Voltage-sensing phosphatase modulation by a C2 domain

Directory of Open Access Journals (Sweden)

Paul M. Castle

2015-04-01

Full Text Available The voltage-sensing phosphatase (VSP is the first example of an enzyme controlled by changes in membrane potential. VSP has four distinct regions: the transmembrane voltage-sensing domain (VSD, the inter-domain linker, the cytosolic catalytic domain and the C2 domain. The VSD transmits the changes in membrane potential through the inter-domain linker activating the catalytic domain which then dephosphorylates phosphatidylinositol phosphate lipids. The role of the C2, however, has not been established. In this study, we explore two possible roles for the C2: catalysis and membrane-binding. The Ci-VSP crystal structures show that the C2 residue Y522 lines the active site suggesting a contribution to catalysis. When we mutated Y522 to phenylalanine, we found a shift in the voltage dependence of activity. This suggests hydrogen bonding as a mechanism of action. Going one step further, when we deleted the entire C2 domain, we found voltage-dependent enzyme activity was no longer detectable. This result clearly indicates the entire C2 is necessary for catalysis as well as for modulating activity. As C2s are known membrane-binding domains, we tested whether the VSP C2 interacts with the membrane. We probed a cluster of four positively charged residues lining the top of the C2 and suggested by previous studies to interact with phosphatidylinositol 4,5-bisphosphate (PI(4,5P2 (Kalli et al., 2014. Neutralizing those positive charges significantly shifted the voltage dependence of activity to higher voltages. We tested membrane binding by depleting PI(4,5P2 from the membrane using the 5HT2C receptor and found that the VSD motions as measured by voltage clamp fluorometry were not changed. These results suggest that if the C2 domain interacts with the membrane to influence VSP function it may not occur exclusively through PI(4,5P2. Together, this data advances our understanding of the VSP C2 by demonstrating a necessary and critical role for the C2 domain in

The JH2 domain and SH2-JH2 linker regulate JAK2 activity: A detailed kinetic analysis of wild type and V617F mutant kinase domains.

Science.gov (United States)

Sanz Sanz, Arturo; Niranjan, Yashavanthi; Hammarén, Henrik; Ungureanu, Daniela; Ruijtenbeek, Rob; Touw, Ivo P; Silvennoinen, Olli; Hilhorst, Riet

2014-10-01

JAK2 tyrosine kinase regulates many cellular functions. Its activity is controlled by the pseudokinase (JH2) domain by still poorly understood mechanisms. The V617F mutation in the pseudokinase domain activates JAK2 and causes myeloproliferative neoplasms. We conducted a detailed kinetic analysis of recombinant JAK2 tyrosine kinase domain (JH1) and wild-type and V617F tandem kinase (JH1JH2) domains using peptide microarrays to define the functions of the kinase domains. The results show that i) JAK2 follows a random Bi-Bi reaction mechanism ii) JH2 domain restrains the activity of the JH1 domain by reducing the affinity for ATP and ATP competitive inhibitors iii) V617F decreases affinity for ATP but increases catalytic activity compared to wild-type and iv) the SH2-JH2 linker region participates in controlling activity by reducing the affinity for ATP. Copyright © 2014 Elsevier B.V. All rights reserved.

Crystal structure of the G3BP2 NTF2-like domain in complex with a canonical FGDF motif peptide.

Science.gov (United States)

Kristensen, Ole

2015-11-06

The crystal structure of the NTF2-like domain of the human Ras GTPase SH3 Binding Protein (G3BP), isoform 2, was determined at a resolution of 2.75 Å in complex with a peptide containing a FGDF sequence motif. The overall structure of the protein is highly similar to the homodimeric N-terminal domains of the G3BP1 and Rasputin proteins. Recently, a subset of G3BP interacting proteins was recognized to share a common sequence motif, FGDF. The most studied binding partners, USP10 and viral nsP3, interfere with essential G3BP functions related to assembly of cellular stress granules. Reported molecular modeling suggested that FGDF-motif containing peptides bind in an extended conformation into a hydrophobic groove on the surface of the G3BP NTF2-like domain in a manner similar to the known binding of FxFG nucleoporin repeats. The results in this paper provide evidence for a different binding mode. The FGDF peptide binds and changes conformation of the protruding N-terminal residues by providing hydrophobic interactions to a symmetry related molecule that facilitated crystallization of the G3BP2 isoform. Copyright © 2015 Elsevier Inc. All rights reserved.

Molecular determinants of transport stimulation of EAAT2 are located at interface between the trimerization and substrate transport domains.

Science.gov (United States)

Mortensen, Ole V; Liberato, José L; Coutinho-Netto, Joaquim; Dos Santos, Wagner F; Fontana, Andréia C K

2015-04-01

Excitatory amino acid transporters (EAATs) regulate glutamatergic signal transmission by clearing extracellular glutamate. Dysfunction of these transporters has been implicated in the pathogenesis of various neurological disorders. Previous studies have shown that venom from the spider Parawixia bistriata and a purified compound (Parawixin1) stimulate EAAT2 activity and protect retinal tissue from ischemic damage. In the present study, the EAAT2 subtype specificity of this compound was explored, employing chimeric proteins between EAAT2 and EAAT3 transporter subtypes and mutants to characterize the structural region targeted by the compound. This identified a critical residue (Histidine-71 in EAAT2 and Serine-45 in EAAT3) in transmembrane domain 2 (TM2) to be important for the selectivity between EAAT2 and EAAT3 and for the activity of the venom. Using the identified residue in TM2 as a structural anchor, several neighboring amino acids within TM5 and TM8 were identified to also be important for the activity of the venom. This structural domain of the transporter lies at the interface of the rigid trimerization domain and the central substrate-binding transport domain. Our studