag or changes to the G-rich NC-binding regions of Psi RNA significantly reduce the nonelectrostatic component of binding, leading to an increase in Zeff. These results show that Gag interacts with gRNA using different binding modes; both the NC and MA domains are bound to RNA in the case of TARPolyA, whereas binding to Psi RNA involves only the NC domain. Taken together, these results suggest a novel mechanism for selective gRNA encapsidation.

Calcium-dependent and -independent binding of the pentraxin serum amyloid P component to glycosaminoglycans and amyloid proteins

DEFF Research Database (Denmark)

Danielsen, B; Sørensen, I J; Nybo, Mads

1997-01-01

precursor protein beta2M was observed. This binding was also enhanced at slightly acid pH, most pronounced at pH 5.0. The results of this study indicate that SAP can exhibit both Ca2(+)-dependent and -independent binding to ligands involved in amyloid fibril formation and that the binding is enhanced under...... and beta2M) by ELISA. An increase in the dose-dependent binding of SAP to heparan sulfate, AA-protein and beta2M was observed as the pH decreased from 8.0 to 5.0. Furthermore, a lower, but significant Ca2(+)-independent binding of SAP to heparan sulfate, dermatan sulfate, AA protein and the amyloid...

Structural analysis of peptides capable of binding to more than one Ia antigen

DEFF Research Database (Denmark)

Sette, A; Buus, S; Colon, S

1989-01-01

The Ia binding regions were analyzed for three unrelated peptide Ag (sperm whale myoglobin 106-118, influenza hemagglutinin 130-142, and lambda repressor protein 12-26) for which binding to more than one Ia molecule has previously been demonstrated. By determining the binding profile of three...... separate series of truncated synthetic peptides, it was found that in all three cases the different Ia reactivities mapped to largely overlapping regions of the peptides; although, for two of the peptides, the regions involved in binding the different Ia specificities were distinct. Moreover, subtle...... differences were found to dramatically influence some, but not other, Ia reactivities. Using a large panel of synthetic peptides it was found that a significant correlation exists between the capacity of peptides to interact with different alleles of the same molecule (i.e., IAd and IAk), but no correlation...

Ubiquitin-coated nanodiamonds bind to autophagy receptors for entry into the selective autophagy pathway.

Science.gov (United States)

Liu, Kuang-Kai; Qiu, Wei-Ru; Naveen Raj, Emmanuel; Liu, Huei-Fang; Huang, Hou-Syun; Lin, Yu-Wei; Chang, Chien-Jen; Chen, Ting-Hua; Chen, Chinpiao; Chang, Huan-Cheng; Hwang, Jenn-Kang; Chao, Jui-I

2017-01-02

Selective macroautophagy/autophagy plays a pivotal role in the processing of foreign pathogens and cellular components to maintain homeostasis in human cells. To date, numerous studies have demonstrated the uptake of nanoparticles by cells, but their intracellular processing through selective autophagy remains unclear. Here we show that carbon-based nanodiamonds (NDs) coated with ubiquitin (Ub) bind to autophagy receptors (SQSTM1 [sequestosome 1], OPTN [optineurin], and CALCOCO2/NDP52 [calcium binding and coiled-coil domain 2]) and are then linked to MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) for entry into the selective autophagy pathway. NDs are ultimately delivered to lysosomes. Ectopically expressed SQSTM1-green fluorescence protein (GFP) could bind to the Ub-coated NDs. By contrast, the Ub-associated domain mutant of SQSTM1 (ΔUBA)-GFP did not bind to the Ub-coated NDs. Chloroquine, an autophagy inhibitor, prevented the ND-containing autophagosomes from fusing with lysosomes. Furthermore, autophagy receptors OPTN and CALCOCO2/NDP52, involved in the processing of bacteria, were found to be involved in the selective autophagy of NDs. However, ND particles located in the lysosomes of cells did not induce mitotic blockage, senescence, or cell death. Single ND clusters in the lysosomes of cells were observed in the xenografted human lung tumors of nude mice. This study demonstrated for the first time that Ub-coated nanoparticles bind to autophagy receptors for entry into the selective autophagy pathway, facilitating their delivery to lysosomes.

Characterization of a human coagulation factor Xa-binding site on Viperidae snake venom phospholipases A2 by affinity binding studies and molecular bioinformatics

Directory of Open Access Journals (Sweden)

Gowda Veerabasappa T

2007-12-01

Full Text Available Abstract Background The snake venom group IIA secreted phospholipases A2 (SVPLA2, present in the Viperidae snake family exhibit a wide range of toxic and pharmacological effects. They exert their different functions by catalyzing the hydrolysis of phospholipids (PL at the membrane/water interface and by highly specific direct binding to: (i presynaptic membrane-bound or intracellular receptors; (ii natural PLA2-inhibitors from snake serum; and (iii coagulation factors present in human blood. Results Using surface plasmon resonance (SPR protein-protein interaction measurements and an in vitro biological test of inhibition of prothrombinase activity, we identify a number of Viperidae venom SVPLA2s that inhibit blood coagulation through direct binding to human blood coagulation factor Xa (FXa via a non-catalytic, PL-independent mechanism. We classify the SVPLA2s in four groups, depending on the strength of their binding. Molecular electrostatic potentials calculated at the surface of 3D homology-modeling models show a correlation with inhibition of prothrombinase activity. In addition, molecular docking simulations between SVPLA2 and FXa guided by the experimental data identify the potential FXa binding site on the SVPLA2s. This site is composed of the following regions: helices A and B, the Ca2+ loop, the helix C-β-wing loop, and the C-terminal fragment. Some of the SVPLA2 binding site residues belong also to the interfacial binding site (IBS. The interface in FXa involves both, the light and heavy chains. Conclusion We have experimentally identified several strong FXa-binding SVPLA2s that disrupt the function of the coagulation cascade by interacting with FXa by the non-catalytic PL-independent mechanism. By theoretical methods we mapped the interaction sites on both, the SVPLA2s and FXa. Our findings may lead to the design of novel, non-competitive FXa inhibitors.

Introduction of lysine and clot binding properties in the kringle one domain of tissue-type plasminogen activator

NARCIS (Netherlands)

Bakker, A.H.F.; Greef, W. van der; Rehberg, E.F.; Marotti, K.R.; Verheijen, J.H.

1993-01-01

Despite the high overall similarity in primary structure between kringle one (K1) and kringle two (K2) of tissue-type plasminogen activator (t-PA) there exists an enormous functional difference. It is thought that, in contrast to K1, K2 mediates lysine binding and fibrin binding and is involved in

PASTA in Penicillin Binding Proteins and Serine/Threonine Kinases: A Recipe of Structural, Dynamic and Binding Properties.

Science.gov (United States)

Calvanese, Luisa; Falcigno, Lucia; Squeglia, Flavia; D'Auria, Gabriella; Berisio, Rita

2017-11-24

Penicillin binding proteins (PBPs) and Serine Threonine kinases (STPKs) are two classes of bacterial enzymes whose involvement in a series of vital processes in bacterial growth and division is well assessed. Many PBPs and STPKs show linked an ancillary domain named PASTA, whose functional role is not completely deciphered so far. It has been proposed that PASTAs are sensor modules that by binding opportune ligands (i.e. muropeptides) activate the cognate proteins to their functions. However, based on recent data, the sensor annotation sounds true for PASTA from STPKs, and false for PASTA from PBPs. Different PASTA domains, belonging or not to different protein classes, sharing or not appreciable sequence identities, always show identical folds. This survey of the structural, binding and dynamic properties of PASTA domains pursues the reasons why identical topologies may turn in different roles. Amino acid compositions, total charges and distribution of the hydrophobic/hydrophilic patches on the surface, significantly vary among PASTAs from STPKs and PBPs and appear to correlate with different functions. A possible criterion to discriminate between PASTA modules of STPKs or PBPs solely based on their sequences is proposed. Possibly reflecting different species as well as functional roles and evolutionary profile, our routine represents a fast even though approximate method to distinguish between PASTA belonging to different classes. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Agonist and antagonist binding to rat brain muscarinic receptors: influence of aging

International Nuclear Information System (INIS)

Gurwitz, D.; Egozi, Y.; Henis, Y.I.; Kloog, Y.; Sokolovsky, M.

1987-01-01

The objective of the present study was to determine the binding properties of muscarinic receptors in six brain regions in mature and old rats of both sexes by employing direct binding of [ 3 H]-antagonist as well as of the labeled natural neurotransmitter, [ 3 H]-acetylcholine [( 3 H]-AcCh). In addition, age-related factors were evaluated in the modulation processes involved in agonist binding. The results indicate that as the rat ages the density of the muscarinic receptors is altered differently in the various brain regions: it is decreased in the cerebral cortex, hippocampus, striatum and olfactory bulb of both male and female rats, but is increased (58%) in the brain stem of senescent males while no significant change is observed for females. The use of the highly sensitive technique measuring direct binding of [ 3 H]-AcCh facilitated the separate detection of age-related changes in the two classes (high- and low-affinity) of muscarinic agonist binding sites. In old female rats the density of high-affinity [ 3 H]-AcCh binding sites was preserved in all tissues studied, indicating that the decreases in muscarinic receptor density observed with [ 3 H]-antagonist represent a loss of low-affinity agonist binding sites. In contrast, [ 3 H]-AcCh binding is decreased in the hypothalamus and increased in the brain stem of old male rats. These data imply sexual dimorphism of the aging process in central cholinergic mechanisms

Decipher the mechanisms of protein conformational changes induced by nucleotide binding through free-energy landscape analysis: ATP binding to Hsp70.

Directory of Open Access Journals (Sweden)

Adrien Nicolaï

Full Text Available ATP regulates the function of many proteins in the cell by transducing its binding and hydrolysis energies into protein conformational changes by mechanisms which are challenging to identify at the atomic scale. Based on molecular dynamics (MD simulations, a method is proposed to analyze the structural changes induced by ATP binding to a protein by computing the effective free-energy landscape (FEL of a subset of its coordinates along its amino-acid sequence. The method is applied to characterize the mechanism by which the binding of ATP to the nucleotide-binding domain (NBD of Hsp70 propagates a signal to its substrate-binding domain (SBD. Unbiased MD simulations were performed for Hsp70-DnaK chaperone in nucleotide-free, ADP-bound and ATP-bound states. The simulations revealed that the SBD does not interact with the NBD for DnaK in its nucleotide-free and ADP-bound states whereas the docking of the SBD was found in the ATP-bound state. The docked state induced by ATP binding found in MD is an intermediate state between the initial nucleotide-free and final ATP-bound states of Hsp70. The analysis of the FEL projected along the amino-acid sequence permitted to identify a subset of 27 protein internal coordinates corresponding to a network of 91 key residues involved in the conformational change induced by ATP binding. Among the 91 residues, 26 are identified for the first time, whereas the others were shown relevant for the allosteric communication of Hsp70 s in several experiments and bioinformatics analysis. The FEL analysis revealed also the origin of the ATP-induced structural modifications of the SBD recently measured by Electron Paramagnetic Resonance. The pathway between the nucleotide-free and the intermediate state of DnaK was extracted by applying principal component analysis to the subset of internal coordinates describing the transition. The methodology proposed is general and could be applied to analyze allosteric communication in

Identification of binding peptides of the ADAM15 disintegrin domain ...

Indian Academy of Sciences (India)

Madhsudhan

ADAM15 disintegrin domain (RADD) that could inhibit melanoma cell adhesion by using Escherichia coli. Second, four specific binding peptides (peptides A, B, C, and D) were selected using a phage display 12-mer peptide library. The screening protocol involved 4 rounds of positive panning on RADD and 2 rounds of ...

Synthesis, DNA Binding, and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives

Directory of Open Access Journals (Sweden)

Sinara Mônica Vitalino de Almeida

2015-06-01

Full Text Available In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z-2-(acridin-9-ylmethylene-N-phenylhydrazinecarbothioamide derivatives (3a–h were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. The calculated binding constants ranged from 1.74 × 104 to 1.0 × 106 M−1 and quenching constants from −0.2 × 104 to 2.18 × 104 M−1 indicating high affinity to ctDNA base pairs. The most efficient compound in binding to ctDNA in vitro was (Z-2-(acridin-9-ylmethylene-N- (4-chlorophenyl hydrazinecarbothioamide (3f, while the most active compound in antiproliferative assay was (Z-2-(acridin-9-ylmethylene-N-phenylhydrazinecarbothioamide (3a. There was no correlation between DNA-binding and in vitro antiproliferative activity, but the results suggest that DNA binding can be involved in the biological activity mechanism. This study may guide the choice of the size and shape of the intercalating part of the ligand and the strategic selection of substituents that increase DNA-binding or antiproliferative properties.

YY1 binding association with sex-biased transcription revealed through X-linked transcript levels and allelic binding analyses.

Science.gov (United States)

Chen, Chih-Yu; Shi, Wenqiang; Balaton, Bradley P; Matthews, Allison M; Li, Yifeng; Arenillas, David J; Mathelier, Anthony; Itoh, Masayoshi; Kawaji, Hideya; Lassmann, Timo; Hayashizaki, Yoshihide; Carninci, Piero; Forrest, Alistair R R; Brown, Carolyn J; Wasserman, Wyeth W

2016-11-18

Sex differences in susceptibility and progression have been reported in numerous diseases. Female cells have two copies of the X chromosome with X-chromosome inactivation imparting mono-allelic gene silencing for dosage compensation. However, a subset of genes, named escapees, escape silencing and are transcribed bi-allelically resulting in sexual dimorphism. Here we conducted in silico analyses of the sexes using human datasets to gain perspectives into such regulation. We identified transcription start sites of escapees (escTSSs) based on higher transcription levels in female cells using FANTOM5 CAGE data. Significant over-representations of YY1 transcription factor binding motif and ChIP-seq peaks around escTSSs highlighted its positive association with escapees. Furthermore, YY1 occupancy is significantly biased towards the inactive X (Xi) at long non-coding RNA loci that are frequent contacts of Xi-specific superloops. Our study suggests a role for YY1 in transcriptional activity on Xi in general through sequence-specific binding, and its involvement at superloop anchors.

Leishmania replication protein A-1 binds in vivo single-stranded telomeric DNA

International Nuclear Information System (INIS)

Neto, J.L. Siqueira; Lira, C.B.B.; Giardini, M.A.; Khater, L.; Perez, A.M.; Peroni, L.A.; Reis, J.R.R. dos; Freitas-Junior, L.H.; Ramos, C.H.I.; Cano, M.I.N.

2007-01-01

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in different events of DNA metabolism. In yeast, subunits 1 (RPA-1) and 2 (RPA-2) work also as telomerase recruiters and, in humans, the complex unfolds G-quartet structures formed by the 3' G-rich telomeric strand. In most eukaryotes, RPA-1 and RPA-2 bind DNA using multiple OB fold domains. In trypanosomatids, including Leishmania, RPA-1 has a canonical OB fold and a truncated RFA-1 structural domain. In Leishmania amazonensis, RPA-1 alone can form a complex in vitro with the telomeric G-rich strand. In this work, we show that LaRPA-1 is a nuclear protein that associates in vivo with Leishmania telomeres. We mapped the boundaries of the OB fold DNA-binding domain using deletion mutants. Since Leishmania and other trypanosomatids lack homologues of known telomere end binding proteins, our results raise questions about the function of RPA-1 in parasite telomeres

Depressed patients have decreased binding of tritiated imipramine to platelet serotonin ''transporter''

International Nuclear Information System (INIS)

Paul, S.M.; Rehavi, M.; Skolnick, P.; Ballenger, J.C.; Goodwin, F.K.

1981-01-01

The high-affinity tritiated (3H) imipramine binding sites are functionally (and perhaps structurally) associated with the presynaptic neuronal and platelet uptake sites for serotonin. Since there is an excellent correlation between the relative potencies of a series of antidepressants in displacing 3H-imipramine from binding sites in human brain and platelet, we have examined the binding of 3H-imipramine to platelets from 14 depressed patients and 28 age- and sex-matched controls. A highly significant decrease in the number of 3H-imipramine binding sites, with no significant change in the apparent affinity constants, was observed in platelets from the depressed patients compared with the controls. These results, coupled with previous studies showing a significant decrease in the maximal uptake of serotonin in platelets from depressed patients, suggest that an inherited or acquired deficiency of the serotonin transport protein or proteins may be involved in the pathogenesis of depression

[3H]QNB binding and contraction of rabbit colonic smooth muscle cells

International Nuclear Information System (INIS)

Ringer, M.J.; Hyman, P.E.; Kao, H.W.; Hsu, C.T.; Tomomasa, T.; Snape, W.J. Jr.

1987-01-01

The authors used radioligand binding and studies of cell contraction to characterize muscarinic receptors on dispersed smooth muscle cells from rabbit proximal and distal colon. Cells obtained after serial incubations in collagenase were used to measure binding of tritiated quinuclidinyl benzilate ([ 3 H]QNB). At 37 degree C, specific [ 3 H]QNB binding was saturable and linearly related to cell number. Nonlinear regression analysis was used to determine the affinity of [ 3 H]QNB for its receptor. The IC 50 for the muscarinic agonists bethanechol and oxotremorine were 80 and 0.57 μM, respectively. Hill coefficients were 0.67 for both, suggesting more complex interaction involving receptors of different affinities. In studies of cell contraction, bethanechol stimulated a dose-dependent decrease in cell length with half the maximal contraction occurring at 100 pM. These results suggest that (1) contraction is mediated by binding of bethanechol to M 2 -muscarinic receptors and that (2) there are a large number of spare receptors in colonic smooth muscle

Activation of the ATR kinase by the RPA-binding protein ETAA1

DEFF Research Database (Denmark)

Haahr, Peter; Hoffmann, Saskia; Tollenaere, Maxim A X

2016-01-01

Activation of the ATR kinase following perturbations to DNA replication relies on a complex mechanism involving ATR recruitment to RPA-coated single-stranded DNA via its binding partner ATRIP and stimulation of ATR kinase activity by TopBP1. Here, we discovered an independent ATR activation pathway...... in vertebrates, mediated by the uncharacterized protein ETAA1 (Ewing's tumour-associated antigen 1). Human ETAA1 accumulates at DNA damage sites via dual RPA-binding motifs and promotes replication fork progression and integrity, ATR signalling and cell survival after genotoxic insults. Mechanistically...

Fluorometric and molecular docking investigation on the binding characteristics of SB202190 to human serum albumin

International Nuclear Information System (INIS)

Nasruddin, Ahmad N.; Feroz, Shevin R.; Mukarram, Abdul K.; Mohamad, Saharuddin B.; Tayyab, Saad

2016-01-01

The interaction of SB202190, a p38 mitogen-activated protein kinase inhibitor with the main drug transporter in human circulation, human serum albumin (HSA) was studied using fluorescence spectroscopy and in silico docking methods. The association constant, K a of the binding reaction was determined to be 3.24±0.07×10 4 M −1 at 25 °C based on fluorescence quenching titration results. The values of enthalpy change and entropy change for the interaction were found as −8.54 kJ mol −1 and 58.01 J mol −1 K −1 , respectively. Both thermodynamic data and docking results suggested the involvement of hydrophobic and van der Waals forces in the complex formation. Three-dimensional fluorescence data of SB202190–HSA complex demonstrated significant changes in the microenvironment around the protein fluorophores upon drug binding. Comparison of HSA thermograms obtained in the absence and the presence of SB202190 suggested improved protein thermal stability upon complexation with the drug. Competitive drug displacement results as well as modeling data concluded the preferred binding site of SB202190 on HSA as Sudlow's site I. - Highlights: • SB202190 interacts with HSA with moderate affinity. • Involvement of hydrophobic and van der Waals forces in SB202190 binding. • SB202190 binding results in microenvironmental changes around fluorophores. • Sudlow's site I is the preferred binding site of SB202190.

Fluorometric and molecular docking investigation on the binding characteristics of SB202190 to human serum albumin

Energy Technology Data Exchange (ETDEWEB)

Nasruddin, Ahmad N.; Feroz, Shevin R. [Biomolecular Research Group, Biochemistry Programme, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur (Malaysia); Mukarram, Abdul K. [Bioinformatics Programme, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur (Malaysia); Mohamad, Saharuddin B. [Bioinformatics Programme, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur (Malaysia); Centre of Research for Computational Sciences and Informatics for Biology, Bioindustry, Environment, Agriculture and Healthcare, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur (Malaysia); Tayyab, Saad, E-mail: saadtayyab2004@yahoo.com [Biomolecular Research Group, Biochemistry Programme, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur (Malaysia); Centre of Research for Computational Sciences and Informatics for Biology, Bioindustry, Environment, Agriculture and Healthcare, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur (Malaysia)

2016-06-15

The interaction of SB202190, a p38 mitogen-activated protein kinase inhibitor with the main drug transporter in human circulation, human serum albumin (HSA) was studied using fluorescence spectroscopy and in silico docking methods. The association constant, K{sub a} of the binding reaction was determined to be 3.24±0.07×10{sup 4} M{sup −1} at 25 °C based on fluorescence quenching titration results. The values of enthalpy change and entropy change for the interaction were found as −8.54 kJ mol{sup −1} and 58.01 J mol{sup −1} K{sup −1}, respectively. Both thermodynamic data and docking results suggested the involvement of hydrophobic and van der Waals forces in the complex formation. Three-dimensional fluorescence data of SB202190–HSA complex demonstrated significant changes in the microenvironment around the protein fluorophores upon drug binding. Comparison of HSA thermograms obtained in the absence and the presence of SB202190 suggested improved protein thermal stability upon complexation with the drug. Competitive drug displacement results as well as modeling data concluded the preferred binding site of SB202190 on HSA as Sudlow's site I. - Highlights: • SB202190 interacts with HSA with moderate affinity. • Involvement of hydrophobic and van der Waals forces in SB202190 binding. • SB202190 binding results in microenvironmental changes around fluorophores. • Sudlow's site I is the preferred binding site of SB202190.

Serum albumin promotes ATP-binding cassette transporter-dependent sterol uptake in yeast

DEFF Research Database (Denmark)

Marek, Magdalena; Silvestro, Daniele; Fredslund, Maria D.

2014-01-01

Sterol uptake in fungi is a multistep process that involves interaction between external sterols and the cell wall, incorporation of sterol molecules into the plasma membrane, and subsequent integration into intracellular membranes for turnover. ATP-binding cassette (ABC) transporters have been...

Binding of polycyclic and nitropolycyclic aromatic hydrocarbons to specific fractions of rat lung chromatin

International Nuclear Information System (INIS)

Mitchell, C.E.; Akkaraju, S.

1988-01-01

Binding of polycyclic aromatic hydrocarbons and nitropolycyclic aromatic hydrocarbons (NPAH) to rat lung nuclei was investigated. Following carcinogen exposure, nuclei were fractionated into active chromatin, nuclear matrix, low salt, and high salt fractions. Preferential binding to active chromatin and nuclear matrix fractions was observed for benzo(a)pyrene (BP), 6-nitro benzo(a)pyrene, 1,6-dinitropyrene (1,6-DNP), and 1-nitropyrene. Incubation of nuclei with BP, benzo(a)pyrene diolepoxide (BPDE), and 1,6-DNP showed that the selective binding was dependent upon the concentration of chemical with less selectivity at higher concentrations. This study shows that NPAH should be considered as another class of compounds that may exert their biological effects by binding to selected regions of chromatin that are involved in DNA replication and translation. (author)

Ligand photo-isomerization triggers conformational changes in iGluR2 ligand binding domain.

Directory of Open Access Journals (Sweden)

Tino Wolter

Full Text Available Neurological glutamate receptors bind a variety of artificial ligands, both agonistic and antagonistic, in addition to glutamate. Studying their small molecule binding properties increases our understanding of the central nervous system and a variety of associated pathologies. The large, oligomeric multidomain membrane protein contains a large and flexible ligand binding domains which undergoes large conformational changes upon binding different ligands. A recent application of glutamate receptors is their activation or inhibition via photo-switchable ligands, making them key systems in the emerging field of optochemical genetics. In this work, we present a theoretical study on the binding mode and complex stability of a novel photo-switchable ligand, ATA-3, which reversibly binds to glutamate receptors ligand binding domains (LBDs. We propose two possible binding modes for this ligand based on flexible ligand docking calculations and show one of them to be analogues to the binding mode of a similar ligand, 2-BnTetAMPA. In long MD simulations, it was observed that transitions between both binding poses involve breaking and reforming the T686-E402 protein hydrogen bond. Simulating the ligand photo-isomerization process shows that the two possible configurations of the ligand azo-group have markedly different complex stabilities and equilibrium binding modes. A strong but slow protein response is observed after ligand configuration changes. This provides a microscopic foundation for the observed difference in ligand activity upon light-switching.

A novel-type phosphatidylinositol phosphate-interactive, Ca-binding protein PCaP1 in Arabidopsis thaliana: stable association with plasma membrane and partial involvement in stomata closure.

Science.gov (United States)

Nagata, Chisako; Miwa, Chika; Tanaka, Natsuki; Kato, Mariko; Suito, Momoe; Tsuchihira, Ayako; Sato, Yori; Segami, Shoji; Maeshima, Masayoshi

2016-05-01

The Ca(2+)-binding protein-1 (PCaP1) of Arabidopsis thaliana is a new type protein that binds to phosphatidylinositol phosphates and Ca(2+)-calmodulin complex as well as free Ca(2+). Although biochemical properties, such as binding to ligands and N-myristoylation, have been revealed, the intracellular localization, tissue and cell specificity, integrity of membrane association and physiological roles of PCaP1 are unknown. We investigated the tissue and intracellular distribution of PCaP1 by using transgenic lines expressing PCaP1 linked with a green fluorescence protein (GFP) at the carboxyl terminus of PCaP1. GFP fluorescence was obviously detected in most tissues including root, stem, leaf and flower. In these tissues, PCaP1-GFP signal was observed predominantly in the plasma membrane even under physiological stress conditions but not in other organelles. The fluorescence was detected in the cytosol when the 25-residue N-terminal sequence was deleted from PCaP1 indicating essential contribution of N-myristoylation to the plasma membrane anchoring. Fluorescence intensity of PCaP1-GFP in roots was slightly decreased in seedlings grown in medium supplemented with high concentrations of iron for 1 week and increased in those grown with copper. In stomatal guard cells, PCaP1-GFP was strictly, specifically localized to the plasma membrane at the epidermal-cell side but not at the pore side. A T-DNA insertion mutant line of PCaP1 did not show marked phenotype in a life cycle except for well growth under high CO2 conditions. However, stomata of the mutant line did not close entirely even in high osmolarity, which usually induces stomata closure. These results suggest that PCaP1 is involved in the stomatal movement, especially closure process, in leaves and response to excessive copper in root and leaf as a mineral nutrient as a physiological role.

Ebolavirus VP35 uses a bimodal strategy to bind dsRNA for innate immune suppression

Energy Technology Data Exchange (ETDEWEB)

Kimberlin, Christopher R.; Bornholdt, Zachary A.; Li, Sheng; Woods, Jr., Virgil L.; MacRae, Ian J.; Saphire, Erica Ollmann (Scripps); (UCSD)

2010-03-12

Ebolavirus causes a severe hemorrhagic fever and is divided into five distinct species, of which Reston ebolavirus is uniquely nonpathogenic to humans. Disease caused by ebolavirus is marked by early immunosuppression of innate immune signaling events, involving silencing and sequestration of double-stranded RNA (dsRNA) by the viral protein VP35. Here we present unbound and dsRNA-bound crystal structures of the dsRNA-binding domain of Reston ebolavirus VP35. The structures show that VP35 forms an unusual, asymmetric dimer on dsRNA binding, with each of the monomers binding dsRNA in a different way: one binds the backbone whereas the other caps the terminus. Additional SAXS, DXMS, and dsRNA-binding experiments presented here support a model of cooperative dsRNA recognition in which binding of the first monomer assists binding of the next monomer of the oligomeric VP35 protein. This work illustrates how ebolavirus VP35 could mask key recognition sites of molecules such as RIG-I, MDA-5, and Dicer to silence viral dsRNA in infection.

Equilibrium expert: an add-in to Microsoft Excel for multiple binding equilibrium simulations and parameter estimations.

Science.gov (United States)

Raguin, Olivier; Gruaz-Guyon, Anne; Barbet, Jacques

2002-11-01

An add-in to Microsoft Excel was developed to simulate multiple binding equilibriums. A partition function, readily written even when the equilibrium is complex, describes the experimental system. It involves the concentrations of the different free molecular species and of the different complexes present in the experiment. As a result, the software is not restricted to a series of predefined experimental setups but can handle a large variety of problems involving up to nine independent molecular species. Binding parameters are estimated by nonlinear least-square fitting of experimental measurements as supplied by the user. The fitting process allows user-defined weighting of the experimental data. The flexibility of the software and the way it may be used to describe common experimental situations and to deal with usual problems such as tracer reactivity or nonspecific binding is demonstrated by a few examples. The software is available free of charge upon request.

The LXCXE Retinoblastoma Protein-Binding Motif of FOG-2 Regulates Adipogenesis.

Science.gov (United States)

Goupille, Olivier; Penglong, Tipparat; Kadri, Zahra; Granger-Locatelli, Marine; Denis, Raphaël; Luquet, Serge; Badoual, Cécile; Fucharoen, Suthat; Maouche-Chrétien, Leila; Leboulch, Philippe; Chrétien, Stany

2017-12-19

GATA transcription factors and their FOG cofactors play a key role in tissue-specific development and differentiation, from worms to humans. Mammals have six GATA and two FOG factors. We recently demonstrated that interactions between retinoblastoma protein (pRb) and GATA-1 are crucial for erythroid proliferation and differentiation. We show here that the LXCXE pRb-binding site of FOG-2 is involved in adipogenesis. Unlike GATA-1, which inhibits cell division, FOG-2 promotes proliferation. Mice with a knockin of a Fog2 gene bearing a mutated LXCXE pRb-binding site are resistant to obesity and display higher rates of white-to-brown fat conversion. Thus, each component of the GATA/FOG complex (GATA-1 and FOG-2) is involved in pRb/E2F regulation, but these molecules have markedly different roles in the control of tissue homeostasis. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Binding of MCM-interacting proteins to ATP-binding site in MCM6

Directory of Open Access Journals (Sweden)

Hosoi A

2016-03-01

Full Text Available Atsutoshi Hosoi, Taku Sakairi, Yukio Ishimi Graduate School of Science and Engineering, Ibaraki University, Mito, Ibaraki, Japan Abstract: The function of MCM2–7 complex that is a DNA helicase in DNA replication may be regulated by various MCM-interacting proteins, including CDC45, RPA, TIM, TIPIN, Claspin, MCM10, and MCM-BP. It has been shown by immunoprecipitation that human MCM6 interacts with all these proteins in coexpressed insect cells. To determine the region in MCM6 to interact with these proteins, we prepared various truncated forms of MCM6 and examined the interaction of these MCM6 fragments with the MCM-interacting proteins. All these proteins bound to C-terminal half of MCM6, and CDC45, RPA2, TIM, TIPIN, MCM-BP, and MCM10 bound to the fragments containing ATP-binding motifs. CDC45 and RPA2 bound to the smallest fragment containing Walker motif A. Only MCM-BP is bound to the N-terminal half of MCM6. Site-directed mutagenesis study suggests that hydrophobic interaction is involved in the interaction of MCM6 with CDC45 and TIM. These results suggest a possibility that MCM-interacting proteins regulate MCM2–7 function by modulating the ATP-binding ability of the MCM2–7. Keywords: DNA helicase, DNA replication, checkpoint, MCM2–7 proteins

Structure of Drosophila Oskar reveals a novel RNA binding protein

Science.gov (United States)

Yang, Na; Yu, Zhenyu; Hu, Menglong; Wang, Mingzhu; Lehmann, Ruth; Xu, Rui-Ming

2015-01-01

Oskar (Osk) protein plays critical roles during Drosophila germ cell development, yet its functions in germ-line formation and body patterning remain poorly understood. This situation contrasts sharply with the vast knowledge about the function and mechanism of osk mRNA localization. Osk is predicted to have an N-terminal LOTUS domain (Osk-N), which has been suggested to bind RNA, and a C-terminal hydrolase-like domain (Osk-C) of unknown function. Here, we report the crystal structures of Osk-N and Osk-C. Osk-N shows a homodimer of winged-helix–fold modules, but without detectable RNA-binding activity. Osk-C has a lipase-fold structure but lacks critical catalytic residues at the putative active site. Surprisingly, we found that Osk-C binds the 3′UTRs of osk and nanos mRNA in vitro. Mutational studies identified a region of Osk-C important for mRNA binding. These results suggest possible functions of Osk in the regulation of stability, regulation of translation, and localization of relevant mRNAs through direct interaction with their 3′UTRs, and provide structural insights into a novel protein–RNA interaction motif involving a hydrolase-related domain. PMID:26324911

The human intestinal fatty acid binding protein (hFABP2) gene is regulated by HNF-4α

International Nuclear Information System (INIS)

Klapper, Maja; Boehme, Mike; Nitz, Inke; Doering, Frank

2007-01-01

The cytosolic human intestinal fatty acid binding protein (hFABP2) is proposed to be involved in intestinal absorption of long-chain fatty acids. The aim of this study was to investigate the regulation of hFABP2 by the endodermal hepatocyte nuclear factor 4α (HNF-4α), involved in regulation of genes of fatty acid metabolism and differentiation. Electromobility shift assays demonstrated that HNF-4α binds at position -324 to -336 within the hFABP2 promoter. Mutation of this HNF-4 binding site abolished the luciferase reporter activity of hFABP2 in postconfluent Caco-2 cells. In HeLa cells, this mutation reduced the activation of the hFABP2 promoter by HNF-4α by about 50%. Thus, binding element at position -336/-324 essentially determines the transcriptional activity of promoter and may be important in control of hFABP2 expression by dietary lipids and differentiation. Studying genotype interactions of hFABP2 and HNF-4α, that are both candidate genes for diabetes type 2, may be a powerful approach

The human intestinal fatty acid binding protein (hFABP2) gene is regulated by HNF-4{alpha}

Energy Technology Data Exchange (ETDEWEB)

Klapper, Maja [Molecular Nutrition, Institute of Human Nutrition and Food Science, Christian-Albrechts-University of Kiel, Heinrich-Hecht-Platz 10, D-24118 Kiel (Germany); Boehme, Mike [Molecular Nutrition, Institute of Human Nutrition and Food Science, Christian-Albrechts-University of Kiel, Heinrich-Hecht-Platz 10, D-24118 Kiel (Germany); Nitz, Inke [Molecular Nutrition, Institute of Human Nutrition and Food Science, Christian-Albrechts-University of Kiel, Heinrich-Hecht-Platz 10, D-24118 Kiel (Germany); Doering, Frank [Molecular Nutrition, Institute of Human Nutrition and Food Science, Christian-Albrechts-University of Kiel, Heinrich-Hecht-Platz 10, D-24118 Kiel (Germany)

2007-04-27

The cytosolic human intestinal fatty acid binding protein (hFABP2) is proposed to be involved in intestinal absorption of long-chain fatty acids. The aim of this study was to investigate the regulation of hFABP2 by the endodermal hepatocyte nuclear factor 4{alpha} (HNF-4{alpha}), involved in regulation of genes of fatty acid metabolism and differentiation. Electromobility shift assays demonstrated that HNF-4{alpha} binds at position -324 to -336 within the hFABP2 promoter. Mutation of this HNF-4 binding site abolished the luciferase reporter activity of hFABP2 in postconfluent Caco-2 cells. In HeLa cells, this mutation reduced the activation of the hFABP2 promoter by HNF-4{alpha} by about 50%. Thus, binding element at position -336/-324 essentially determines the transcriptional activity of promoter and may be important in control of hFABP2 expression by dietary lipids and differentiation. Studying genotype interactions of hFABP2 and HNF-4{alpha}, that are both candidate genes for diabetes type 2, may be a powerful approach.

The RNA-binding protein xCIRP2 is involved in apoptotic tail regression during metamorphosis in Xenopus laevis tadpoles.

Science.gov (United States)

Eto, Ko; Iwama, Tomoyuki; Tajima, Tatsuya; Abe, Shin-ichi

2012-10-01

Frog metamorphosis induced by thyroid hormone (TH) involves not only cell proliferation and differentiation in reconstituted organs such as limbs, but also apoptotic cell death in degenerated organs such as tails. However, the molecular mechanisms directing the TH-dependent cell fate determination remain unclear. We have previously identified from newts an RNA-binding protein (nRBP) acting as the regulator governing survival and death in germ cells during spermatogenesis. To investigate the molecular events leading the tail resorption during metamorphosis, we analyzed the expression, the functional role in apoptosis, and the regulation of xCIRP2, a frog homolog of nRBP, in tails of Xenopus laevis tadpoles. At the prometamorphic stage, xCIRP2 protein is expressed in fibroblast, epidermal, nerve, and muscular cells and localized in their cytoplasm. When spontaneous metamorphosis progressed, the level of xCIRP2 mRNA remained unchanged but the amount of the protein decreased. In organ cultures of tails at the prometamorphic stage, xCIRP2 protein decreased before their lengths shortened during TH-dependent metamorphosis. The inhibition of calpain or proteasome attenuated the TH-induced decrease of xCIRP2 protein in tails, impairing their regression. These results suggest that xCIRP2 protein is downregulated through calpain- and proteasome-mediated proteolysis in response to TH at the onset of metamorphosis, inducing apoptosis in tails and thereby degenerating them. Copyright © 2012 Elsevier Inc. All rights reserved.

Lysozyme binding ability toward psychoactive stimulant drugs: Modulatory effect of colloidal metal nanoparticles.

Science.gov (United States)

Sonu, Vikash K; Islam, Mullah Muhaiminul; Rohman, Mostofa Ataur; Mitra, Sivaprasad

2016-10-01

The interaction and binding behavior of the well-known psychoactive stimulant drugs theophylline (THP) and theobromine (THB) with lysozyme (LYS) was monitored by in-vitro fluorescence titration and molecular docking calculations under physiological condition. The quenching of protein fluorescence on addition of the drugs is due to the formation of protein-drug complex in the ground state in both the cases. However, the binding interaction is almost three orders of magnitude stronger in THP, which involves mostly hydrogen bonding interaction in comparison with THB where hydrophobic binding plays the predominant role. The mechanism of fluorescence quenching (static type) remains same also in presence of gold and silver nanoparticles (NPs); however, the binding capacity of LYS with the drugs changes drastically in comparison with that in aqueous buffer medium. While the binding affinity of LYS to THB increases ca. 100 times in presence of both the NPs, it is seen to decrease drastically (by almost 1000 fold) for THP. This significant modulation in binding behavior indicates that the drug transportation capacity of LYS can be controlled significantly with the formation protein-NP noncovalent assembly system as an efficient delivery channel. Copyright © 2016 Elsevier B.V. All rights reserved.

Interaction of the anaphase-promoting complex/cyclosome and proteasome protein complexes with multiubiquitin chain-binding proteins

DEFF Research Database (Denmark)

Seeger, Michael; Hartmann-Petersen, Rasmus; Wilkinson, Caroline R M

2003-01-01

Fission yeast Rhp23 and Pus1 represent two families of multiubiquitin chain-binding proteins that associate with the proteasome. We show that both proteins bind to different regions of the proteasome subunit Mts4. The binding site for Pus1 was mapped to a cluster of repetitive sequences also found...... in the proteasome subunit SpRpn2 and the anaphase-promoting complex/cyclosome (APC/C) subunit Cut4. The putative role of Pus1 as a factor involved in allocation of ubiquitinylated substrates for the proteasome is discussed....

Proteome scale identification, classification and structural analysis of iron-binding proteins in bread wheat.

Science.gov (United States)

Verma, Shailender Kumar; Sharma, Ankita; Sandhu, Padmani; Choudhary, Neha; Sharma, Shailaja; Acharya, Vishal; Akhter, Yusuf

2017-05-01

Bread wheat is one of the major staple foods of worldwide population and iron plays a significant role in growth and development of the plant. In this report, we are presenting the genome wide identification of iron-binding proteins in bread wheat. The wheat genome derived putative proteome was screened for identification of iron-binding sequence motifs. Out of 602 putative iron-binding proteins, 130 were able to produce reliable structural models by homology techniques and further analyzed for the presence of iron-binding structural motifs. The computationally identified proteins appear to bind to ferrous and ferric ions and showed diverse coordination geometries. Glu, His, Asp and Cys amino acid residues were found to be mostly involved in iron binding. We have classified these proteins on the basis of their localization in the different cellular compartments. The identified proteins were further classified into their protein folds, families and functional classes ranging from structure maintenance of cellular components, regulation of gene expression, post translational modification, membrane proteins, enzymes, signaling and storage proteins. This comprehensive report regarding structural iron binding proteome provides useful insights into the diversity of iron binding proteins of wheat plants and further utilized to study their roles in plant growth, development and physiology. Copyright © 2017 Elsevier Inc. All rights reserved.

Competitive protein binding assay

International Nuclear Information System (INIS)

Kaneko, Toshio; Oka, Hiroshi

1975-01-01

The measurement of cyclic GMP (cGMP) by competitive protein binding assay was described and discussed. The principle of binding assay was represented briefly. Procedures of our method by binding protein consisted of preparation of cGMP binding protein, selection of 3 H-cyclic GMP on market, and measurement procedures. In our method, binding protein was isolated from the chrysalis of silk worm. This method was discussed from the points of incubation medium, specificity of binding protein, the separation of bound cGMP from free cGMP, and treatment of tissue from which cGMP was extracted. cGMP existing in the tissue was only one tenth or one scores of cGMP, and in addition, cGMP competed with cGMP in binding with binding protein. Therefore, Murad's technique was applied to the isolation of cGMP. This method provided the measurement with sufficient accuracy; the contamination by cAMP was within several per cent. (Kanao, N.)

Elucidating the evolutionary conserved DNA-binding specificities of WRKY transcription factors by molecular dynamics and in vitro binding assays

Science.gov (United States)

Brand, Luise H.; Fischer, Nina M.; Harter, Klaus; Kohlbacher, Oliver; Wanke, Dierk

2013-01-01

WRKY transcription factors constitute a large protein family in plants that is involved in the regulation of developmental processes and responses to biotic or abiotic stimuli. The question arises how stimulus-specific responses are mediated given that the highly conserved WRKY DNA-binding domain (DBD) exclusively recognizes the ‘TTGACY’ W-box consensus. We speculated that the W-box consensus might be more degenerate and yet undetected differences in the W-box consensus of WRKYs of different evolutionary descent exist. The phylogenetic analysis of WRKY DBDs suggests that they evolved from an ancestral group IIc-like WRKY early in the eukaryote lineage. A direct descent of group IIc WRKYs supports a monophyletic origin of all other group II and III WRKYs from group I by loss of an N-terminal DBD. Group I WRKYs are of paraphyletic descent and evolved multiple times independently. By homology modeling, molecular dynamics simulations and in vitro DNA–protein interaction-enzyme-linked immunosorbent assay with AtWRKY50 (IIc), AtWRKY33 (I) and AtWRKY11 (IId) DBDs, we revealed differences in DNA-binding specificities. Our data imply that other components are essentially required besides the W-box-specific binding to DNA to facilitate a stimulus-specific WRKY function. PMID:23975197

Synthesis of an 125I analog of MK-0591 and characterization of a 5-lipoxygenase activating protein binding assay

International Nuclear Information System (INIS)

Eggler, J.F.; Cheng, J.B.; Cooper, K.; Hanak, L.M.; Pillar, J.S.

1994-01-01

The 125 I analog of MK-0591,1, has been prepared for use as a radioligand for developing a 5-lipoxygenase activating protein (FLAP) binding assay. The radiosynthesis involves a two step oxidative iododestannylation-saponification procedure. A FLAP binding assay has been developed in human neutrophil membranes. The binding of 1 to human neutrophil FLAP is rapid, reversible, of high affinity, saturable and selective for FLAP inhibitors. (author)

Thermodynamic fingerprints of ligand binding to human telomeric G-quadruplexes.

Science.gov (United States)

Bončina, Matjaž; Podlipnik, Črtomir; Piantanida, Ivo; Eilmes, Julita; Teulade-Fichou, Marie-Paule; Vesnaver, Gorazd; Lah, Jurij

2015-12-02

Thermodynamic studies of ligand binding to human telomere (ht) DNA quadruplexes, as a rule, neglect the involvement of various ht-DNA conformations in the binding process. Therefore, the thermodynamic driving forces and the mechanisms of ht-DNA G-quadruplex-ligand recognition remain poorly understood. In this work we characterize thermodynamically and structurally binding of netropsin (Net), dibenzotetraaza[14]annulene derivatives (DP77, DP78), cationic porphyrin (TMPyP4) and two bisquinolinium ligands (Phen-DC3, 360A-Br) to the ht-DNA fragment (Tel22) AGGG(TTAGGG)3 using isothermal titration calorimetry, CD and fluorescence spectroscopy, gel electrophoresis and molecular modeling. By global thermodynamic analysis of experimental data we show that the driving forces characterized by contributions of specific interactions, changes in solvation and conformation differ significantly for binding of ligands with low quadruplex selectivity over duplexes (Net, DP77, DP78, TMPyP4; KTel22 ≈ KdsDNA). These contributions are in accordance with the observed structural features (changes) and suggest that upon binding Net, DP77, DP78 and TMPyP4 select hybrid-1 and/or hybrid-2 conformation while Phen-DC3 and 360A-Br induce the transition of hybrid-1 and hybrid-2 to the structure with characteristics of antiparallel or hybrid-3 type conformation. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Genes Involved in Human Ribosome Biogenesis areTranscriptionally Upregulated in Colorectal Cancer

DEFF Research Database (Denmark)

Mansilla, Francisco; Lamy, Philippe; Ørntoft, Torben Falck

2009-01-01

Microarray gene expression profiling comprising 168 colorectal adenocarcinomas and 10 normal mucosas showed that over 79% of the genes involved in human ribosome biogenesis are significantly upregulated (log2>0.5, p<10-3) when compared to normal mucosa. Overexpression was independent of microsate......Microarray gene expression profiling comprising 168 colorectal adenocarcinomas and 10 normal mucosas showed that over 79% of the genes involved in human ribosome biogenesis are significantly upregulated (log2>0.5, p... of microsatellite status. The promoters of the genes studied showed a significant enrichment for several transcription factor binding sites. There was a significant correlation between the number of binding site targets for these transcription factors and the observed gene transcript upregulation. The upregulation...

Cholinergic receptor binding in the frontal cortex of suicide victims

International Nuclear Information System (INIS)

Stanley, M.

1986-01-01

Because there is a high incidence of individuals diagnosed as having an affective disorder who subsequently commit suicide, the author thought it would be of interest to determine QNB binding in the brains of a large sample of suicide victims, and to compare the findings with a well-matched control group. Brain samples were obtained at autopsy from 22 suicide victims and 22 controls. Frontal cortex samples were diseected, frozen, and stored until assayed. Samples of tissue homogenate were incubated in duplicate with 10 concentrations of tritium-QNB. Specific binding was determined with and without atropine. The results confirmed previous studies in which no changes were noted in suicide versus control brains. While the findings neither disprove nor support the cholinergic hypothesis of depression, they do suggest that the neurochemical basis for the in vivo observations of increased responsivity of depressed individuals to muscarinic cholinergic agents might not involve changes in receptors estimated by QNB binding

Different characteristics and nucleotide binding properties of inosine monophosphate dehydrogenase (IMPDH isoforms.

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Elaine C Thomas

Full Text Available We recently reported that Inosine Monophosphate Dehydrogenase (IMPDH, a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, clustered into macrostructures in response to decreased nucleotide levels and that there were differences between the IMPDH isoforms, IMPDH1 and IMPDH2. We hypothesised that the Bateman domains, which are present in both isoforms and serve as energy-sensing/allosteric modules in unrelated proteins, would contribute to isoform-specific differences and that mutations situated in and around this domain in IMPDH1 which give rise to retinitis pigmentosa (RP would compromise regulation. We employed immuno-electron microscopy to investigate the ultrastructure of IMPDH macrostructures and live-cell imaging to follow clustering of an IMPDH2-GFP chimera in real-time. Using a series of IMPDH1/IMPDH2 chimera we demonstrated that the propensity to cluster was conferred by the N-terminal 244 amino acids, which includes the Bateman domain. A protease protection assay suggested isoform-specific purine nucleotide binding characteristics, with ATP protecting IMPDH1 and AMP protecting IMPDH2, via a mechanism involving conformational changes upon nucleotide binding to the Bateman domain without affecting IMPDH catalytic activity. ATP binding to IMPDH1 was confirmed in a nucleotide binding assay. The RP-causing mutation, R224P, abolished ATP binding and nucleotide protection and this correlated with an altered propensity to cluster. Collectively these data demonstrate that (i the isoforms are differentially regulated by AMP and ATP by a mechanism involving the Bateman domain, (ii communication occurs between the Bateman and catalytic domains and (iii the RP-causing mutations compromise such regulation. These findings support the idea that the IMPDH isoforms are subject to distinct regulation and that regulatory defects contribute to human disease.

Resistance to Linezolid Caused by Modifications at Its Binding Site on the Ribosome

DEFF Research Database (Denmark)

Long, Katherine S.; Vester, Birte

2012-01-01

Linezolid is an oxazolidinone antibiotic in clinical use for the treatment of serious infections of resistant Gram-positive bacteria. It inhibits protein synthesis by binding to the peptidyl transferase center on the ribosome. Almost all known resistance mechanisms involve small alterations...... to the linezolid binding site, so this review will therefore focus on the various changes that can adversely affect drug binding and confer resistance. High-resolution structures of linezolid bound to the 50S ribosomal subunit show that it binds in a deep cleft that is surrounded by 23S rRNA nucleotides. Mutation...... of 23S rRNA has for some time been established as a linezolid resistance mechanism. Although ribosomal proteins L3 and L4 are located further away from the bound drug, mutations in specific regions of these proteins are increasingly being associated with linezolid resistance. However, very little...

Selective metal binding to Cys-78 within endonuclease V causes an inhibition of catalytic activities without altering nontarget and target DNA binding

International Nuclear Information System (INIS)

Prince, M.A.; Friedman, B.; Gruskin, E.A.; Schrock, R.D. III; Lloyd, R.S.

1991-01-01

T4 endonuclease V is a pyrimidine dimer-specific DNA repair enzyme which has been previously shown not to require metal ions for either of its two catalytic activities or its DNA binding function. However, we have investigated whether the single cysteine within the enzyme was able to bind metal salts and influence the various activities of this repair enzyme. A series of metals (Hg2+, Ag+, Cu+) were shown to inactivate both endonuclease Vs pyrimidine dimer-specific DNA glycosylase activity and the subsequent apurinic nicking activity. The binding of metal to endonuclease V did not interfere with nontarget DNA scanning or pyrimidine dimer-specific binding. The Cys-78 codon within the endonuclease V gene was changed by oligonucleotide site-directed mutagenesis to Thr-78 and Ser-78 in order to determine whether the native cysteine was directly involved in the enzyme's DNA catalytic activities and whether the cysteine was primarily responsible for the metal binding. The mutant enzymes were able to confer enhanced ultraviolet light (UV) resistance to DNA repair-deficient Escherichia coli at levels equal to that conferred by the wild type enzyme. The C78T mutant enzyme was purified to homogeneity and shown to be catalytically active on pyrimidine dimer-containing DNA. The catalytic activities of the C78T mutant enzyme were demonstrated to be unaffected by the addition of Hg2+ or Ag+ at concentrations 1000-fold greater than that required to inhibit the wild type enzyme. These data suggest that the cysteine is not required for enzyme activity but that the binding of certain metals to that amino acid block DNA incision by either preventing a conformational change in the enzyme after it has bound to a pyrimidine dimer or sterically interfering with the active site residue's accessibility to the pyrimidine dimer

Regulatory Interactions of Csr Components: the RNA Binding Protein CsrA Activates csrB Transcription in Escherichia coli

OpenAIRE

Gudapaty, Seshagirirao; Suzuki, Kazushi; Wang, Xin; Babitzke, Paul; Romeo, Tony

2002-01-01

The global regulator CsrA (carbon storage regulator) of Escherichia coli is a small RNA binding protein that represses various metabolic pathways and processes that are induced in the stationary phase of growth, while it activates certain exponential phase functions. Both repression and activation by CsrA involve posttranscriptional mechanisms, in which CsrA binding to mRNA leads to decreased or increased transcript stability, respectively. CsrA also binds to a small untranslated RNA, CsrB, f...

Ethanol intake and 3H-serotonin uptake II: A study in alcoholic patients using platelets 3H-paroxetine binding

International Nuclear Information System (INIS)

Daoust, M.; Boucly, P.; Ernouf, D.; Breton, P.; Lhuintre, J.P.

1991-01-01

The kinetic parameters of 3 H-paroxetine binding and 3 H-serotonin uptake were studied in platelets of alcoholic patients. There was no difference between alcoholic and non alcoholic subjects in 3 H-paroxetine binding. When binding and 3 H-serotonin uptake were studied, in the same plasma of the same subjects, the Vmax of serotonin uptake was increased in alcoholics. The data confirm the involvement of serotonin uptake system in alcohol dependance and suggest that serotonin uptake and paroxetine binding sites may be regulated independently in this pathology

Involvement of sigma-1 receptors in the antidepressant-like effects of dextromethorphan.

Science.gov (United States)

Nguyen, Linda; Robson, Matthew J; Healy, Jason R; Scandinaro, Anna L; Matsumoto, Rae R

2014-01-01

Dextromethorphan is an antitussive with a high margin of safety that has been hypothesized to display rapid-acting antidepressant activity based on pharmacodynamic similarities to the N-methyl-D-aspartate (NMDA) receptor antagonist ketamine. In addition to binding to NMDA receptors, dextromethorphan binds to sigma-1 (σ1) receptors, which are believed to be protein targets for a potential new class of antidepressant medications. The purpose of this study was to determine whether dextromethorphan elicits antidepressant-like effects and the involvement of σ1 receptors in mediating its antidepressant-like actions. The antidepressant-like effects of dextromethorphan were assessed in male, Swiss Webster mice using the forced swim test. Next, σ1 receptor antagonists (BD1063 and BD1047) were evaluated in conjunction with dextromethorphan to determine the involvement of σ receptors in its antidepressant-like effects. Quinidine, a cytochrome P450 (CYP) 2D6 inhibitor, was also evaluated in conjunction with dextromethorphan to increase the bioavailability of dextromethorphan and reduce exposure to additional metabolites. Finally, saturation binding assays were performed to assess the manner in which dextromethorphan interacts at the σ1 receptor. Our results revealed dextromethorphan displays antidepressant-like effects in the forced swim test that can be attenuated by pretreatment with σ1 receptor antagonists, with BD1063 causing a shift to the right in the dextromethorphan dose response curve. Concomitant administration of quinidine potentiated the antidepressant-like effects of dextromethorphan. Saturation binding assays revealed that a Ki concentration of dextromethorphan reduces both the Kd and the Bmax of [(3)H](+)-pentazocine binding to σ1 receptors. Taken together, these data suggest that dextromethorphan exerts some of its antidepressant actions through σ1 receptors.

Pharmacophore screening of the protein data bank for specific binding site chemistry.

Science.gov (United States)

Campagna-Slater, Valérie; Arrowsmith, Andrew G; Zhao, Yong; Schapira, Matthieu

2010-03-22

A simple computational approach was developed to screen the Protein Data Bank (PDB) for putative pockets possessing a specific binding site chemistry and geometry. The method employs two commonly used 3D screening technologies, namely identification of cavities in protein structures and pharmacophore screening of chemical libraries. For each protein structure, a pocket finding algorithm is used to extract potential binding sites containing the correct types of residues, which are then stored in a large SDF-formatted virtual library; pharmacophore filters describing the desired binding site chemistry and geometry are then applied to screen this virtual library and identify pockets matching the specified structural chemistry. As an example, this approach was used to screen all human protein structures in the PDB and identify sites having chemistry similar to that of known methyl-lysine binding domains that recognize chromatin methylation marks. The selected genes include known readers of the histone code as well as novel binding pockets that may be involved in epigenetic signaling. Putative allosteric sites were identified on the structures of TP53BP1, L3MBTL3, CHEK1, KDM4A, and CREBBP.

Accurate Estimation of the Standard Binding Free Energy of Netropsin with DNA

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Hong Zhang

2018-01-01

Full Text Available DNA is the target of chemical compounds (drugs, pollutants, photosensitizers, etc., which bind through non-covalent interactions. Depending on their structure and their chemical properties, DNA binders can associate to the minor or to the major groove of double-stranded DNA. They can also intercalate between two adjacent base pairs, or even replace one or two base pairs within the DNA double helix. The subsequent biological effects are strongly dependent on the architecture of the binding motif. Discriminating between the different binding patterns is of paramount importance to predict and rationalize the effect of a given compound on DNA. The structural characterization of DNA complexes remains, however, cumbersome at the experimental level. In this contribution, we employed all-atom molecular dynamics simulations to determine the standard binding free energy of DNA with netropsin, a well-characterized antiviral and antimicrobial drug, which associates to the minor groove of double-stranded DNA. To overcome the sampling limitations of classical molecular dynamics simulations, which cannot capture the large change in configurational entropy that accompanies binding, we resort to a series of potentials of mean force calculations involving a set of geometrical restraints acting on collective variables.

Relational and conjunctive binding functions dissociate in short-term memory.

Science.gov (United States)

Parra, Mario A; Fabi, Katia; Luzzi, Simona; Cubelli, Roberto; Hernandez Valdez, Maria; Della Sala, Sergio

2015-02-01

Remembering complex events requires binding features within unified objects (conjunctions) and holding associations between objects (relations). Recent studies suggest that the two functions dissociate in long-term memory (LTM). Less is known about their functional organization in short-term memory (STM). The present study investigated this issue in patient AE affected by a stroke which caused damage to brain regions known to be relevant for relational functions both in LTM and in STM (i.e., the hippocampus). The assessment involved a battery of standard neuropsychological tasks and STM binding tasks. One STM binding task (Experiment 1) presented common objects and common colors forming either pairs (relations) or integrated objects (conjunctions). Free recall of relations or conjunctions was assessed. A second STM binding task used random polygons and non-primary colors instead (Experiment 2). Memory was assessed by selecting the features that made up the relations or the conjunctions from a set of single polygons and a set of single colors. The neuropsychological assessment revealed impaired delayed memory in AE. AE's pronounced relational STM binding deficits contrasted with his completely preserved conjunctive binding functions in both Experiments 1 and 2. Only 2.35% and 1.14% of the population were expected to have a discrepancy more extreme than that presented by AE in Experiments 1 and 2, respectively. Processing relations and conjunctions of very elementary nonspatial features in STM led to dissociating performances in AE. These findings may inform current theories of memory decline such as those linked to cognitive aging.

A novel signal transduction protein: Combination of solute binding and tandem PAS-like sensor domains in one polypeptide chain: Periplasmic Ligand Binding Protein Dret_0059

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Wu, R. [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439; Wilton, R. [Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439; Cuff, M. E. [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439; Structural Biology Center, Argonne National Laboratory, Argonne Illinois 60439; Endres, M. [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Babnigg, G. [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439; Edirisinghe, J. N. [Mathematics and Computer Science Division, Argonne National Laboratory, Argonne Illinois 60439; Computation Institute, University of Chicago, Chicago Illinois 60637; Henry, C. S. [Mathematics and Computer Science Division, Argonne National Laboratory, Argonne Illinois 60439; Computation Institute, University of Chicago, Chicago Illinois 60637; Joachimiak, A. [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439; Structural Biology Center, Argonne National Laboratory, Argonne Illinois 60439; Department of Biochemistry and Molecular Biology, University of Chicago, Chicago Illinois 60637; Schiffer, M. [Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439; Pokkuluri, P. R. [Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439

2017-03-06

We report the structural and biochemical characterization of a novel periplasmic ligand-binding protein, Dret_0059, from Desulfohalobium retbaense DSM 5692, an organism isolated from the Salt Lake Retba in Senegal. The structure of the protein consists of a unique combination of a periplasmic solute binding protein (SBP) domain at the N-terminal and a tandem PAS-like sensor domain at the C-terminal region. SBP domains are found ubiquitously and their best known function is in solute transport across membranes. PAS-like sensor domains are commonly found in signal transduction proteins. These domains are widely observed as parts of many protein architectures and complexes but have not been observed previously within the same polypeptide chain. In the structure of Dret_0059, a ketoleucine moiety is bound to the SBP, whereas a cytosine molecule is bound in the distal PAS-like domain of the tandem PAS-like domain. Differential scanning flourimetry support the binding of ligands observed in the crystal structure. There is significant interaction between the SBP and tandem PAS-like domains, and it is possible that the binding of one ligand could have an effect on the binding of the other. We uncovered three other proteins with this structural architecture in the non-redundant sequence data base, and predict that they too bind the same substrates. The genomic context of this protein did not offer any clues for its function. We did not find any biological process in which the two observed ligands are coupled. The protein Dret_0059 could be involved in either signal transduction or solute transport.

Tyrosine411 and Arginine410 of Human Serum Albumin Play an Important Role in the Binding of Sodium 4-Phenylbutyrate to Site II.

Science.gov (United States)

Enokida, Taisuke; Yamasaki, Keishi; Okamoto, Yuko; Taguchi, Kazuaki; Ishiguro, Takako; Maruyama, Toru; Seo, Hakaru; Otagiri, Masaki

2016-06-01

Sodium 4-phenylbutyrate (PB) has many pharmacological activities; therefore extending its clinical use to the treatment of a wider variety of diseases would be desirable. However, our knowledge of the binding of PB to plasma proteins is not extensive. To address this issue in more detail, we characterized the protein binding of PB. Binding experiments showed that PB mainly binds to human serum albumin (HSA) in plasma. PB was also found to bind to a single site on HSA, which was identified as site II by fluorescent probe displacement experiment. Furthermore, an appropriate alkyl chain length and a carboxylic group in the PB structure were required for PB binding to HSA, suggesting that hydrophobic (and van der Waals) and electrostatic interactions are involved as binding modes. The contributions of hydrogen bonding and/or van der Waals interactions were also indicated by thermodynamic analyses. Tyrosine411 and arginine410 were identified as being involved in the binding of PB to site II, based on binding experiments using chemically modified- and mutant-HSA preparations. In conclusion, the available evidence indicates that PB binds to site II of HSA with assistance by multiple forces and that tyrosine411 and arginine410 both play important roles in this phenomenon. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

To bind or not to bind? Different temporal binding effects from voluntary pressing and releasing actions.

Science.gov (United States)

Zhao, Ke; Chen, Yu-Hsin; Yan, Wen-Jing; Fu, Xiaolan

2013-01-01

Binding effect refers to the perceptual attraction between an action and an outcome leading to a subjective compression of time. Most studies investigating binding effects exclusively employ the "pressing" action without exploring other types of actions. The present study addresses this issue by introducing another action, releasing action or the voluntary lifting of the finger/wrist, to investigate the differences between voluntary pressing and releasing actions. Results reveal that releasing actions led to robust yet short-lived temporal binding effects, whereas pressing condition had steady temporal binding effects up to super-seconds. The two actions also differ in sensitivity to changes in temporal contiguity and contingency, which could be attributed to the difference in awareness of action. Extending upon current models of "willed action," our results provide insights from a temporal point of view and support the concept of a dual system consisting of predictive motor control and top-down mechanisms.

A protein-binding domain, EH, identified in the receptor tyrosine kinase substrate Eps15 and conserved in evolution

DEFF Research Database (Denmark)

Wong, W T; Schumacher, C; Salcini, A E

1995-01-01

In this report we structurally and functionally define a binding domain that is involved in protein association and that we have designated EH (for Eps15 homology domain). This domain was identified in the tyrosine kinase substrate Eps15 on the basis of regional conservation with several heteroge......In this report we structurally and functionally define a binding domain that is involved in protein association and that we have designated EH (for Eps15 homology domain). This domain was identified in the tyrosine kinase substrate Eps15 on the basis of regional conservation with several...... heterogeneous proteins of yeast and nematode. The EH domain spans about 70 amino acids and shows approximately 60% overall amino acid conservation. We demonstrated the ability of the EH domain to specifically bind cytosolic proteins in normal and malignant cells of mesenchymal, epithelial, and hematopoietic...... (for Eps15-related). Structural comparison of Eps15 and Eps15r defines a family of signal transducers possessing extensive networking abilities including EH-mediated binding and association with Src homology 3-containing proteins....

Binding of (/sup 3/H)imipramine to human platelet membranes with compensation for saturable binding to filters and its implication for binding studies with brain membranes

Energy Technology Data Exchange (ETDEWEB)

Phillips, O.M.; Wood, K.M.; Williams, D.C.

1984-08-01

Apparent specific binding of (/sup 3/H)imipramine to human platelet membranes at high concentrations of imipramine showed deviation from that expected of a single binding site, a result consistent with a low-affinity binding site. The deviation was due to displaceable, saturable binding to the glass fibre filters used in the assays. Imipramine, chloripramine, desipramine, and fluoxetine inhibited binding to filters whereas 5-hydroxytryptamine and ethanol were ineffective. Experimental conditions were developed that eliminated filter binding, allowing assay of high- and low-affinity binding to membranes. Failure to correct for filter binding may lead to overestimation of binding parameters, Bmax and KD for high-affinity binding to membranes, and may also be misinterpreted as indicating a low-affinity binding component in both platelet and brain membranes. Low-affinity binding (KD less than 2 microM) of imipramine to human platelet membranes was demonstrated and its significance discussed.

Serotonin 1B Receptor Binding Is Associated With Trait Anger and Level of Psychopathy in Violent Offenders

DEFF Research Database (Denmark)

da Cunha-Bang, Sofi; Hjordt, Liv Vadskjaer; Perfalk, Erik

2017-01-01

anger (difference in slopes, pcorrected = .04). In the violent offender group, striatal 5-HT1BR binding was positively correlated with self-reported trait anger (p = .0004), trait psychopathy (p = .008), and level of psychopathy according to the Psychopathy Checklist-Revised (p = .02). We found no group...... differences in 5-HT1BR binding. CONCLUSIONS: Our data demonstrate for the first time in humans a specific involvement of 5-HT1BR binding in anger and psychopathy. 5-HT1BRs putatively represent a molecular target for development of pharmacologic antiaggressive treatments....

Identification of the bile salt binding site on IpaD from Shigella flexneri and the influence of ligand binding on IpaD structure.

Science.gov (United States)

Barta, Michael L; Guragain, Manita; Adam, Philip; Dickenson, Nicholas E; Patil, Mrinalini; Geisbrecht, Brian V; Picking, Wendy L; Picking, William D

2012-03-01

Type III secretion (TTS) is an essential virulence factor for Shigella flexneri, the causative agent of shigellosis. The Shigella TTS apparatus (TTSA) is an elegant nanomachine that is composed of a basal body, an external needle to deliver effectors into human cells, and a needle tip complex that controls secretion activation. IpaD is at the tip of the nascent TTSA needle where it controls the first step of TTS activation. The bile salt deoxycholate (DOC) binds to IpaD to induce recruitment of the translocator protein IpaB into the maturing tip complex. We recently used spectroscopic analyses to show that IpaD undergoes a structural rearrangement that accompanies binding to DOC. Here, we report a crystal structure of IpaD with DOC bound and test the importance of the residues that make up the DOC binding pocket on IpaD function. IpaD binds DOC at the interface between helices α3 and α7, with concomitant movement in the orientation of helix α7 relative to its position in unbound IpaD. When the IpaD residues involved in DOC binding are mutated, some are found to lead to altered invasion and secretion phenotypes. These findings suggest that adoption of a DOC bound structural state for IpaD primes the Shigella TTSA for contact with host cells. The data presented here and in the studies leading up to this work provide the foundation for developing a model of the first step in Shigella TTS activation.

MIT domain of Vps4 is a Ca2+-dependent phosphoinositide-binding domain.

Science.gov (United States)

Iwaya, Naoko; Takasu, Hirotoshi; Goda, Natsuko; Shirakawa, Masahiro; Tanaka, Toshiki; Hamada, Daizo; Hiroaki, Hidekazu

2013-05-01

The microtubule interacting and trafficking (MIT) domain is a small protein module that is conserved in proteins of diverged function, such as Vps4, spastin and sorting nexin 15 (SNX15). The molecular function of the MIT domain is protein-protein interaction, in which the domain recognizes peptides containing MIT-interacting motifs. Recently, we identified an evolutionarily related domain, 'variant' MIT domain at the N-terminal region of the microtubule severing enzyme katanin p60. We found that the domain was responsible for binding to microtubules and Ca(2+). Here, we have examined whether the authentic MIT domains also bind Ca(2+). We found that the loop between the first and second α-helices of the MIT domain binds a Ca(2+) ion. Furthermore, the MIT domains derived from Vps4b and SNX15a showed phosphoinositide-binding activities in a Ca(2+)-dependent manner. We propose that the MIT domain is a novel membrane-associating domain involved in endosomal trafficking.

Inhibition of benzodiazepine binding in vitro by amentoflavone, a constituent of various species of Hypericum.

Science.gov (United States)

Baureithel, K H; Büter, K B; Engesser, A; Burkard, W; Schaffner, W

1997-06-01

Flower extracts of Hypericum perforatum, Hypericum hirsutum, Hypericum patulum and Hypericum olympicum efficiently inhibited binding of [3H]flumazenil to rat brain benzodiazepine binding sites of the GABAA-receptor in vitro with IC50 values of 6.83, 6.97, 13.2 and 6.14 micrograms/ml, respectively. Single constituents of the extracts like hypericin, the flavones quercetin and luteolin, the glycosylated flavonoides rutin, hyperoside and quercitrin and the biflavone 13, II8-biapigenin did not inhibit binding up to concentrations of 1 microM. In contrast, amentoflavone revealed an IC50 = 14.9 +/- 1.9 nM on benzodiazepine binding in vitro. Comparative HPLC analyses of hypericin and amentoflavone in extracts of different Hypericum species revealed a possible correlation between the amentoflavone concentration and the inhibition of flumazenil binding. For hypericin no such correlation was observed. Our experimental data demonstrate that amentoflavone, in contrast to hypericin, presents a very active compound with regard to the inhibition of [3H]-flumazenil binding in vitro and thus might be involved in the antidepressant effects of Hypericum perforatum extracts.

Critical role of γ-phosphate in structural transition of Na,K-ATPase upon ATP binding

Science.gov (United States)

Petrushanko, Irina Yu.; Mitkevich, Vladimir A.; Anashkina, Anastasia A.; Klimanova, Elizaveta A.; Dergousova, Elena A.; Lopina, Olga D.; Makarov, Alexander A.

2014-06-01

Active transport of sodium and potassium ions by Na,K-ATPase is accompanied by the enzyme conformational transition between E1 and E2 states. ATP and ADP bind to Na,K-ATPase in the E1 conformation with similar affinity but the properties of enzyme in complexes with these nucleotides are different. We have studied thermodynamics of Na,K-ATPase binding with adenine nucleotides at different temperatures using isothermal titration calorimetry. Our data indicate that β-phosphate is involved in complex formation by increasing the affinity of adenine nucleotides to Na,K-ATPase by an order of magnitude, while γ-phosphate does not affect it. ATP binding to Na,K-ATPase in contrast to ADP binding generates a structural transition in the enzyme, which is consistent with the movement of a significant portion of the surface area to a solvent-protected state. We propose that ATP binding leads to convergence of the nucleotide-binding and phosphorylation domains transferring the enzyme from the ``E1-open'' to ``E1-closed'' conformation ready for phosphorylation.

Antibody binding constants from Farr test and other radioimmunoassays. A theoretical and experimental analysis

International Nuclear Information System (INIS)

Engel, J.; Schalch, W.

1980-01-01

For the reaction of monovalently reacting antibody (116-700pIEF) with its antigen (streptococcal group A-variant polysaccharide), an apparent binding constant Ksub(a) was derived by the ammonium sulfate precipitation technique (Farr assay) which was 40 times larger than the true binding constant K = 10 6 M -1 determined by fluorescence titration and equilibrium dialysis. For monovalently reacting antibodies the time needed for re-equilibration of the binding reaction is short as compared to the time of ammonium sulfate incubation. A thermodynamic analysis was therefore performed for the case of complete equilibration of all components in solution and in the ammonium sulfate precipitate. It was found that in this limiting case Ksub(a)/K is equal to the ratio of the solubilities of the antibody and the antibody complex corrected by the activity coefficients of the components in the precipitate. For other antibody-antigen reactions in which the antibody reacts with both binding sites to the same antigen molecule, re-equilibration of the binding reaction in solution is much slower. For such systems a disturbance of the binding reaction by the precipitation is less likely and correct binding constants may be obtained by the Farr technique or other radioimmunoassays involving precipitation. (author)

C5a binding to human polymorphonuclear leukocyte plasma membrane (PMNLM) receptors

International Nuclear Information System (INIS)

Conway, R.G.; Mollison, K.W.; Carter, G.W.; Lane, B.

1986-01-01

Previous investigations of the C5a receptor have been performed using intact human PMNL. To circumvent some of the potential problems with such whole cell assays (e.g. internalization or metabolism of radioligand) the authors have developed a PMNLM binding assay. Human PMNLM were prepared by nitrogen cavitation and Percoll gradient centrifugation. Specific binding of [ 125 I]C5a to PMNLM was: high affinity, K/sub D/ = 0.6 nM; saturable, B/sub max/ = 8.7 pmol/mg protein; and reversible. Kinetic measurements agree with the K/sub D/ value obtained by Scatchard analysis. Furthermore, the binding activity of C5a correlates with biological activity as measured by myeloperoxidase release from human PMNL. Human serum C5a and recombinant C5a bind with similar affinities when measured by competition or direct binding and label the same number of sites in human PMNLM. The nonhydrolyzable GTP analog, GppNHp, induces a low affinity state of the C5a receptor (4-6 fold shift in K/sub D/) with little effect on B/sub max/. In summary, the criteria have been satisfied for identification of a biologically relevant C5a binding site in human PMNLM. Regulation of the C5a receptor and its membrane transduction mechanism(s) appears to involve guanyl nucleotides, as has been found for other chemoattractant receptors

Involvement of serotonin system in bullimia

International Nuclear Information System (INIS)

Marazziti, D.; Macchi, E.; Rotondo, A.; Placidi, G.F.; Cassano, G.B.

1988-01-01

Platelet 3 H-imipramine binding was investigated in 8 patients affected by bulimia according to DSM III criteria, and in 7 health volunteers. The Bmax /+ -/SD (fmol/mg protein) was 356 /+ -/ 53 in patients, and 1144 /+ -/ 134 in controls. The Kd /+ -/ SD (nM) was 1.35 /+ -/ 0.44 in patients, and 1.90 /+ -/ 0.72 in controls. There was a significant difference in Bmax values in the two groups, whereas no significant difference was observed in Kd values. This study suggests the possible involvement of the indoleamine system in bullimia

Involvement of serotonin system in bullimia

Energy Technology Data Exchange (ETDEWEB)

Marazziti, D.; Macchi, E.; Rotondo, A.; Placidi, G.F.; Cassano, G.B.

1988-01-01

Platelet /sup 3/H-imipramine binding was investigated in 8 patients affected by bulimia according to DSM III criteria, and in 7 health volunteers. The Bmax /+ -/SD (fmol/mg protein) was 356 /+ -/ 53 in patients, and 1144 /+ -/ 134 in controls. The Kd /+ -/ SD (nM) was 1.35 /+ -/ 0.44 in patients, and 1.90 /+ -/ 0.72 in controls. There was a significant difference in Bmax values in the two groups, whereas no significant difference was observed in Kd values. This study suggests the possible involvement of the indoleamine system in bullimia.

Trans-acting translational regulatory RNA binding proteins.

Science.gov (United States)

Harvey, Robert F; Smith, Tom S; Mulroney, Thomas; Queiroz, Rayner M L; Pizzinga, Mariavittoria; Dezi, Veronica; Villenueva, Eneko; Ramakrishna, Manasa; Lilley, Kathryn S; Willis, Anne E

2018-05-01

The canonical molecular machinery required for global mRNA translation and its control has been well defined, with distinct sets of proteins involved in the processes of translation initiation, elongation and termination. Additionally, noncanonical, trans-acting regulatory RNA-binding proteins (RBPs) are necessary to provide mRNA-specific translation, and these interact with 5' and 3' untranslated regions and coding regions of mRNA to regulate ribosome recruitment and transit. Recently it has also been demonstrated that trans-acting ribosomal proteins direct the translation of specific mRNAs. Importantly, it has been shown that subsets of RBPs often work in concert, forming distinct regulatory complexes upon different cellular perturbation, creating an RBP combinatorial code, which through the translation of specific subsets of mRNAs, dictate cell fate. With the development of new methodologies, a plethora of novel RNA binding proteins have recently been identified, although the function of many of these proteins within mRNA translation is unknown. In this review we will discuss these methodologies and their shortcomings when applied to the study of translation, which need to be addressed to enable a better understanding of trans-acting translational regulatory proteins. Moreover, we discuss the protein domains that are responsible for RNA binding as well as the RNA motifs to which they bind, and the role of trans-acting ribosomal proteins in directing the translation of specific mRNAs. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Translation > Translation Regulation Translation > Translation Mechanisms. © 2018 Medical Research Council and University of Cambridge. WIREs RNA published by Wiley Periodicals, Inc.

Distinct ubiquitin binding modes exhibited by SH3 domains: molecular determinants and functional implications.

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Jose L Ortega Roldan

Full Text Available SH3 domains constitute a new type of ubiquitin-binding domains. We previously showed that the third SH3 domain (SH3-C of CD2AP binds ubiquitin in an alternative orientation. We have determined the structure of the complex between first CD2AP SH3 domain and ubiquitin and performed a structural and mutational analysis to decipher the determinants of the SH3-C binding mode to ubiquitin. We found that the Phe-to-Tyr mutation in CD2AP and in the homologous CIN85 SH3-C domain does not abrogate ubiquitin binding, in contrast to previous hypothesis and our findings for the first two CD2AP SH3 domains. The similar alternative binding mode of the SH3-C domains of these related adaptor proteins is characterised by a higher affinity to C-terminal extended ubiquitin molecules. We conclude that CD2AP/CIN85 SH3-C domain interaction with ubiquitin constitutes a new ubiquitin-binding mode involved in a different cellular function and thus changes the previously established mechanism of EGF-dependent CD2AP/CIN85 mono-ubiquitination.

Predicting protein-binding RNA nucleotides with consideration of binding partners.

Science.gov (United States)

Tuvshinjargal, Narankhuu; Lee, Wook; Park, Byungkyu; Han, Kyungsook

2015-06-01

In recent years several computational methods have been developed to predict RNA-binding sites in protein. Most of these methods do not consider interacting partners of a protein, so they predict the same RNA-binding sites for a given protein sequence even if the protein binds to different RNAs. Unlike the problem of predicting RNA-binding sites in protein, the problem of predicting protein-binding sites in RNA has received little attention mainly because it is much more difficult and shows a lower accuracy on average. In our previous study, we developed a method that predicts protein-binding nucleotides from an RNA sequence. In an effort to improve the prediction accuracy and usefulness of the previous method, we developed a new method that uses both RNA and protein sequence data. In this study, we identified effective features of RNA and protein molecules and developed a new support vector machine (SVM) model to predict protein-binding nucleotides from RNA and protein sequence data. The new model that used both protein and RNA sequence data achieved a sensitivity of 86.5%, a specificity of 86.2%, a positive predictive value (PPV) of 72.6%, a negative predictive value (NPV) of 93.8% and Matthews correlation coefficient (MCC) of 0.69 in a 10-fold cross validation; it achieved a sensitivity of 58.8%, a specificity of 87.4%, a PPV of 65.1%, a NPV of 84.2% and MCC of 0.48 in independent testing. For comparative purpose, we built another prediction model that used RNA sequence data alone and ran it on the same dataset. In a 10 fold-cross validation it achieved a sensitivity of 85.7%, a specificity of 80.5%, a PPV of 67.7%, a NPV of 92.2% and MCC of 0.63; in independent testing it achieved a sensitivity of 67.7%, a specificity of 78.8%, a PPV of 57.6%, a NPV of 85.2% and MCC of 0.45. In both cross-validations and independent testing, the new model that used both RNA and protein sequences showed a better performance than the model that used RNA sequence data alone in

Titration calorimetry of anesthetic-protein interaction: negative enthalpy of binding and anesthetic potency.

Science.gov (United States)

Ueda, I; Yamanaka, M

1997-04-01

Anesthetic potency increases at lower temperatures. In contrast, the transfer enthalpy of volatile anesthetics from water to macromolecules is usually positive. The transfer decreases at lower temperature. It was proposed that a few selective proteins bind volatile anesthetics with negative delta H, and these proteins are involved in signal transduction. There has been no report on direct estimation of binding delta H of anesthetics to proteins. This study used isothermal titration calorimetry to analyze chloroform binding to bovine serum albumin. The calorimetrically measured delta H cal was -10.37 kJ.mol-1. Thus the negative delta H of anesthetic binding is not limited to signal transduction proteins. The binding was saturable following Fermi-Dirac statistics and is characterized by the Langmuir adsorption isotherms, which is interfacial. The high-affinity association constant, K, was 2150 +/- 132 M-1 (KD = 0.47 mM) with the maximum binding number, Bmax = 3.7 +/- 0.2. The low-affinity K was 189 +/- 3.8 M-1 (KD = 5.29 mM), with a Bmax of 13.2 +/- 0.3. Anesthetic potency is a function of the activity of anesthetic molecules, not the concentration. Because the sign of delta H determines the temperature dependence of distribution of anesthetic molecules, it is irrelevant to the temperature dependence of anesthetic potency.

Photoaffinity labeling of the oxysterol binding protein

International Nuclear Information System (INIS)

Taylor, F.R.; Kandutsch, A.A.; Anzalone, L.; Spencer, T.A.

1986-01-01

A cytosolic receptor protein for oxygenated sterols, that is thought to be involved in the regulation of HMG-CoA reductase and cholesterol biosynthesis, can be labeled covalently by the photoactivated affinity compound [5,6- 3 H]-7,7'-azocholestane-3β,25-diol (I). Several other compounds were tested including 25-hydroxycholesta-4,6-dien-3-one, 25-azido-27-norcholest-5-en-3β-ol,3β,25-dihydroxycholest-5-en-7-one and 3β-hydroxycholesta-8(14),9(11)-dien-15-one. However, these sterols either did not bind to the receptor with adequate affinity or did not react covalently with the receptor during photolysis. Compound I binds to the receptor with very high affinity (K/sub d/ = 30 nM). After activation with long wavelength UV, two tritium labeled proteins, M/sub r/ approximately 95K and 65K daltons, are found upon SDS gel electrophoresis. No labeling occurs when the binding reaction is carried out in the presence of a large excess of 25-hydroxycholesterol. It is possible that the smaller polypeptide is a degradation product. Under the reaction conditions investigated so far labeling is relatively inefficient (< 1% of bound sterol). These results are generally consistent with previous information suggesting that the M/sub r/ of the receptor subunit is 97,000. Covalent labeling of the receptor should greatly facilitate its further purification and characterization

Familial Risk for Major Depression is Associated with Lower Striatal 5-HT4 Receptor Binding

DEFF Research Database (Denmark)

Madsen, Karine; Torstensen, Eva; Holst, Klaus K

2014-01-01

was to determine whether familial risk for MDD is associated with cerebral 5-HT4 receptor binding as measured with [(11)C]SB207145 brain PET imaging. Familial risk is the most potent risk factor of MDD. METHODS: We studied 57 healthy individuals (mean age 36 yrs, range 20-86; 21 women), 26 of which had first......-degree relatives treated for MDD. RESULTS: We found that having a family history of MDD was associated with lower striatal 5-HT4 receptor binding (p = 0.038; in individuals below 40 years, p = 0.013). Further, we found evidence for a "risk-dose effect" on 5-HT4 receptor binding, since the number of first......-degree relatives with a history of MDD binding correlated negatively with 5-HT4 receptor binding in both the striatum (p = 0.001) and limbic regions (p = 0.012). CONCLUSIONS: Our data suggest that the 5-HT4 receptor is involved in the neurobiological mechanism underlying familial risk for depression...

Vaccine-Elicited Tier 2 HIV-1 Neutralizing Antibodies Bind to Quaternary Epitopes Involving Glycan-Deficient Patches Proximal to the CD4 Binding Site.

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Ema T Crooks

2015-05-01

Full Text Available Eliciting broad tier 2 neutralizing antibodies (nAbs is a major goal of HIV-1 vaccine research. Here we investigated the ability of native, membrane-expressed JR-FL Env trimers to elicit nAbs. Unusually potent nAb titers developed in 2 of 8 rabbits immunized with virus-like particles (VLPs expressing trimers (trimer VLP sera and in 1 of 20 rabbits immunized with DNA expressing native Env trimer, followed by a protein boost (DNA trimer sera. All 3 sera neutralized via quaternary epitopes and exploited natural gaps in the glycan defenses of the second conserved region of JR-FL gp120. Specifically, trimer VLP sera took advantage of the unusual absence of a glycan at residue 197 (present in 98.7% of Envs. Intriguingly, removing the N197 glycan (with no loss of tier 2 phenotype rendered 50% or 16.7% (n = 18 of clade B tier 2 isolates sensitive to the two trimer VLP sera, showing broad neutralization via the surface masked by the N197 glycan. Neutralizing sera targeted epitopes that overlap with the CD4 binding site, consistent with the role of the N197 glycan in a putative "glycan fence" that limits access to this region. A bioinformatics analysis suggested shared features of one of the trimer VLP sera and monoclonal antibody PG9, consistent with its trimer-dependency. The neutralizing DNA trimer serum took advantage of the absence of a glycan at residue 230, also proximal to the CD4 binding site and suggesting an epitope similar to that of monoclonal antibody 8ANC195, albeit lacking tier 2 breadth. Taken together, our data show for the first time that strain-specific holes in the glycan fence can allow the development of tier 2 neutralizing antibodies to native spikes. Moreover, cross-neutralization can occur in the absence of protecting glycan. Overall, our observations provide new insights that may inform the future development of a neutralizing antibody vaccine.

Mycobacterial laminin-binding histone-like protein mediates collagen-dependent cytoadherence

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André Alves Dias

2012-12-01

Full Text Available When grown in the presence of exogenous collagen I, Mycobacterium bovis BCG was shown to form clumps. Scanning electron microscopy examination of these clumps revealed the presence of collagen fibres cross-linking the bacilli. Since collagen is a major constituent of the eukaryotic extracellular matrices, we assayed BCG cytoadherence in the presence of exogenous collagen I. Collagen increased the interaction of the bacilli with A549 type II pneumocytes or U937 macrophages, suggesting that BCG is able to recruit collagen to facilitate its attachment to host cells. Using an affinity chromatography approach, we have isolated a BCG collagen-binding protein corresponding to the previously described mycobacterial laminin-binding histone-like protein (LBP/Hlp, a highly conserved protein associated with the mycobacterial cell wall. Moreover, Mycobacterium leprae LBP/Hlp, a well-characterized adhesin, was also able to bind collagen I. Finally, using recombinant fragments of M. leprae LBP/Hlp, we mapped the collagen-binding activity within the C-terminal domain of the adhesin. Since this protein was already shown to be involved in the recognition of laminin and heparan sulphate-containing proteoglycans, the present observations reinforce the adhesive activities of LBP/Hlp, which can be therefore considered as a multifaceted mycobacterial adhesin, playing an important role in both leprosy and tuberculosis pathogenesis.

The role of pectin in Cd binding by orange peel biosorbents: A comparison of peels, depectinated peels and pectic acid

International Nuclear Information System (INIS)

Schiewer, Silke; Iqbal, Muhammad

2010-01-01

Biosorption by cheaply and abundantly available materials such as citrus peels can be a cost efficient method for removing heavy metals from wastewater. To investigate the role pectin plays in metal binding by citrus peels, native orange peels, protonated peels, depectinated peels, and extracted pectic acid were compared. Kinetic experiments showed that equilibrium was achieved in 1 h. The 1st-order model was more effective in describing the kinetics than the 2nd-order model. Titrations showed two acidic sites with pK a values around 4 (carboxyl) and 10.5 (hydroxyl), respectively. The pH dependent surface charge was described well by a two-site model. Sorption isotherms were best modeled assuming a 1:2 binding stoichiometry, followed by the Langmuir and the Freundlich model. The binding capacity was highest for pectic acid (2.9 mequiv./g) followed by protonated peels and native peels, being lowest for depectinated peels (1.7 mequiv./g). This showed the importance of pectin in metal binding by citrus peels. However, even depectinated peels were still good sorbents which still provided carboxyl groups that were involved in metal binding. FTIR spectra confirmed the presence of carboxyl and hydroxyl groups in all materials and their involvement in metal binding.

The role of pectin in Cd binding by orange peel biosorbents: A comparison of peels, depectinated peels and pectic acid

Energy Technology Data Exchange (ETDEWEB)

Schiewer, Silke, E-mail: sschiewer@alaska.edu [Department of Civil and Environmental Engineering, University of Alaska Fairbanks, PO Box 755900, Fairbanks, AK 99775 (United States); Iqbal, Muhammad, E-mail: iqbalmdr@brain.net.pk [Department of Civil and Environmental Engineering, University of Alaska Fairbanks, PO Box 755900, Fairbanks, AK 99775 (United States)

2010-05-15

Biosorption by cheaply and abundantly available materials such as citrus peels can be a cost efficient method for removing heavy metals from wastewater. To investigate the role pectin plays in metal binding by citrus peels, native orange peels, protonated peels, depectinated peels, and extracted pectic acid were compared. Kinetic experiments showed that equilibrium was achieved in 1 h. The 1st-order model was more effective in describing the kinetics than the 2nd-order model. Titrations showed two acidic sites with pK{sub a} values around 4 (carboxyl) and 10.5 (hydroxyl), respectively. The pH dependent surface charge was described well by a two-site model. Sorption isotherms were best modeled assuming a 1:2 binding stoichiometry, followed by the Langmuir and the Freundlich model. The binding capacity was highest for pectic acid (2.9 mequiv./g) followed by protonated peels and native peels, being lowest for depectinated peels (1.7 mequiv./g). This showed the importance of pectin in metal binding by citrus peels. However, even depectinated peels were still good sorbents which still provided carboxyl groups that were involved in metal binding. FTIR spectra confirmed the presence of carboxyl and hydroxyl groups in all materials and their involvement in metal binding.

A strategy for interaction site prediction between phospho-binding modules and their partners identified from proteomic data.

Science.gov (United States)

Aucher, Willy; Becker, Emmanuelle; Ma, Emilie; Miron, Simona; Martel, Arnaud; Ochsenbein, Françoise; Marsolier-Kergoat, Marie-Claude; Guerois, Raphaël

2010-12-01

Small and large scale proteomic technologies are providing a wealth of potential interactions between proteins bearing phospho-recognition modules and their substrates. Resulting interaction maps reveal such a dense network of interactions that the functional dissection and understanding of these networks often require to break specific interactions while keeping the rest intact. Here, we developed a computational strategy, called STRIP, to predict the precise interaction site involved in an interaction with a phospho-recognition module. The method was validated by a two-hybrid screen carried out using the ForkHead Associated (FHA)1 domain of Rad53, a key protein of Saccharomyces cerevisiae DNA checkpoint, as a bait. In this screen we detected 11 partners, including Cdc7 and Cdc45, essential components of the DNA replication machinery. FHA domains are phospho-threonine binding modules and the threonines involved in both interactions could be predicted using the STRIP strategy. The threonines T484 and T189 in Cdc7 and Cdc45, respectively, were mutated and loss of binding could be monitored experimentally with the full-length proteins. The method was further tested for the analysis of 63 known Rad53 binding partners and provided several key insights regarding the threonines likely involved in these interactions. The STRIP method relies on a combination of conservation, phosphorylation likelihood, and binding specificity criteria and can be accessed via a web interface at http://biodev.extra.cea.fr/strip/.

A Strategy for Interaction Site Prediction between Phospho-binding Modules and their Partners Identified from Proteomic Data*

Science.gov (United States)

Aucher, Willy; Becker, Emmanuelle; Ma, Emilie; Miron, Simona; Martel, Arnaud; Ochsenbein, Françoise; Marsolier-Kergoat, Marie-Claude; Guerois, Raphaël

2010-01-01

Small and large scale proteomic technologies are providing a wealth of potential interactions between proteins bearing phospho-recognition modules and their substrates. Resulting interaction maps reveal such a dense network of interactions that the functional dissection and understanding of these networks often require to break specific interactions while keeping the rest intact. Here, we developed a computational strategy, called STRIP, to predict the precise interaction site involved in an interaction with a phospho-recognition module. The method was validated by a two-hybrid screen carried out using the ForkHead Associated (FHA)1 domain of Rad53, a key protein of Saccharomyces cerevisiae DNA checkpoint, as a bait. In this screen we detected 11 partners, including Cdc7 and Cdc45, essential components of the DNA replication machinery. FHA domains are phospho-threonine binding modules and the threonines involved in both interactions could be predicted using the STRIP strategy. The threonines T484 and T189 in Cdc7 and Cdc45, respectively, were mutated and loss of binding could be monitored experimentally with the full-length proteins. The method was further tested for the analysis of 63 known Rad53 binding partners and provided several key insights regarding the threonines likely involved in these interactions. The STRIP method relies on a combination of conservation, phosphorylation likelihood, and binding specificity criteria and can be accessed via a web interface at http://biodev.extra.cea.fr/strip/. PMID:20733106

CaMELS: In silico prediction of calmodulin binding proteins and their binding sites.

Science.gov (United States)

Abbasi, Wajid Arshad; Asif, Amina; Andleeb, Saiqa; Minhas, Fayyaz Ul Amir Afsar

2017-09-01

Due to Ca 2+ -dependent binding and the sequence diversity of Calmodulin (CaM) binding proteins, identifying CaM interactions and binding sites in the wet-lab is tedious and costly. Therefore, computational methods for this purpose are crucial to the design of such wet-lab experiments. We present an algorithm suite called CaMELS (CalModulin intEraction Learning System) for predicting proteins that interact with CaM as well as their binding sites using sequence information alone. CaMELS offers state of the art accuracy for both CaM interaction and binding site prediction and can aid biologists in studying CaM binding proteins. For CaM interaction prediction, CaMELS uses protein sequence features coupled with a large-margin classifier. CaMELS models the binding site prediction problem using multiple instance machine learning with a custom optimization algorithm which allows more effective learning over imprecisely annotated CaM-binding sites during training. CaMELS has been extensively benchmarked using a variety of data sets, mutagenic studies, proteome-wide Gene Ontology enrichment analyses and protein structures. Our experiments indicate that CaMELS outperforms simple motif-based search and other existing methods for interaction and binding site prediction. We have also found that the whole sequence of a protein, rather than just its binding site, is important for predicting its interaction with CaM. Using the machine learning model in CaMELS, we have identified important features of protein sequences for CaM interaction prediction as well as characteristic amino acid sub-sequences and their relative position for identifying CaM binding sites. Python code for training and evaluating CaMELS together with a webserver implementation is available at the URL: http://faculty.pieas.edu.pk/fayyaz/software.html#camels. © 2017 Wiley Periodicals, Inc.

Involvement of sigma-1 receptors in the antidepressant-like effects of dextromethorphan.

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Linda Nguyen

Full Text Available Dextromethorphan is an antitussive with a high margin of safety that has been hypothesized to display rapid-acting antidepressant activity based on pharmacodynamic similarities to the N-methyl-D-aspartate (NMDA receptor antagonist ketamine. In addition to binding to NMDA receptors, dextromethorphan binds to sigma-1 (σ1 receptors, which are believed to be protein targets for a potential new class of antidepressant medications. The purpose of this study was to determine whether dextromethorphan elicits antidepressant-like effects and the involvement of σ1 receptors in mediating its antidepressant-like actions. The antidepressant-like effects of dextromethorphan were assessed in male, Swiss Webster mice using the forced swim test. Next, σ1 receptor antagonists (BD1063 and BD1047 were evaluated in conjunction with dextromethorphan to determine the involvement of σ receptors in its antidepressant-like effects. Quinidine, a cytochrome P450 (CYP 2D6 inhibitor, was also evaluated in conjunction with dextromethorphan to increase the bioavailability of dextromethorphan and reduce exposure to additional metabolites. Finally, saturation binding assays were performed to assess the manner in which dextromethorphan interacts at the σ1 receptor. Our results revealed dextromethorphan displays antidepressant-like effects in the forced swim test that can be attenuated by pretreatment with σ1 receptor antagonists, with BD1063 causing a shift to the right in the dextromethorphan dose response curve. Concomitant administration of quinidine potentiated the antidepressant-like effects of dextromethorphan. Saturation binding assays revealed that a Ki concentration of dextromethorphan reduces both the Kd and the Bmax of [(3H](+-pentazocine binding to σ1 receptors. Taken together, these data suggest that dextromethorphan exerts some of its antidepressant actions through σ1 receptors.

Functional consequences of piceatannol binding to glyceraldehyde-3-phosphate dehydrogenase.

Science.gov (United States)

Gerszon, Joanna; Serafin, Eligiusz; Buczkowski, Adam; Michlewska, Sylwia; Bielnicki, Jakub Antoni; Rodacka, Aleksandra

2018-01-01

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the key redox-sensitive proteins whose activity is largely affected by oxidative modifications at its highly reactive cysteine residue in the enzyme's active site (Cys149). Prolonged exposure to oxidative stress may cause, inter alia, the formation of intermolecular disulfide bonds leading to accumulation of GAPDH aggregates and ultimately to cell death. Recently these anomalies have been linked with the pathogenesis of Alzheimer's disease. Novel evidences indicate that low molecular compounds may be effective inhibitors potentially preventing the GAPDH translocation to the nucleus, and inhibiting or slowing down its aggregation and oligomerization. Therefore, we decided to establish the ability of naturally occurring compound, piceatannol, to interact with GAPDH and to reveal its effect on functional properties and selected parameters of the dehydrogenase structure. The obtained data revealed that piceatannol binds to GAPDH. The ITC analysis indicated that one molecule of the tetrameric enzyme may bind up to 8 molecules of polyphenol (7.3 ± 0.9). Potential binding sites of piceatannol to the GAPDH molecule were analyzed using the Ligand Fit algorithm. Conducted analysis detected 11 ligand binding positions. We indicated that piceatannol decreases GAPDH activity. Detailed analysis allowed us to presume that this effect is due to piceatannol ability to assemble a covalent binding with nucleophilic cysteine residue (Cys149) which is directly involved in the catalytic reaction. Consequently, our studies strongly indicate that piceatannol would be an exceptional inhibitor thanks to its ability to break the aforementioned pathologic disulfide linkage, and therefore to inhibit GAPDH aggregation. We demonstrated that by binding with GAPDH piceatannol blocks cysteine residue and counteracts its oxidative modifications, that induce oligomerization and GAPDH aggregation.

Search for Partner Proteins of A. thaliana Immunophilins Involved in the Control of Plant Immunity

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Inna A. Abdeeva

2018-04-01

Full Text Available The involvement of plant immunophilins in multiple essential processes such as development, various ways of adapting to biotic and abiotic stresses, and photosynthesis has already been established. Previously, research has demonstrated the involvement of three immunophilin genes (AtCYP19-1/ROC3, AtFKBP65/ROF2, and AtCYP57 in the control of plant response to invasion by various pathogens. Current research attempts to identify host target proteins for each of the selected immunophilins. As a result, candidate interactors have been determined and confirmed using a yeast 2-hybrid (Y2H system for protein–protein interaction assays. The generation of mutant isoforms of ROC3 and AtCYP57 harboring substituted amino acids in the in silico-predicted active sites became essential to achieving significant binding to its target partners. This data shows that ROF2 targets calcium-dependent lipid-binding domain-containing protein (At1g70790; AT1 and putative protein phosphatase (At2g30020; АТ2, whereas ROC3 interacts with GTP-binding protein (At1g30580; ENGD-1 and RmlC-like cupin (At5g39120. The immunophilin AtCYP57 binds to putative pyruvate decarboxylase-1 (Pdc1 and clathrin adaptor complex-related protein (At5g05010. Identified interactors confirm our previous findings that immunophilins ROC3, ROF2, and AtCYP57 are directly involved with stress response control. Further, these findings extend our understanding of the molecular functional pathways of these immunophilins.

DNA-binding polarity of human replication protein A positions nucleases in nucleotide excision repair.

Science.gov (United States)

de Laat, W L; Appeldoorn, E; Sugasawa, K; Weterings, E; Jaspers, N G; Hoeijmakers, J H

1998-08-15

The human single-stranded DNA-binding replication A protein (RPA) is involved in various DNA-processing events. By comparing the affinity of hRPA for artificial DNA hairpin structures with 3'- or 5'-protruding single-stranded arms, we found that hRPA binds ssDNA with a defined polarity; a strong ssDNA interaction domain of hRPA is positioned at the 5' side of its binding region, a weak ssDNA-binding domain resides at the 3' side. Polarity appears crucial for positioning of the excision repair nucleases XPG and ERCC1-XPF on the DNA. With the 3'-oriented side of hRPA facing a duplex ssDNA junction, hRPA interacts with and stimulates ERCC1-XPF, whereas the 5'-oriented side of hRPA at a DNA junction allows stable binding of XPG to hRPA. Our data pinpoint hRPA to the undamaged strand during nucleotide excision repair. Polarity of hRPA on ssDNA is likely to contribute to the directionality of other hRPA-dependent processes as well.

Pulmonary toxicity of trichloroethylene in mice. Covalent binding and morphological manifestations

International Nuclear Information System (INIS)

Forkert, P.G.; Birch, D.W.

1989-01-01

We examined the time course of trichloroethylene (TCE)-induced pulmonary injury and focused on morphological changes and covalent binding of [ 14 C]TCE soon after administration of a single dose of TCE (2000 mg/kg) to CD-1 male mice. At 1 hr after chemical treatment, Clara cells of the bronchiolar epithelium exhibited necrotic changes involving the mitochondria and endoplasmic reticulum. Dilatation of the endoplasmic reticulum became more severe at 2 hr after TCE administration and, by 4 hr, distended cisternae coalesced to form small vacuoles within the cytoplasmic matrix of the Clara cell. The severity of cellular damage increased progressively between 8 and 12 hr and, by 24 hr, the majority of Clara cells within an airway were severely vacuolated. Covalent binding of [ 14 C]TCE to lung macromolecules was evident at 1 hr, peaked at 4 hr, declined thereafter, and reached a plateau between 12 and 24 hr. Peak binding (142.6 +/- 31.8 nmol/g of wet weight) represented approximately 20% of [ 14 C]TCE distributed to the lung. Although the levels of binding in the liver were at all times greater than those in the lung, liver injury was relatively insignificant. The results demonstrate a positive correlation between the onset of Clara cell injury and the formation of reactive metabolites, as assessed by covalent binding of [ 14 C]TCE

An allosteric binding site at the human serotonin transporter mediates the inhibition of escitalopram by R-citalopram: kinetic binding studies with the ALI/VFL-SI/TT mutant.

Science.gov (United States)

Zhong, Huailing; Hansen, Kasper B; Boyle, Noel J; Han, Kiho; Muske, Galina; Huang, Xinyan; Egebjerg, Jan; Sánchez, Connie

2009-10-25

The human serotonin transporter (hSERT) has primary and allosteric binding sites for escitalopram and R-citalopram. Previous studies have established that the interaction of these two compounds at a low affinity allosteric binding site of hSERT can affect the dissociation of [(3)H]escitalopram from hSERT. The allosteric binding site involves a series of residues in the 10th, 11th, and 12th trans-membrane domains of hSERT. The low affinity allosteric activities of escitalopram and R-citalopram are essentially eliminated in a mutant hSERT with changes in some of these residues, namely A505V, L506F, I507L, S574T, I575T, as measured in dissociation binding studies. We confirm that in association binding experiments, R-citalopram at clinically relevant concentrations reduces the association rate of [(3)H]escitalopram as a ligand to wild type hSERT. We demonstrate that the ability of R-citalopram to reduce the association rate of escitalopram is also abolished in the mutant hSERT (A505V, L506F, I507L, S574T, I575T), along with the expected disruption the low affinity allosteric function on dissociation binding. This suggests that the allosteric binding site mediates both the low affinity and higher affinity interactions between R-citalopram, escitalopram, and hSERT. Our data add an additional structural basis for the different efficacies of escitalopram compared to racemic citalopram reported in animal studies and clinical trials, and substantiate the hypothesis that hSERT has complex allosteric mechanisms underlying the unexplained in vivo activities of its inhibitors.

Computational Approaches to the Chemical Equilibrium Constant in Protein-ligand Binding.

Science.gov (United States)

Montalvo-Acosta, Joel José; Cecchini, Marco

2016-12-01

The physiological role played by protein-ligand recognition has motivated the development of several computational approaches to the ligand binding affinity. Some of them, termed rigorous, have a strong theoretical foundation but involve too much computation to be generally useful. Some others alleviate the computational burden by introducing strong approximations and/or empirical calibrations, which also limit their general use. Most importantly, there is no straightforward correlation between the predictive power and the level of approximation introduced. Here, we present a general framework for the quantitative interpretation of protein-ligand binding based on statistical mechanics. Within this framework, we re-derive self-consistently the fundamental equations of some popular approaches to the binding constant and pinpoint the inherent approximations. Our analysis represents a first step towards the development of variants with optimum accuracy/efficiency ratio for each stage of the drug discovery pipeline. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tritium NMR spectroscopy of ligand binding to maltose-binding protein

International Nuclear Information System (INIS)

Gehring, K.; Williams, P.G.; Pelton, J.G.; Morimoto, H.; Wemmer, D.E.

1991-01-01

Tritium-labeled α- and β-maltodextrins have been used to study their complexes with maltose-binding protein (MBP), a 40-kDa bacterial protein. Five substrates, from maltose to maltohexaose, were labeled at their reducing ends and their binding studied. Tritium NMR specctroscopy of the labeled sugars showed large upfield chamical shift changes upon binding and strong anomeric specficity. At 10 degrees C, MBP bound α-maltose with 2.7 ± 0.5-fold higher affinity than β-maltose, and, for longer maltodextrins, the ratio of affinities was even larger. The maximum chemical shift change was 2.2 ppm, suggesting that the reducing end of bound α-maltodextrin makes close contact with an aromatic residue in the MBP-binding site. Experiments with maltotriose (and longer maltodextrins) also revealed the presence of two bound β-maltotriose resonances in rapid exchange. The authors interpret these two resonances as arising from two distinct sugar-protein complexes. In one complex, the β-maltodextrin is bound by its reducing end, and, in the other complex, the β-maltodextrin is bound by the middle glucose residue(s). This interpretation also suggests how MBP is able to bind both linear and circular maltodextrins

Structural characterization of copper(II) binding to α-synuclein: Insights into the bioinorganic chemistry of Parkinson's disease

Science.gov (United States)

Rasia, Rodolfo M.; Bertoncini, Carlos W.; Marsh, Derek; Hoyer, Wolfgang; Cherny, Dmitry; Zweckstetter, Markus; Griesinger, Christian; Jovin, Thomas M.; Fernández, Claudio O.

2005-01-01

The aggregation of α-synuclein (AS) is characteristic of Parkinson's disease and other neurodegenerative synucleinopathies. We demonstrate here that Cu(II) ions are effective in accelerating AS aggregation at physiologically relevant concentrations without altering the resultant fibrillar structures. By using numerous spectroscopic techniques (absorption, CD, EPR, and NMR), we have located the primary binding for Cu(II) to a specific site in the N terminus, involving His-50 as the anchoring residue and other nitrogen/oxygen donor atoms in a square planar or distorted tetragonal geometry. The carboxylate-rich C terminus, originally thought to drive copper binding, is able to coordinate a second Cu(II) equivalent, albeit with a 300-fold reduced affinity. The NMR analysis of AS–Cu(II) complexes reveals the existence of conformational restrictions in the native state of the protein. The metallobiology of Cu(II) in Parkinson's disease is discussed by a comparative analysis with other Cu(II)-binding proteins involved in neurodegenerative disorders. PMID:15767574

Blue news update: BODIPY-GTP binds to the blue-light receptor YtvA while GTP does not.

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Matthias Dorn

Full Text Available Light is an important environmental factor for almost all organisms. It is mainly used as an energy source but it is also a key factor for the regulation of multiple cellular functions. Light as the extracellular stimulus is thereby converted into an intracellular signal by photoreceptors that act as signal transducers. The blue-light receptor YtvA, a bacterial counterpart of plant phototropins, is involved in the stress response of Bacillus subtilis. The mechanism behind its activation, however, remains unknown. It was suggested based on fluorescence spectroscopic studies that YtvA function involves GTP binding and that this interaction is altered by absorption of light. We have investigated this interaction by several biophysical methods and show here using fluorescence spectroscopy, ITC titrations, and three NMR spectroscopic assays that while YtvA interacts with BODIPY-GTP as a fluorescent GTP analogue originally used for the detection of GTP binding, it does not bind GTP.

The Role of Metal Binding in the Amyotrophic Lateral Sclerosis-Related Aggregation of Copper-Zinc Superoxide Dismutase

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Ivana Sirangelo

2017-08-01

Full Text Available Protein misfolding and conformational changes are common hallmarks in many neurodegenerative diseases involving formation and deposition of toxic protein aggregates. Although many players are involved in the in vivo protein aggregation, physiological factors such as labile metal ions within the cellular environment are likely to play a key role. In this review, we elucidate the role of metal binding in the aggregation process of copper-zinc superoxide dismutase (SOD1 associated to amyotrophic lateral sclerosis (ALS. SOD1 is an extremely stable Cu-Zn metalloprotein in which metal binding is crucial for folding, enzymatic activity and maintenance of the native conformation. Indeed, demetalation in SOD1 is known to induce misfolding and aggregation in physiological conditions in vitro suggesting that metal binding could play a key role in the pathological aggregation of SOD1. In addition, this study includes recent advances on the role of aberrant metal coordination in promoting SOD1 aggregation, highlighting the influence of metal ion homeostasis in pathologic aggregation processes.

Crystal structure of glucose isomerase in complex with xylitol inhibitor in one metal binding mode.

Science.gov (United States)

Bae, Ji-Eun; Kim, In Jung; Nam, Ki Hyun

2017-11-04

Glucose isomerase (GI) is an intramolecular oxidoreductase that interconverts aldoses and ketoses. These characteristics are widely used in the food, detergent, and pharmaceutical industries. In order to obtain an efficient GI, identification of novel GI genes and substrate binding/inhibition have been studied. Xylitol is a well-known inhibitor of GI. In Streptomyces rubiginosus, two crystal structures have been reported for GI in complex with xylitol inhibitor. However, a structural comparison showed that xylitol can have variable conformation at the substrate binding site, e.g., a nonspecific binding mode. In this study, we report the crystal structure of S. rubiginosus GI in a complex with xylitol and glycerol. Our crystal structure showed one metal binding mode in GI, which we presumed to represent the inactive form of the GI. The metal ion was found only at the M1 site, which was involved in substrate binding, and was not present at the M2 site, which was involved in catalytic function. The O 2 and O 4 atoms of xylitol molecules contributed to the stable octahedral coordination of the metal in M1. Although there was no metal at the M2 site, no large conformational change was observed for the conserved residues coordinating M2. Our structural analysis showed that the metal at the M2 site was not important when a xylitol inhibitor was bound to the M1 site in GI. Thus, these findings provided important information for elucidation or engineering of GI functions. Copyright © 2017 Elsevier Inc. All rights reserved.

Solute-vacancy binding in aluminum

International Nuclear Information System (INIS)

Wolverton, C.

2007-01-01

Previous efforts to understand solute-vacancy binding in aluminum alloys have been hampered by a scarcity of reliable, quantitative experimental measurements. Here, we report a large database of solute-vacancy binding energies determined from first-principles density functional calculations. The calculated binding energies agree well with accurate measurements where available, and provide an accurate predictor of solute-vacancy binding in other systems. We find: (i) some common solutes in commercial Al alloys (e.g., Cu and Mg) possess either very weak (Cu), or even repulsive (Mg), binding energies. Hence, we assert that some previously reported large binding energies for these solutes are erroneous. (ii) Large binding energies are found for Sn, Cd and In, confirming the proposed mechanism for the reduced natural aging in Al-Cu alloys containing microalloying additions of these solutes. (iii) In addition, we predict that similar reduction in natural aging should occur with additions of Si, Ge and Au. (iv) Even larger binding energies are found for other solutes (e.g., Pb, Bi, Sr, Ba), but these solutes possess essentially no solubility in Al. (v) We have explored the physical effects controlling solute-vacancy binding in Al. We find that there is a strong correlation between binding energy and solute size, with larger solute atoms possessing a stronger binding with vacancies. (vi) Most transition-metal 3d solutes do not bind strongly with vacancies, and some are even energetically strongly repelled from vacancies, particularly for the early 3d solutes, Ti and V

Effects on prolactin secretion and binding to dopaminergic receptors in sleep-deprived lupus-prone mice

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B.D. Palma

2009-03-01

Full Text Available Sleep disturbances have far-reaching effects on the neuroendocrine and immune systems and may be linked to disease manifestation. Sleep deprivation can accelerate the onset of lupus in NZB/NZWF1 mice, an animal model of severe systemic lupus erythematosus. High prolactin (PRL concentrations are involved in the pathogenesis of systemic lupus erythematosus in human beings, as well as in NZB/NZWF1 mice. We hypothesized that PRL could be involved in the earlier onset of the disease in sleep-deprived NZB/NZWF1 mice. We also investigated its binding to dopaminergic receptors, since PRL secretion is mainly controlled by dopamine. Female NZB/NZWF1 mice aged 9 weeks were deprived of sleep using the multiple platform method. Blood samples were taken for the determination of PRL concentrations and quantitative receptor autoradiography was used to map binding of the tritiated dopaminergic receptor ligands [³H]-SCH23390, [³H]-raclopride and [³H]-WIN35,428 to D1 and D2 dopaminergic receptors and dopamine transporter sites throughout the brain, respectively. Sleep deprivation induced a significant decrease in plasma PRL secretion (2.58 ± 0.95 ng/mL compared with the control group (25.25 ± 9.18 ng/mL. The binding to D1 and D2 binding sites was not significantly affected by sleep deprivation; however, dopamine transporter binding was significantly increased in subdivisions of the caudate-putamen - posterior (16.52 ± 0.5 vs 14.44 ± 0.6, dorsolateral (18.84 ± 0.7 vs 15.97 ± 0.7 and ventrolateral (24.99 ± 0.5 vs 22.54 ± 0.7 µCi/g, in the sleep-deprived mice when compared to the control group. These results suggest that PRL is not the main mechanism involved in the earlier onset of the disease observed in sleep-deprived NZB/NZWF1 mice and the reduction of PRL concentrations after sleep deprivation may be mediated by modifications in the dopamine transporter sites of the caudate-putamen.

Crystal structure of the botulinum neurotoxin type G binding domain: insight into cell surface binding.

Science.gov (United States)

Stenmark, Pål; Dong, Min; Dupuy, Jérôme; Chapman, Edwin R; Stevens, Raymond C

2010-04-16

Botulinum neurotoxins (BoNTs) typically bind the neuronal cell surface via dual interactions with both protein receptors and gangliosides. We present here the 1.9-A X-ray structure of the BoNT serotype G (BoNT/G) receptor binding domain (residues 868-1297) and a detailed view of protein receptor and ganglioside binding regions. The ganglioside binding motif (SxWY) has a conserved structure compared to the corresponding regions in BoNT serotype A and BoNT serotype B (BoNT/B), but several features of interactions with the hydrophilic face of the ganglioside are absent at the opposite side of the motif in the BoNT/G ganglioside binding cleft. This may significantly reduce the affinity between BoNT/G and gangliosides. BoNT/G and BoNT/B share the protein receptor synaptotagmin (Syt) I/II. The Syt binding site has a conserved hydrophobic plateau located centrally in the proposed protein receptor binding interface (Tyr1189, Phe1202, Ala1204, Pro1205, and Phe1212). Interestingly, only 5 of 14 residues that are important for binding between Syt-II and BoNT/B are conserved in BoNT/G, suggesting that the means by which BoNT/G and BoNT/B bind Syt diverges more than previously appreciated. Indeed, substitution of Syt-II Phe47 and Phe55 with alanine residues had little effect on the binding of BoNT/G, but strongly reduced the binding of BoNT/B. Furthermore, an extended solvent-exposed hydrophobic loop, located between the Syt binding site and the ganglioside binding cleft, may serve as a third membrane association and binding element to contribute to high-affinity binding to the neuronal membrane. While BoNT/G and BoNT/B are homologous to each other and both utilize Syt-I/Syt-II as their protein receptor, the precise means by which these two toxin serotypes bind to Syt appears surprisingly divergent. Copyright (c) 2010. Published by Elsevier Ltd.

Study on dopamine D2 binding capacity in vascular parkinsonism

International Nuclear Information System (INIS)

Terashi, Hiroo; Nagata, Ken; Hirata, Yutaka; Hatazawa, Jun; Utsumi, Hiroya

2001-01-01

To investigate whether the striatal dopamine receptor function is involved in the development of vascular parkinsonism (VP), a positron emission tomography (PET) study was conducted on 9 patients with VP by using [ 11 C] N-methylspiperone as the tracer. The rate of binding availability in the striatal dopamine D 2 receptor (k 3 ) was determined semiquantitatively, and the values were compared to the predicted normal values based on the results from 7 normal volunteers. Of 9 patients with VP, the normalized D 2 receptor binding [%k 3 ] was more than 90% in 5 patients, 89 to 87% in 3, and 75% in one. These values showed no evident correlation with the Hoehn and Yahr stage. The laterality of the striatal %k 3 did not correspond to that of the parkinsonism. Thus, the striatal dopamine D 2 receptor binding was not severely impaired and did not correlate with the neurological status in patients with VP. This may indicate that striatal dopamine D 2 receptor function is not primarily associated with the development of the parkinsonism in VP. (author)

Prediction of Nucleotide Binding Peptides Using Star Graph Topological Indices.

Science.gov (United States)

Liu, Yong; Munteanu, Cristian R; Fernández Blanco, Enrique; Tan, Zhiliang; Santos Del Riego, Antonino; Pazos, Alejandro

2015-11-01

The nucleotide binding proteins are involved in many important cellular processes, such as transmission of genetic information or energy transfer and storage. Therefore, the screening of new peptides for this biological function is an important research topic. The current study proposes a mixed methodology to obtain the first classification model that is able to predict new nucleotide binding peptides, using only the amino acid sequence. Thus, the methodology uses a Star graph molecular descriptor of the peptide sequences and the Machine Learning technique for the best classifier. The best model represents a Random Forest classifier based on two features of the embedded and non-embedded graphs. The performance of the model is excellent, considering similar models in the field, with an Area Under the Receiver Operating Characteristic Curve (AUROC) value of 0.938 and true positive rate (TPR) of 0.886 (test subset). The prediction of new nucleotide binding peptides with this model could be useful for drug target studies in drug development. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

-NH-dansyl isocolchicine exhibits a significantly improved tubulin-binding affinity and microtubule inhibition in comparison to isocolchicine by binding tubulin through its A and B rings.

Science.gov (United States)

Das, Lalita; Datta, Ajit B; Gupta, Suvroma; Poddar, Asim; Sengupta, Suparna; Janik, Mark E; Bhattacharyya, Bhabatarak

2005-03-08

Structure-activity relationship studies have established that the A and C rings of colchicine comprise the minimum structural feature necessary for high affinity drug-tubulin binding. Thus, colchicine acts as a bifunctional ligand by making two points of attachment to the protein. Furthermore, analogues belonging to the iso series of colchicine are virtually inactive in binding to tubulin and inhibiting microtubule assembly. In the present study, we found that the substitution of a hydrophobic dansyl group on the B-ring side chain (C7 position) of isocolchicine reverses the structural alterations at the C ring and the newly synthesized -NH-dansyl isocolchicine restores the lost biological activity of the compound. It inhibits microtubule assembly efficiently with an IC(50) value of 10 microM and competes with [(3)H]colchicine for binding to tubulin. Moreover, although -NH-dansyl colchicine binding to tubulin involves two steps, the -NH-dansyl isocolchicine-tubulin interaction has been found to occur via a one-step process. Also, the affinity constant of the -NH-dansyl isocolchicine-tubulin interaction is roughly only 3 times lower than that of the -NH-dansyl colchicine-tubulin interaction. These results suggest that the enhanced microtubule inhibitory ability of -NH-dansyl isocolchicine is therefore related to the affinity of the drug-tubulin interaction and not to any conformational changes upon binding tubulin. We also observed that the competition of -NH-dansyl isocolchicine with [(3)H]colchicine for binding to tubulin was dependent on the tubulin concentration. In conclusion, this paper for the first time indicates that a biologically active bifuntional colchicine analogue can be designed where the drug binds tubulin through its A and B rings, while the C ring remains inactive.

Leptospiral outer membrane protein microarray, a novel approach to identification of host ligand-binding proteins.

Science.gov (United States)

Pinne, Marija; Matsunaga, James; Haake, David A

2012-11-01

Leptospirosis is a zoonosis with worldwide distribution caused by pathogenic spirochetes belonging to the genus Leptospira. The leptospiral life cycle involves transmission via freshwater and colonization of the renal tubules of their reservoir hosts. Infection requires adherence to cell surfaces and extracellular matrix components of host tissues. These host-pathogen interactions involve outer membrane proteins (OMPs) expressed on the bacterial surface. In this study, we developed an Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 OMP microarray containing all predicted lipoproteins and transmembrane OMPs. A total of 401 leptospiral genes or their fragments were transcribed and translated in vitro and printed on nitrocellulose-coated glass slides. We investigated the potential of this protein microarray to screen for interactions between leptospiral OMPs and fibronectin (Fn). This approach resulted in the identification of the recently described fibronectin-binding protein, LIC10258 (MFn8, Lsa66), and 14 novel Fn-binding proteins, denoted Microarray Fn-binding proteins (MFns). We confirmed Fn binding of purified recombinant LIC11612 (MFn1), LIC10714 (MFn2), LIC11051 (MFn6), LIC11436 (MFn7), LIC10258 (MFn8, Lsa66), and LIC10537 (MFn9) by far-Western blot assays. Moreover, we obtained specific antibodies to MFn1, MFn7, MFn8 (Lsa66), and MFn9 and demonstrated that MFn1, MFn7, and MFn9 are expressed and surface exposed under in vitro growth conditions. Further, we demonstrated that MFn1, MFn4 (LIC12631, Sph2), and MFn7 enable leptospires to bind fibronectin when expressed in the saprophyte, Leptospira biflexa. Protein microarrays are valuable tools for high-throughput identification of novel host ligand-binding proteins that have the potential to play key roles in the virulence mechanisms of pathogens.

Social Justice and Social Order: Binding Moralities across the Political Spectrum.

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Janoff-Bulman, Ronnie; Carnes, Nate C

2016-01-01

Two studies explored the relationship between political ideology and endorsement of a range of moral principles. Political liberals and conservatives did not differ on intrapersonal or interpersonal moralities, which require self-regulation. However differences emerged on collective moralities, which involve social regulation. Contrary to Moral Foundations Theory, both liberals and conservatives endorsed a group-focused binding morality, specifically Social Justice and Social Order respectively. Libertarians were the group without a binding morality. Although Social Justice and Social Order appear conflictual, analyses based on earlier cross-cultural work on societal tightness-looseness suggest that countries actually benefit in terms of economic success and societal well-being when these group-based moralities co-exist and serve as counterweights in social regulation.

Human-Phosphate-Binding-Protein inhibits HIV-1 gene transcription and replication

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Candolfi Ermanno

2011-07-01

Full Text Available Abstract The Human Phosphate-Binding protein (HPBP is a serendipitously discovered lipoprotein that binds phosphate with high affinity. HPBP belongs to the DING protein family, involved in various biological processes like cell cycle regulation. We report that HPBP inhibits HIV-1 gene transcription and replication in T cell line, primary peripherical blood lymphocytes and primary macrophages. We show that HPBP is efficient in naïve and HIV-1 AZT-resistant strains. Our results revealed HPBP as a new and potent anti HIV molecule that inhibits transcription of the virus, which has not yet been targeted by HAART and therefore opens new strategies in the treatment of HIV infection.

Crystal structure of human importin-α1 (Rch1, revealing a potential autoinhibition mode involving homodimerization.

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Hideyuki Miyatake

Full Text Available In this study, we determined the crystal structure of N-terminal importin-β-binding domain (IBB-truncated human importin-α1 (ΔIBB-h-importin-α1 at 2.63 Å resolution. The crystal structure of ΔIBB-h-importin-α1 reveals a novel closed homodimer. The homodimer exists in an autoinhibited state in which both the major and minor nuclear localization signal (NLS binding sites are completely buried in the homodimerization interface, an arrangement that restricts NLS binding. Analytical ultracentrifugation studies revealed that ΔIBB-h-importin-α1 is in equilibrium between monomers and dimers and that NLS peptides shifted the equilibrium toward the monomer side. This finding suggests that the NLS binding sites are also involved in the dimer interface in solution. These results show that when the IBB domain dissociates from the internal NLS binding sites, e.g., by binding to importin-β, homodimerization possibly occurs as an autoinhibition state.

Neuroticism Associates with Cerebral in Vivo Serotonin Transporter Binding Differently in Males and Females

DEFF Research Database (Denmark)

Tuominen, Lauri; Miettunen, Jouko; Cannon, Dara M

2017-01-01

scores from 91 healthy males and 56 healthy females. We specifically tested if the association between neuroticism and serotonin transporter is different in females and males. Results: We found that neuroticism and thalamic serotonin transporter binding potentials were associated in both males......). Conclusions: The finding is in agreement with recent studies showing that the serotonergic system is involved in affective disorders differently in males and females and suggests that contribution of thalamic serotonin transporter to the risk of affective disorders depends on sex....... and females, but with opposite directionality. Higher neuroticism associated with higher serotonin transporter binding potential in males (standardized beta 0.292, P=.008), whereas in females, higher neuroticism associated with lower serotonin transporter binding potential (standardized beta -0.288, P=.014...

Quantifying high-affinity binding of hydrophobic ligands by isothermal titration calorimetry.

Science.gov (United States)

Krainer, Georg; Broecker, Jana; Vargas, Carolyn; Fanghänel, Jörg; Keller, Sandro

2012-12-18

A fast and reliable quantification of the binding thermodynamics of hydrophobic high-affinity ligands employing a new calorimetric competition experiment is described. Although isothermal titration calorimetry is the method of choice for a quantitative characterization of intermolecular interactions in solution, a reliable determination of a dissociation constant (K(D)) is typically limited to the range 100 μM > K(D) > 1 nM. Interactions displaying higher or lower K(D) values can be assessed indirectly, provided that a suitable competing ligand is available whose K(D) falls within the directly accessible affinity window. This established displacement assay, however, requires the high-affinity ligand to be soluble at high concentrations in aqueous buffer and, consequently, poses serious problems in the study of protein binding involving small-molecule ligands dissolved in organic solvents--a familiar case in many drug-discovery projects relying on compound libraries. The calorimetric competition assay introduced here overcomes this limitation, thus allowing for a detailed thermodynamic description of high-affinity receptor-ligand interactions involving poorly water-soluble compounds. Based on a single titration of receptor into a dilute mixture of the two competing ligands, this competition assay provides accurate and precise values for the dissociation constants and binding enthalpies of both high- and moderate-affinity ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation and high-affinity protein-inhibitor interactions, and explore its potential and limitations with the aid of simulations and statistical analyses.

Identification of clam plasma proteins that bind its pathogen Quahog Parasite Unknown.

Science.gov (United States)

Hartman, Rachel; Pales Espinosa, Emmanuelle; Allam, Bassem

2018-06-01

The hard clam (Mercenaria mercenaria) is among the most economically-important marine species along the east coast of the United States, representing the first marine resource in several Northeastern states. The species is rather resilient to infections and the only important disease of hard clams results from an infection caused by Quahog Parasite Unknown (QPX), a protistan parasite that can lead to significant mortality events in wild and aquacultured clam stocks. Though the presence of QPX disease has been documented since the 1960s, little information is available on cellular and molecular interactions between the parasite and the host. This study examined the interactions between the clam immune system and QPX cells. First, the effect of clam plasma on the binding of hemocytes to parasite cells was evaluated. Second, clam plasma proteins that bind QPX cells were identified through proteomic (LC-MS/MS) analyses. Finally, the effect of prior clam exposure to QPX on the abundance of QPX-reactive proteins in the plasma was evaluated. Results showed that plasma factors enhance the attachment of hemocytes to QPX. Among the proteins that specifically bind to QPX cells, several lectins were identified, as well as complement component proteins and proteolytic enzymes. Furthermore, results showed that some of these lectins and complement-related proteins are inducible as their abundance significantly increased following QPX challenge. These results shed light on plasma proteins involved in the recognition and binding of parasite cells and provide molecular targets for future investigations of factors involved in clam resistance to the disease, and ultimately for the selection of resistant clam stocks. Copyright © 2018 Elsevier Ltd. All rights reserved.

An energy conservation approach to adsorbate-induced surface stress and the extraction of binding energy using nanomechanics

Science.gov (United States)

Pinnaduwage, Lal A.; Boiadjiev, Vassil I.; Hawk, John E.; Gehl, Anthony C.; Fernando, Gayanath W.; Rohana Wijewardhana, L. C.

2008-03-01

Surface stress induced by molecular adsorption in three different binding processes has been studied experimentally using a microcantilever sensor. A comprehensive free-energy analysis based on an energy conservation approach is proposed to explain the experimental observations. We show that when guest molecules bind to atoms/molecules on a microcantilever surface, the released binding energy is retained in the host surface, leading to a metastable state where the excess energy on the surface is manifested as an increase in surface stress leading to the bending of the microcantilever. The released binding energy appears to be almost exclusively channeled to the surface energy, and energy distribution to other channels, including heat, appears to be inactive for this micromechanical system. When this excess surface energy is released, the microcantilever relaxes back to the original state, and the relaxation time depends on the particular binding process involved. Such vapor phase experiments were conducted for three binding processes: physisorption, hydrogen bonding, and chemisorption. Binding energies for these three processes were also estimated.

An energy conservation approach to adsorbate-induced surface stress and the extraction of binding energy using nanomechanics

Energy Technology Data Exchange (ETDEWEB)

Pinnaduwage, Lal A; Boiadjiev, Vassil I; Hawk, John E; Gehl, Anthony C [Oak Ridge National Laboratory, PO Box 2008, Oak Ridge, TN 37831-6122 (United States); Fernando, Gayanath W [Physics Department, University of Connecticut, Storrs, CT 06269 (United States); Wijewardhana, L C Rohana [Physics Department, University of Cincinnati, Cincinnati, OH 45221 (United States)

2008-03-12

Surface stress induced by molecular adsorption in three different binding processes has been studied experimentally using a microcantilever sensor. A comprehensive free-energy analysis based on an energy conservation approach is proposed to explain the experimental observations. We show that when guest molecules bind to atoms/molecules on a microcantilever surface, the released binding energy is retained in the host surface, leading to a metastable state where the excess energy on the surface is manifested as an increase in surface stress leading to the bending of the microcantilever. The released binding energy appears to be almost exclusively channeled to the surface energy, and energy distribution to other channels, including heat, appears to be inactive for this micromechanical system. When this excess surface energy is released, the microcantilever relaxes back to the original state, and the relaxation time depends on the particular binding process involved. Such vapor phase experiments were conducted for three binding processes: physisorption, hydrogen bonding, and chemisorption. Binding energies for these three processes were also estimated.

Mediator binds to boundaries of chromosomal interaction domains and to proteins involved in DNA looping, RNA metabolism, chromatin remodeling, and actin assembly.

Science.gov (United States)

Chereji, Razvan V; Bharatula, Vasudha; Elfving, Nils; Blomberg, Jeanette; Larsson, Miriam; Morozov, Alexandre V; Broach, James R; Björklund, Stefan

2017-09-06

Mediator is a multi-unit molecular complex that plays a key role in transferring signals from transcriptional regulators to RNA polymerase II in eukaryotes. We have combined biochemical purification of the Saccharomyces cerevisiae Mediator from chromatin with chromatin immunoprecipitation in order to reveal Mediator occupancy on DNA genome-wide, and to identify proteins interacting specifically with Mediator on the chromatin template. Tandem mass spectrometry of proteins in immunoprecipitates of mediator complexes revealed specific interactions between Mediator and the RSC, Arp2/Arp3, CPF, CF 1A and Lsm complexes in chromatin. These factors are primarily involved in chromatin remodeling, actin assembly, mRNA 3'-end processing, gene looping and mRNA decay, but they have also been shown to enter the nucleus and participate in Pol II transcription. Moreover, we have found that Mediator, in addition to binding Pol II promoters, occupies chromosomal interacting domain (CID) boundaries and that Mediator in chromatin associates with proteins that have been shown to interact with CID boundaries, such as Sth1, Ssu72 and histone H4. This suggests that Mediator plays a significant role in higher-order genome organization. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Multidrug ATP-binding cassette transporters are essential for hepatic development of Plasmodium sporozoites

NARCIS (Netherlands)

Rijpma, S.R.; Velden, M. van der; Gonzalez-Pons, M.; Annoura, T.; Schaijk, B.C.L. van; Gemert, G.J.A. van; Heuvel, J.M.W. van den; Ramesar, J.; Chevalley-Maurel, S.; Ploemen, I.H.; Khan, S.M.; Franetich, J.F.; Mazier, D.; Wilt, J.H.W. de; Serrano, A.E.; Russel, F.G.; Janse, C.J.; Sauerwein, R.W.; Koenderink, J.B.; Franke-Fayard, B.M.

2016-01-01

Multidrug resistance-associated proteins (MRPs) belong to the C-family of ATP-binding cassette (ABC) transport proteins and are known to transport a variety of physiologically important compounds and to be involved in the extrusion of pharmaceuticals. Rodent malaria parasites encode a single ABC

A CESA from Griffithsia monilis (Rhodophyta, Florideophyceae) has a family 48 carbohydrate-binding module.

Science.gov (United States)

Matthews, Peter R; Schindler, Michael; Howles, Paul; Arioli, Tony; Williamson, Richard E

2010-10-01

Cellulose synthases form rosette terminal complexes in the plasma membranes of Streptophyta and various linear terminal complexes in other taxa. The sequence of a putative CESA from Griffithsia monilis (Rhodophyta, Floridiophyceae) was deduced using a cloning strategy involving degenerate primers, a cDNA library screen, and 5' and 3' rapid amplification of cDNA ends (RACE). RACE identified two alternative transcriptional starts and four alternative polyadenylation sites. The first translation start codon provided an open reading frame of 2610 bp encoding 870 amino acids and was PCR amplified without introns from genomic DNA. Southern hybridization indicated one strongly hybridizing gene with possible weakly related genes or pseudogenes. Amino acid sequence analysis identified a family 48 carbohydrate-binding module (CBM) upstream of the protein's first predicted transmembrane domain. There are broad similarities in predicted 3D structures of the family 48 modules from CESA, from several glycogen- and starch-binding enzymes, and from protein kinases, but there are substitutions at some residues thought to be involved in ligand binding. The module in G. monilis CESA will be on the cytoplasmic face of the plasma membrane so that it could potentially bind either low molecular weight ligands or starch which is cytosolic rather than inside membrane-bound plastids in red algae. Possible reasons why red algal CESAs have evolved family 48 modules perhaps as part of a system to regulate cellulose synthase activity in relation to cellular carbohydrate status are briefly discussed.

A CESA from Griffithsia monilis (Rhodophyta, Florideophyceae) has a family 48 carbohydrate-binding module

Science.gov (United States)

Matthews, Peter R.; Schindler, Michael; Howles, Paul; Arioli, Tony; Williamson, Richard E.

2010-01-01

Cellulose synthases form rosette terminal complexes in the plasma membranes of Streptophyta and various linear terminal complexes in other taxa. The sequence of a putative CESA from Griffithsia monilis (Rhodophyta, Floridiophyceae) was deduced using a cloning strategy involving degenerate primers, a cDNA library screen, and 5′ and 3′ rapid amplification of cDNA ends (RACE). RACE identified two alternative transcriptional starts and four alternative polyadenylation sites. The first translation start codon provided an open reading frame of 2610 bp encoding 870 amino acids and was PCR amplified without introns from genomic DNA. Southern hybridization indicated one strongly hybridizing gene with possible weakly related genes or pseudogenes. Amino acid sequence analysis identified a family 48 carbohydrate-binding module (CBM) upstream of the protein's first predicted transmembrane domain. There are broad similarities in predicted 3D structures of the family 48 modules from CESA, from several glycogen- and starch-binding enzymes, and from protein kinases, but there are substitutions at some residues thought to be involved in ligand binding. The module in G. monilis CESA will be on the cytoplasmic face of the plasma membrane so that it could potentially bind either low molecular weight ligands or starch which is cytosolic rather than inside membrane-bound plastids in red algae. Possible reasons why red algal CESAs have evolved family 48 modules perhaps as part of a system to regulate cellulose synthase activity in relation to cellular carbohydrate status are briefly discussed. PMID:20702566

Chronic cobalt-induced epilepsy: noradrenaline ionophoresis and adrenoceptor binding studies in the rat cerebral cortex

International Nuclear Information System (INIS)

Bregman, B.; Le Saux, F.; Maurin, Y.; Trottier, S.; Chauvel, P.

1985-01-01

Several studies indicate that brain noradrenaline (NA) depletion facilitates the occurrence of epileptogenic syndromes in various animal models. In cobalt-induced epilepsy in the rat, seizure activity is associated with a cortical NA denervation. In order to search for cortical adrenoceptor modifications, inonophoretic studies and adrenoceptor binding assays were performed. At the period of maximal seizure activity, there was a significant supersensitivity of cortial neurons to the ionophoretic application of NA. An increase in the density of β-adrenoceptor binding sites was observed. No modification in α 1 - and α 2 -adrenoceptor binding sites was found. This suggests that in cobalt-induced epilepsy there is a denervation supersensitivity which rests on a selective involvement of β-adrenoceptors. (Author)

Recent improvements to Binding MOAD: a resource for protein–ligand binding affinities and structures

Science.gov (United States)

Ahmed, Aqeel; Smith, Richard D.; Clark, Jordan J.; Dunbar, James B.; Carlson, Heather A.

2015-01-01

For over 10 years, Binding MOAD (Mother of All Databases; http://www.BindingMOAD.org) has been one of the largest resources for high-quality protein–ligand complexes and associated binding affinity data. Binding MOAD has grown at the rate of 1994 complexes per year, on average. Currently, it contains 23 269 complexes and 8156 binding affinities. Our annual updates curate the data using a semi-automated literature search of the references cited within the PDB file, and we have recently upgraded our website and added new features and functionalities to better serve Binding MOAD users. In order to eliminate the legacy application server of the old platform and to accommodate new changes, the website has been completely rewritten in the LAMP (Linux, Apache, MySQL and PHP) environment. The improved user interface incorporates current third-party plugins for better visualization of protein and ligand molecules, and it provides features like sorting, filtering and filtered downloads. In addition to the field-based searching, Binding MOAD now can be searched by structural queries based on the ligand. In order to remove redundancy, Binding MOAD records are clustered in different families based on 90% sequence identity. The new Binding MOAD, with the upgraded platform, features and functionalities, is now equipped to better serve its users. PMID:25378330

Synthetic LPS-Binding Polymer Nanoparticles

Science.gov (United States)

Jiang, Tian

Lipopolysaccharide (LPS), one of the principal components of most gram-negative bacteria's outer membrane, is a type of contaminant that can be frequently found in recombinant DNA products. Because of its strong and even lethal biological effects, selective LPS removal from bioproducts solution is of particular importance in the pharmaceutical and health care industries. In this thesis, for the first time, a proof-of-concept study on preparing LPS-binding hydrogel-like NPs through facile one-step free-radical polymerization was presented. With the incorporation of various hydrophobic (TBAm), cationic (APM, GUA) monomers and cross-linkers (BIS, PEG), a small library of NPs was constructed. Their FITC-LPS binding behaviors were investigated and compared with those of commercially available LPS-binding products. Moreover, the LPS binding selectivity of the NPs was also explored by studying the NPs-BSA interactions. The results showed that all NPs obtained generally presented higher FITC-LPS binding capacity in lower ionic strength buffer than higher ionic strength. However, unlike commercial poly-lysine cellulose and polymyxin B agarose beads' nearly linear increase of FITC-LPS binding with particle concentration, NPs exhibited serious aggregation and the binding quickly saturated or even decreased at high particle concentration. Among various types of NPs, higher FITC-LPS binding capacity was observed for those containing more hydrophobic monomers (TBAm). However, surprisingly, more cationic NPs with higher content of APM exhibited decreased FITC-LPS binding in high ionic strength conditions. Additionally, when new cationic monomer and cross-linker, GUA and PEG, were applied to replace APM and BIS, the obtained NPs showed improved FITC-LPS binding capacity at low NP concentration. But compared with APM- and BIS-containing NPs, the FITC-LPS binding capacity of GUA- and PEG-containing NPs saturated earlier. To investigate the NPs' binding to proteins, we tested the NPs

RBPmap: a web server for mapping binding sites of RNA-binding proteins.

Science.gov (United States)

Paz, Inbal; Kosti, Idit; Ares, Manuel; Cline, Melissa; Mandel-Gutfreund, Yael

2014-07-01

Regulation of gene expression is executed in many cases by RNA-binding proteins (RBPs) that bind to mRNAs as well as to non-coding RNAs. RBPs recognize their RNA target via specific binding sites on the RNA. Predicting the binding sites of RBPs is known to be a major challenge. We present a new webserver, RBPmap, freely accessible through the website http://rbpmap.technion.ac.il/ for accurate prediction and mapping of RBP binding sites. RBPmap has been developed specifically for mapping RBPs in human, mouse and Drosophila melanogaster genomes, though it supports other organisms too. RBPmap enables the users to select motifs from a large database of experimentally defined motifs. In addition, users can provide any motif of interest, given as either a consensus or a PSSM. The algorithm for mapping the motifs is based on a Weighted-Rank approach, which considers the clustering propensity of the binding sites and the overall tendency of regulatory regions to be conserved. In addition, RBPmap incorporates a position-specific background model, designed uniquely for different genomic regions, such as splice sites, 5' and 3' UTRs, non-coding RNA and intergenic regions. RBPmap was tested on high-throughput RNA-binding experiments and was proved to be highly accurate. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Insulin-like growth factors and insulin-like growth factor binding proteins in mammary gland function

International Nuclear Information System (INIS)

Marshman, Emma; Streuli, Charles H

2002-01-01

Insulin-like growth factor (IGF)-mediated proliferation and survival are essential for normal development in the mammary gland during puberty and pregnancy. IGFs interact with IGF-binding proteins and regulate their function. The present review focuses on the role of IGFs and IGF-binding proteins in the mammary gland and describes how modulation of their actions occurs by association with hormones, other growth factors and the extracellular matrix. The review will also highlight the involvement of the IGF axis in breast cancer

Computer modelling reveals new conformers of the ATP binding loop of Na+/K+-ATPase involved in the transphosphorylation process of the sodium pump.

Science.gov (United States)

Tejral, Gracian; Sopko, Bruno; Necas, Alois; Schoner, Wilhelm; Amler, Evzen

2017-01-01

Hydrolysis of ATP by Na + /K + -ATPase, a P-Type ATPase, catalyzing active Na + and K + transport through cellular membranes leads transiently to a phosphorylation of its catalytical α -subunit. Surprisingly, three-dimensional molecular structure analysis of P-type ATPases reveals that binding of ATP to the N-domain connected by a hinge to the P-domain is much too far away from the Asp 369 to allow the transfer of ATP's terminal phosphate to its aspartyl-phosphorylation site. In order to get information for how the transfer of the γ -phosphate group of ATP to the Asp 369 is achieved, analogous molecular modeling of the M 4 -M 5 loop of ATPase was performed using the crystal data of Na + /K + -ATPase of different species. Analogous molecular modeling of the cytoplasmic loop between Thr 338 and Ile 760 of the α 2 -subunit of Na + /K + -ATPase and the analysis of distances between the ATP binding site and phosphorylation site revealed the existence of two ATP binding sites in the open conformation; the first one close to Phe 475 in the N-domain, the other one close to Asp 369 in the P-domain. However, binding of Mg 2+ •ATP to any of these sites in the "open conformation" may not lead to phosphorylation of Asp 369 . Additional conformations of the cytoplasmic loop were found wobbling between "open conformation"  "semi-open conformation  "closed conformation" in the absence of 2Mg 2+ •ATP. The cytoplasmic loop's conformational change to the "semi-open conformation"-characterized by a hydrogen bond between Arg 543 and Asp 611 -triggers by binding of 2Mg 2+ •ATP to a single ATP site and conversion to the "closed conformation" the phosphorylation of Asp 369 in the P-domain, and hence the start of Na + /K + -activated ATP hydrolysis.

Trimeric autotransporter DsrA is a major mediator of fibrinogen binding in Haemophilus ducreyi.

Science.gov (United States)

Fusco, William G; Elkins, Christopher; Leduc, Isabelle

2013-12-01

Haemophilus ducreyi is the etiologic agent of the sexually transmitted genital ulcer disease chancroid. In both natural and experimental chancroid, H. ducreyi colocalizes with fibrin at the base of the ulcer. Fibrin is obtained by cleavage of the serum glycoprotein fibrinogen (Fg) by thrombin to initiate formation of the blood clot. Fg binding proteins are critical virulence factors in medically important Gram-positive bacteria. H. ducreyi has previously been shown to bind Fg in an agglutination assay, and the H. ducreyi Fg binding protein FgbA was identified in ligand blotting with denatured proteins. To better characterize the interaction of H. ducreyi with Fg, we examined Fg binding to intact, viable H. ducreyi bacteria and identified a novel Fg binding protein. H. ducreyi bound unlabeled Fg in a dose-dependent manner, as measured by two different methods. In ligand blotting with total denatured cellular proteins, digoxigenin (DIG)-Fg bound only two H. ducreyi proteins, the trimeric autotransporter DsrA and the lectin DltA; however, only the isogenic dsrA mutant had significantly less cell-associated Fg than parental strains in Fg binding assays with intact bacteria. Furthermore, expression of DsrA, but not DltA or an empty vector, rendered the non-Fg-binding H. influenzae strain Rd capable of binding Fg. A 13-amino-acid sequence in the C-terminal section of the passenger domain of DsrA appears to be involved in Fg binding by H. ducreyi. Taken together, these data suggest that the trimeric autotransporter DsrA is a major determinant of Fg binding at the surface of H. ducreyi.

Structural insights into Cydia pomonella pheromone binding protein 2 mediated prediction of potentially active semiochemicals

Science.gov (United States)

Tian, Zhen; Liu, Jiyuan; Zhang, Yalin

2016-03-01

Given the advantages of behavioral disruption application in pest control and the damage of Cydia pomonella, due progresses have not been made in searching active semiochemicals for codling moth. In this research, 31 candidate semiochemicals were ranked for their binding potential to Cydia pomonella pheromone binding protein 2 (CpomPBP2) by simulated docking, and this sorted result was confirmed by competitive binding assay. This high predicting accuracy of virtual screening led to the construction of a rapid and viable method for semiochemicals searching. By reference to binding mode analyses, hydrogen bond and hydrophobic interaction were suggested to be two key factors in determining ligand affinity, so is the length of molecule chain. So it is concluded that semiochemicals of appropriate chain length with hydroxyl group or carbonyl group at one head tended to be favored by CpomPBP2. Residues involved in binding with each ligand were pointed out as well, which were verified by computational alanine scanning mutagenesis. Progress made in the present study helps establish an efficient method for predicting potentially active compounds and prepares for the application of high-throughput virtual screening in searching semiochemicals by taking insights into binding mode analyses.

The structure of classical swine fever virus N(pro: a novel cysteine Autoprotease and zinc-binding protein involved in subversion of type I interferon induction.

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Keerthi Gottipati

Full Text Available Pestiviruses express their genome as a single polypeptide that is subsequently cleaved into individual proteins by host- and virus-encoded proteases. The pestivirus N-terminal protease (N(pro is a cysteine autoprotease that cleaves between its own C-terminus and the N-terminus of the core protein. Due to its unique sequence and catalytic site, it forms its own cysteine protease family C53. After self-cleavage, N(pro is no longer active as a protease. The released N(pro suppresses the induction of the host's type-I interferon-α/β (IFN-α/β response. N(pro binds interferon regulatory factor-3 (IRF3, the key transcriptional activator of IFN-α/β genes, and promotes degradation of IRF3 by the proteasome, thus preventing induction of the IFN-α/β response to pestivirus infection. Here we report the crystal structures of pestivirus N(pro. N(pro is structurally distinct from other known cysteine proteases and has a novel "clam shell" fold consisting of a protease domain and a zinc-binding domain. The unique fold of N(pro allows auto-catalysis at its C-terminus and subsequently conceals the cleavage site in the active site of the protease. Although many viruses interfere with type I IFN induction by targeting the IRF3 pathway, little information is available regarding structure or mechanism of action of viral proteins that interact with IRF3. The distribution of amino acids on the surface of N(pro involved in targeting IRF3 for proteasomal degradation provides insight into the nature of N(pro's interaction with IRF3. The structures thus establish the mechanism of auto-catalysis and subsequent auto-inhibition of trans-activity of N(pro, and its role in subversion of host immune response.

Visual short-term memory binding deficit in familial Alzheimer's disease.

Science.gov (United States)

Liang, Yuying; Pertzov, Yoni; Nicholas, Jennifer M; Henley, Susie M D; Crutch, Sebastian; Woodward, Felix; Leung, Kelvin; Fox, Nick C; Husain, Masud

2016-05-01

Long-term episodic memory deficits in Alzheimer's disease (AD) are well characterised but, until recently, short-term memory (STM) function has attracted far less attention. We employed a recently-developed, delayed reproduction task which requires participants to reproduce precisely the remembered location of items they had seen only seconds previously. This paradigm provides not only a continuous measure of localization error in memory, but also an index of relational binding by determining the frequency with which an object is misplaced to the location of one of the other items held in memory. Such binding errors in STM have previously been found on this task to be sensitive to medial temporal lobe (MTL) damage in focal lesion cases. Twenty individuals with pathological mutations in presenilin 1 or amyloid precursor protein genes for familial Alzheimer's disease (FAD) were tested together with 62 healthy controls. Participants were assessed using the delayed reproduction memory task, a standard neuropsychological battery and structural MRI. Overall, FAD mutation carriers were worse than controls for object identity as well as in gross localization memory performance. Moreover, they showed greater misbinding of object identity and location than healthy controls. Thus they would often mislocalize a correctly-identified item to the location of one of the other items held in memory. Significantly, asymptomatic gene carriers - who performed similarly to healthy controls on standard neuropsychological tests - had a specific impairment in object-location binding, despite intact memory for object identity and location. Consistent with the hypothesis that the hippocampus is critically involved in relational binding regardless of memory duration, decreased hippocampal volume across FAD participants was significantly associated with deficits in object-location binding but not with recall precision for object identity or localization. Object-location binding may therefore

Functional consequences of piceatannol binding to glyceraldehyde-3-phosphate dehydrogenase.

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Joanna Gerszon

Full Text Available Glyceraldehyde-3-phosphate dehydrogenase (GAPDH is one of the key redox-sensitive proteins whose activity is largely affected by oxidative modifications at its highly reactive cysteine residue in the enzyme's active site (Cys149. Prolonged exposure to oxidative stress may cause, inter alia, the formation of intermolecular disulfide bonds leading to accumulation of GAPDH aggregates and ultimately to cell death. Recently these anomalies have been linked with the pathogenesis of Alzheimer's disease. Novel evidences indicate that low molecular compounds may be effective inhibitors potentially preventing the GAPDH translocation to the nucleus, and inhibiting or slowing down its aggregation and oligomerization. Therefore, we decided to establish the ability of naturally occurring compound, piceatannol, to interact with GAPDH and to reveal its effect on functional properties and selected parameters of the dehydrogenase structure. The obtained data revealed that piceatannol binds to GAPDH. The ITC analysis indicated that one molecule of the tetrameric enzyme may bind up to 8 molecules of polyphenol (7.3 ± 0.9. Potential binding sites of piceatannol to the GAPDH molecule were analyzed using the Ligand Fit algorithm. Conducted analysis detected 11 ligand binding positions. We indicated that piceatannol decreases GAPDH activity. Detailed analysis allowed us to presume that this effect is due to piceatannol ability to assemble a covalent binding with nucleophilic cysteine residue (Cys149 which is directly involved in the catalytic reaction. Consequently, our studies strongly indicate that piceatannol would be an exceptional inhibitor thanks to its ability to break the aforementioned pathologic disulfide linkage, and therefore to inhibit GAPDH aggregation. We demonstrated that by binding with GAPDH piceatannol blocks cysteine residue and counteracts its oxidative modifications, that induce oligomerization and GAPDH aggregation.

CopM is a novel copper-binding protein involved in copper resistance in Synechocystis sp. PCC 6803

Science.gov (United States)

Giner-Lamia, Joaquín; López-Maury, Luis; Florencio, Francisco J

2015-01-01

Copper resistance system in the cyanobacterium Synechocystis sp. PCC 6803 comprises two operons, copMRS and copBAC, which are expressed in response to copper in the media. copBAC codes for a heavy-metal efflux–resistance nodulation and division (HME-RND) system, while copMRS codes for a protein of unknown function, CopM, and a two-component system CopRS, which controls the expression of these two operons. Here, we report that CopM is a periplasmic protein able to bind Cu(I) with high affinity (KD ∼3 × 10−16). Mutants lacking copM showed a sensitive copper phenotype similar to mutants affected in copB, but lower than mutants of the two-component system CopRS, suggesting that CopBAC and CopM constitute two independent resistance mechanisms. Moreover, constitutive expression of copM is able to partially suppress the copper sensitivity of the copR mutant strain, pointing out that CopM per se is able to confer copper resistance. Furthermore, constitutive expression of copM was able to reduce total cellular copper content of the copR mutant to the levels determined in the wild-type (WT) strain. Finally, CopM was localized not only in the periplasm but also in the extracellular space, suggesting that CopM can also prevent copper accumulation probably by direct copper binding outside the cell. PMID:25545960

Temporal mapping of CEBPA and CEBPB binding during liver regeneration reveals dynamic occupancy and specific regulatory codes for homeostatic and cell cycle gene batteries

DEFF Research Database (Denmark)

Jakobsen, Janus Schou; Waage, Johannes; Rapin, Nicolas

2013-01-01

quantified the genome-wide binding patterns of two key hepatocyte transcription factors, CEBPA and CEBPB (also known as C/EBPalpha and C/EBPbeta), at multiple time points during the highly dynamic process of liver regeneration elicited by partial hepatectomy in mouse. Combining these profiles with RNA...... polymerase II binding data, we find three temporal classes of transcription factor binding to be associated with distinct sets of regulated genes involved in the acute phase response, metabolic/homeostatic functions, or cell cycle progression. Moreover, we demonstrate a previously unrecognized early phase......IP experiments involving a panel of central transcription factors and/or by comparison to external ChIP-seq data. Our quantitative investigation not only provides in vivo evidence for the involvement of many new factors in liver regeneration but also points to similarities in the circuitries regulating self...

Recent improvements to Binding MOAD: a resource for protein-ligand binding affinities and structures.

Science.gov (United States)

Ahmed, Aqeel; Smith, Richard D; Clark, Jordan J; Dunbar, James B; Carlson, Heather A

2015-01-01

For over 10 years, Binding MOAD (Mother of All Databases; http://www.BindingMOAD.org) has been one of the largest resources for high-quality protein-ligand complexes and associated binding affinity data. Binding MOAD has grown at the rate of 1994 complexes per year, on average. Currently, it contains 23,269 complexes and 8156 binding affinities. Our annual updates curate the data using a semi-automated literature search of the references cited within the PDB file, and we have recently upgraded our website and added new features and functionalities to better serve Binding MOAD users. In order to eliminate the legacy application server of the old platform and to accommodate new changes, the website has been completely rewritten in the LAMP (Linux, Apache, MySQL and PHP) environment. The improved user interface incorporates current third-party plugins for better visualization of protein and ligand molecules, and it provides features like sorting, filtering and filtered downloads. In addition to the field-based searching, Binding MOAD now can be searched by structural queries based on the ligand. In order to remove redundancy, Binding MOAD records are clustered in different families based on 90% sequence identity. The new Binding MOAD, with the upgraded platform, features and functionalities, is now equipped to better serve its users. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

The actin binding cytoskeletal protein Moesin is involved in nuclear mRNA export.

Science.gov (United States)

Kristó, Ildikó; Bajusz, Csaba; Borsos, Barbara N; Pankotai, Tibor; Dopie, Joseph; Jankovics, Ferenc; Vartiainen, Maria K; Erdélyi, Miklós; Vilmos, Péter

2017-10-01

Current models imply that the evolutionarily conserved, actin-binding Ezrin-Radixin-Moesin (ERM) proteins perform their activities at the plasma membrane by anchoring membrane proteins to the cortical actin network. Here we show that beside its cytoplasmic functions, the single ERM protein of Drosophila, Moesin, has a novel role in the nucleus. The activation of transcription by heat shock or hormonal treatment increases the amount of nuclear Moesin, indicating biological function for the protein in the nucleus. The distribution of Moesin in the nucleus suggests a function in transcription and the depletion of mRNA export factors Nup98 or its interacting partner, Rae1, leads to the nuclear accumulation of Moesin, suggesting that the nuclear function of the protein is linked to mRNA export. Moesin localizes to mRNP particles through the interaction with the mRNA export factor PCID2 and knock down of Moesin leads to the accumulation of mRNA in the nucleus. Based on our results we propose that, beyond its well-known, manifold functions in the cytoplasm, the ERM protein of Drosophila is a new, functional component of the nucleus where it participates in mRNA export. Copyright © 2017 Elsevier B.V. All rights reserved.

Mycobacterium tuberculosis cAMP Receptor Protein (Rv3676) Differs from the Escherichia coli Paradigm in Its cAMP Binding and DNA Binding Properties and Transcription Activation Properties*

Science.gov (United States)

Stapleton, Melanie; Haq, Ihtshamul; Hunt, Debbie M.; Arnvig, Kristine B.; Artymiuk, Peter J.; Buxton, Roger S.; Green, Jeffrey

2010-01-01

The pathogen Mycobacterium tuberculosis produces a burst of cAMP upon infection of macrophages. Bacterial cyclic AMP receptor proteins (CRP) are transcription factors that respond to cAMP by binding at target promoters when cAMP concentrations increase. Rv3676 (CRPMt) is a CRP family protein that regulates expression of genes (rpfA and whiB1) that are potentially involved in M. tuberculosis persistence and/or emergence from the dormant state. Here, the CRPMt homodimer is shown to bind two molecules of cAMP (one per protomer) at noninteracting sites. Furthermore, cAMP binding by CRPMt was relatively weak, entropy driven, and resulted in a relatively small enhancement in DNA binding. Tandem CRPMt-binding sites (CRP1 at −58.5 and CRP2 at −37.5) were identified at the whiB1 promoter (PwhiB1). In vitro transcription reactions showed that CRP1 is an activating site and that CRP2, which was only occupied in the presence of cAMP or at high CRPMt concentrations in the absence of cAMP, is a repressing site. Binding of CRPMt to CRP1 was not essential for open complex formation but was required for transcription activation. Thus, these data suggest that binding of CRPMt to the PwhiB1 CRP1 site activates transcription at a step after open complex formation. In contrast, high cAMP concentrations allowed occupation of both CRP1 and CRP2 sites, resulting in inhibition of open complex formation. Thus, M. tuberculosis CRP has evolved several distinct characteristics, compared with the Escherichia coli CRP paradigm, to allow it to regulate gene expression against a background of high concentrations of cAMP. PMID:20028978

The transcriptional repressor DREAM is involved in thyroid gene expression

International Nuclear Information System (INIS)

D'Andrea, Barbara; Di Palma, Tina; Mascia, Anna; Motti, Maria Letizia; Viglietto, Giuseppe; Nitsch, Lucio; Zannini, Mariastella

2005-01-01

Downstream regulatory element antagonistic modulator (DREAM) was originally identified in neuroendocrine cells as a calcium-binding protein that specifically binds to downstream regulatory elements (DRE) on DNA, and represses transcription of its target genes. To explore the possibility that DREAM may regulate the endocrine activity of the thyroid gland, we analyzed its mRNA expression in undifferentiated and differentiated thyroid cells. We demonstrated that DREAM is expressed in the normal thyroid tissue as well as in differentiated thyroid cells in culture while it is absent in FRT poorly differentiated cells. In the present work, we also show that DREAM specifically binds to DRE sites identified in the 5' untranslated region (UTR) of the thyroid-specific transcription factors Pax8 and TTF-2/FoxE1 in a calcium-dependent manner. By gel retardation assays we demonstrated that thapsigargin treatment increases the binding of DREAM to the DRE sequences present in Pax8 and TTF-2/Foxe1 5' UTRs, and this correlates with a significant reduction of the expression of these genes. Interestingly, in poorly differentiated thyroid cells overexpression of exogenous DREAM strongly inhibits Pax8 expression. Moreover, we provide evidence that a mutated form of DREAM unable to bind Ca 2+ interferes with thyroid cell proliferation. Therefore, we propose that in thyroid cells DREAM is a mediator of the calcium-signaling pathway and it is involved in the regulation of thyroid cell function

The structure and binding mode of citrate in the stabilization of gold nanoparticles

KAUST Repository

Al-Johani, Hind; Abou-Hamad, Edy; Jedidi, Abdesslem; Widdifield, Cory M.; Viger-Gravel, Jasmine; Sangaru, Shiv; Gajan, David; Anjum, Dalaver H.; Ould-Chikh, Samy; Hedhili, Mohamed N.; Gurinov, Andrei; Kelly, Michael J.; El Eter, Mohamad; Cavallo, Luigi; Basset, Jean-Marie; Basset, Jean-Marie

2017-01-01

Elucidating the binding mode of carboxylate-containing ligands to gold nanoparticles (AuNPs) is crucial to understand their stabilizing role. A detailed picture of the three-dimensional structure and coordination modes of citrate, acetate, succinate and glutarate to AuNPs is obtained by 13C and 23Na solid-state NMR in combination with computational modelling and electron microscopy. The binding between the carboxylates and the AuNP surface is found to occur in three different modes. These three modes are simultaneously present at low citrate to gold ratios, while a monocarboxylate monodentate (1κO1) mode is favoured at high citrate:gold ratios. The surface AuNP atoms are found to be predominantly in the zero oxidation state after citrate coordination, although trace amounts of Auδ+ are observed. 23Na NMR experiments show that Na+ ions are present near the gold surface, indicating that carboxylate binding occurs as a 2e− L-type interaction for each oxygen atom involved. This approach has broad potential to probe the binding of a variety of ligands to metal nanoparticles.

The structure and binding mode of citrate in the stabilization of gold nanoparticles

KAUST Repository

Al-Johani, Hind

2017-03-27

Elucidating the binding mode of carboxylate-containing ligands to gold nanoparticles (AuNPs) is crucial to understand their stabilizing role. A detailed picture of the three-dimensional structure and coordination modes of citrate, acetate, succinate and glutarate to AuNPs is obtained by 13C and 23Na solid-state NMR in combination with computational modelling and electron microscopy. The binding between the carboxylates and the AuNP surface is found to occur in three different modes. These three modes are simultaneously present at low citrate to gold ratios, while a monocarboxylate monodentate (1κO1) mode is favoured at high citrate:gold ratios. The surface AuNP atoms are found to be predominantly in the zero oxidation state after citrate coordination, although trace amounts of Auδ+ are observed. 23Na NMR experiments show that Na+ ions are present near the gold surface, indicating that carboxylate binding occurs as a 2e− L-type interaction for each oxygen atom involved. This approach has broad potential to probe the binding of a variety of ligands to metal nanoparticles.

Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function

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St-Laurent Georges

2006-11-01

Full Text Available Abstract Background The HIV Rev protein is known to facilitate export of incompletely spliced and unspliced viral transcripts to the cytoplasm, a necessary step in virus life cycle. The Rev-mediated nucleo-cytoplasmic transport of nascent viral transcripts, dependents on interaction of Rev with the RRE RNA structural element present in the target RNAs. The C-terminal variant of dsRNA-binding nuclear protein 90 (NF90ctv has been shown to markedly attenuate viral replication in stably transduced HIV-1 target cell line. Here we examined a mechanism of interference of viral life cycle involving Rev-NF90ctv interaction. Results Since Rev:RRE complex formations depend on protein:RNA and protein:protein interactions, we investigated whether the expression of NF90ctv might interfere with Rev-mediated export of RRE-containing transcripts. When HeLa cells expressed both NF90ctv and Rev protein, we observed that NF90ctv inhibited the Rev-mediated RNA transport. In particular, three regions of NF90ctv protein are involved in blocking Rev function. Moreover, interaction of NF90ctv with the RRE RNA resulted in the expression of a reporter protein coding sequences linked to the RRE structure. Moreover, Rev influenced the subcellular localization of NF90ctv, and this process is leptomycin B sensitive. Conclusion The dsRNA binding protein, NF90ctv competes with HIV Rev function at two levels, by competitive protein:protein interaction involving Rev binding to specific domains of NF90ctv, as well as by its binding to the RRE-RNA structure. Our results are consistent with a model of Rev-mediated HIV-1 RNA export that envisions Rev-multimerization, a process interrupted by NF90ctv.

Opioid binding sites in the guinea pig and rat kidney: Radioligand homogenate binding and autoradiography

Energy Technology Data Exchange (ETDEWEB)

Dissanayake, V.U.; Hughes, J.; Hunter, J.C. (Parke-Davis Research Unit, Addenbrookes Hospital Site, Cambridge (England))

1991-07-01

The specific binding of the selective {mu}-, {delta}-, and {kappa}-opioid ligands (3H)(D-Ala2,MePhe4,Gly-ol5)enkephalin ((3H) DAGOL), (3H)(D-Pen2,D-Pen5)enkephalin ((3H)DPDPE), and (3H)U69593, respectively, to crude membranes of the guinea pig and rat whole kidney, kidney cortex, and kidney medulla was investigated. In addition, the distribution of specific 3H-opioid binding sites in the guinea pig and rat kidney was visualized by autoradiography. Homogenate binding and autoradiography demonstrated the absence of {mu}- and {kappa}-opioid binding sites in the guinea pig kidney. No opioid binding sites were demonstrable in the rat kidney. In the guinea pig whole kidney, cortex, and medulla, saturation studies demonstrated that (3H)DPDPE bound with high affinity (KD = 2.6-3.5 nM) to an apparently homogeneous population of binding sites (Bmax = 8.4-30 fmol/mg of protein). Competition studies using several opioid compounds confirmed the nature of the {delta}-opioid binding site. Autoradiography experiments demonstrated that specific (3H)DPDPE binding sites were distributed radially in regions of the inner and outer medulla and at the corticomedullary junction of the guinea pig kidney. Computer-assisted image analysis of saturation data yielded KD values (4.5-5.0 nM) that were in good agreement with those obtained from the homogenate binding studies. Further investigation of the {delta}-opioid binding site in medulla homogenates, using agonist ((3H)DPDPE) and antagonist ((3H)diprenorphine) binding in the presence of Na+, Mg2+, and nucleotides, suggested that the {delta}-opioid site is linked to a second messenger system via a GTP-binding protein. Further studies are required to establish the precise localization of the {delta} binding site in the guinea pig kidney and to determine the nature of the second messenger linked to the GTP-binding protein in the medulla.

The Complement Binding and Inhibitory Protein CbiA of Borrelia miyamotoi Degrades Extracellular Matrix Components by Interacting with Plasmin(ogen

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Ngoc T. T. Nguyen

2018-02-01

Full Text Available The emerging relapsing fever spirochete Borrelia (B. miyamotoi is transmitted by ixodid ticks and causes the so-called hard tick-borne relapsing fever or B. miyamotoi disease (BMD. More recently, we identified a surface-exposed molecule, CbiA exhibiting complement binding and inhibitory capacity and rendering spirochetes resistant to complement-mediated lysis. To gain deeper insight into the molecular principles of B. miyamotoi-host interaction, we examined CbiA as a plasmin(ogen receptor that enables B. miyamotoi to interact with the serine protease plasmin(ogen. Recombinant CbiA was able to bind plasminogen in a dose-dependent fashion. Moreover, lysine residues appear to play a crucial role in the protein-protein interaction as binding of plasminogen was inhibited by the lysine analog tranexamic acid as well as increasing ionic strength. Of relevance, plasminogen bound to CbiA can be converted by urokinase-type plasminogen activator (uPa to active plasmin which cleaved both, the chromogenic substrate S-2251 and its physiologic substrate fibrinogen. Concerning the involvement of specific amino acids in the interaction with plasminogen, lysine residues located at the C-terminus are frequently involved in the binding as reported for various other plasminogen-interacting proteins of Lyme disease spirochetes. Lysine residues located within the C-terminal domain were substituted with alanine to generate single, double, triple, and quadruple point mutants. However, binding of plasminogen to the mutated CbiA proteins was not affected, suggesting that lysine residues distant from the C-terminus might be involved in the interaction.

Human serum albumin unfolding pathway upon drug binding: A thermodynamic and spectroscopic description

International Nuclear Information System (INIS)

Cheema, Mohammad Arif; Taboada, Pablo; Barbosa, Silvia; Juarez, Josue; Gutierrez-Pichel, Manuel; Siddiq, Mohammad; Mosquera, Victor

2009-01-01

The interest on phenothiazine drugs has been increased during last years due to their proved utility in the treatment of several diseases and biomolecular processes. In the present work, the binding of the amphiphilic phenothiazines promazine and thioridazine hydrochlorides to the carrier protein human serum albumin (HSA) has been examined by ζ-potential, isothermal titration calorimetry (ITC), fluorescence and circular dichorism (CD) spectroscopies, and dynamic light scattering (DLS) at physiological pH with the aim of analyzing the role of the different interactions in the drug complexation process with this protein. The ζ-potential results were used to check the existence of complexation. This is confirmed by a progressive screening of the protein charge up to a reversal point as a consequence of drug binding. On the other hand, binding causes alterations on the tertiary and secondary structures of the protein, which were observed by fluorescence and CD spectroscopies, involving a two-step, three-state transition. The thermodynamics of the binding process was derived from ITC results. The binding enthalpies were negative, which reveal the existence of electrostatic interactions between protein and drug molecules. In addition, increases in entropy are consistent with the predominance of hydrophobic interactions. Two different classes of binding sites were detected, viz. Binding to the first class of binding sites is dominated by an enthalpic contribution due to electrostatic interactions whereas binding to a second class of binding sites is dominated by hydrophobic bonding. In the light of these results, protein conformational change resembles the acid-induced denaturation of HSA with accumulation of an intermediate state. Binding isotherms were derived from microcalorimetric results by using a theoretical model based on the Langmuir isotherm. On the other hand, the population distribution of the different species in solution and their sizes were determined

Human serum albumin unfolding pathway upon drug binding: A thermodynamic and spectroscopic description

Energy Technology Data Exchange (ETDEWEB)

Cheema, Mohammad Arif [Grupo de Fisica de Coloides y Polimeros, Departamento de Fisica de la Materia Condensada, Facultad de Fisica, Universidad de Santiago de Compostela, E-15782, Santiago de Compostela (Spain); Department of Chemistry, Quaid-i-Azam University, Islamabad 45320 (Pakistan); Taboada, Pablo [Grupo de Fisica de Coloides y Polimeros, Departamento de Fisica de la Materia Condensada, Facultad de Fisica, Universidad de Santiago de Compostela, E-15782, Santiago de Compostela (Spain); Department of Chemistry, Quaid-i-Azam University, Islamabad 45320 (Pakistan)], E-mail: pablo.taboada@usc.es; Barbosa, Silvia; Juarez, Josue; Gutierrez-Pichel, Manuel [Grupo de Fisica de Coloides y Polimeros, Departamento de Fisica de la Materia Condensada, Facultad de Fisica, Universidad de Santiago de Compostela, E-15782, Santiago de Compostela (Spain); Department of Chemistry, Quaid-i-Azam University, Islamabad 45320 (Pakistan); Siddiq, Mohammad [Department of Chemistry, Quaid-i-Azam University, Islamabad 45320 (Pakistan); Mosquera, Victor [Grupo de Fisica de Coloides y Polimeros, Departamento de Fisica de la Materia Condensada, Facultad de Fisica, Universidad de Santiago de Compostela, E-15782, Santiago de Compostela (Spain); Department of Chemistry, Quaid-i-Azam University, Islamabad 45320 (Pakistan)

2009-04-15

The interest on phenothiazine drugs has been increased during last years due to their proved utility in the treatment of several diseases and biomolecular processes. In the present work, the binding of the amphiphilic phenothiazines promazine and thioridazine hydrochlorides to the carrier protein human serum albumin (HSA) has been examined by {zeta}-potential, isothermal titration calorimetry (ITC), fluorescence and circular dichorism (CD) spectroscopies, and dynamic light scattering (DLS) at physiological pH with the aim of analyzing the role of the different interactions in the drug complexation process with this protein. The {zeta}-potential results were used to check the existence of complexation. This is confirmed by a progressive screening of the protein charge up to a reversal point as a consequence of drug binding. On the other hand, binding causes alterations on the tertiary and secondary structures of the protein, which were observed by fluorescence and CD spectroscopies, involving a two-step, three-state transition. The thermodynamics of the binding process was derived from ITC results. The binding enthalpies were negative, which reveal the existence of electrostatic interactions between protein and drug molecules. In addition, increases in entropy are consistent with the predominance of hydrophobic interactions. Two different classes of binding sites were detected, viz. Binding to the first class of binding sites is dominated by an enthalpic contribution due to electrostatic interactions whereas binding to a second class of binding sites is dominated by hydrophobic bonding. In the light of these results, protein conformational change resembles the acid-induced denaturation of HSA with accumulation of an intermediate state. Binding isotherms were derived from microcalorimetric results by using a theoretical model based on the Langmuir isotherm. On the other hand, the population distribution of the different species in solution and their sizes were

A Bifunctional Intronic Element Regulates the Expression of the Arginine/Lysine Transporter Cat-1 via Mechanisms Involving the Purine-rich Element Binding Protein A (Purα)*

Science.gov (United States)

Huang, Charlie C.; Chiribau, Calin-Bogdan; Majumder, Mithu; Chiang, Cheng-Ming; Wek, Ronald C.; Kelm, Robert J.; Khalili, Kamel; Snider, Martin D.; Hatzoglou, Maria

2009-01-01

Expression of the arginine/lysine transporter Cat-1 is highly induced in proliferating and stressed cells via mechanisms that include transcriptional activation. A bifunctional INE (intronic element) within the first intron of the Cat-1 gene was identified and characterized in this study. The INE had high sequence homology to an amino acid response element and was shown to act as a transcriptional enhancer in unstressed cells by binding the transcription factor, purine-rich element binding protein A (Purα). During endoplasmic reticulum stress, binding of Purα to the INE decreased; the element acted as a positive regulator in early stress by binding of the transcription factor ATF4 and as a negative regulator in prolonged stress by binding the stress-induced C/EBP family member, CHOP. We conclude that transcriptional control of the Cat-1 gene is tightly controlled by multiple cis-DNA elements, contributing to regulation of cationic amino acid transport for cell growth and proliferation. In addition, we propose that genes may use stress-response elements such as the INE to support basal expression in the absence of stress. PMID:19720825

De novo design of RNA-binding proteins with a prion-like domain related to ALS/FTD proteinopathies.

Science.gov (United States)

Mitsuhashi, Kana; Ito, Daisuke; Mashima, Kyoko; Oyama, Munenori; Takahashi, Shinichi; Suzuki, Norihiro

2017-12-04

Aberrant RNA-binding proteins form the core of the neurodegeneration cascade in spectrums of disease, such as amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Six ALS-related molecules, TDP-43, FUS, TAF15, EWSR1, heterogeneous nuclear (hn)RNPA1 and hnRNPA2 are RNA-binding proteins containing candidate mutations identified in ALS patients and those share several common features, including harboring an aggregation-prone prion-like domain (PrLD) containing a glycine/serine-tyrosine-glycine/serine (G/S-Y-G/S)-motif-enriched low-complexity sequence and rich in glutamine and/or asparagine. Additinally, these six molecules are components of RNA granules involved in RNA quality control and become mislocated from the nucleus to form cytoplasmic inclusion bodies (IBs) in the ALS/FTD-affected brain. To reveal the essential mechanisms involved in ALS/FTD-related cytotoxicity associated with RNA-binding proteins containing PrLDs, we designed artificial RNA-binding proteins harboring G/S-Y-G/S-motif repeats with and without enriched glutamine residues and nuclear-import/export-signal sequences and examined their cytotoxicity in vitro. These proteins recapitulated features of ALS-linked molecules, including insoluble aggregation, formation of cytoplasmic IBs and components of RNA granules, and cytotoxicity instigation. These findings indicated that these artificial RNA-binding proteins mimicked features of ALS-linked molecules and allowed the study of mechanisms associated with gain of toxic functions related to ALS/FTD pathogenesis.

Structure of the C-terminal effector-binding domain of AhrC bound to its corepressor l-arginine

International Nuclear Information System (INIS)

Garnett, James A.; Baumberg, Simon; Stockley, Peter G.; Phillips, Simon E. V.

2007-01-01

The crystal structure of the C-terminal domain hexameric core of AhrC, with bound corepressor (l-arginine), has been solved at 1.95 Å resolution. Binding of l-arginine results in a rotation between the two trimers of the hexamer, leading to the activation of the DNA-binding state. The arginine repressor/activator protein (AhrC) from Bacillus subtilis belongs to a large family of multifunctional transcription factors that are involved in the regulation of bacterial arginine metabolism. AhrC interacts with operator sites in the promoters of arginine biosynthetic and catabolic operons, acting as a transcriptional repressor at biosynthetic sites and an activator of transcription at catabolic sites. AhrC is a hexamer of identical subunits, each having two domains. The C-terminal domains form the core of the protein and are involved in oligomerization and l-arginine binding. The N-terminal domains lie on the outside of the compact core and play a role in binding to 18 bp DNA operators called ARG boxes. The C-terminal domain of AhrC has been expressed, purified and characterized, and also crystallized as a hexamer with the bound corepressor l-arginine. Here, the crystal structure refined to 1.95 Å is presented

Structural Modulation of Phosducin by Phosphorylation and 14-3-3 Protein Binding

Science.gov (United States)

Rezabkova, Lenka; Kacirova, Miroslava; Sulc, Miroslav; Herman, Petr; Vecer, Jaroslav; Stepanek, Miroslav; Obsilova, Veronika; Obsil, Tomas

2012-01-01

Phosducin (Pdc), a highly conserved phosphoprotein, plays an important role in the regulation of G protein signaling, transcriptional control, and modulation of blood pressure. Pdc is negatively regulated by phosphorylation followed by binding to the 14-3-3 protein, whose role is still unclear. To gain insight into the role of 14-3-3 in the regulation of Pdc function, we studied structural changes of Pdc induced by phosphorylation and 14-3-3 protein binding using time-resolved fluorescence spectroscopy. Our data show that the phosphorylation of the N-terminal domain of Pdc at Ser-54 and Ser-73 affects the structure of the whole Pdc molecule. Complex formation with 14-3-3 reduces the flexibility of both the N- and C-terminal domains of phosphorylated Pdc, as determined by time-resolved tryptophan and dansyl fluorescence. Therefore, our data suggest that phosphorylated Pdc undergoes a conformational change when binding to 14-3-3. These changes involve the Gtβγ binding surface within the N-terminal domain of Pdc, and thus could explain the inhibitory effect of 14-3-3 on Pdc function. PMID:23199924

How Cations Can Assist DNase I in DNA Binding and Hydrolysis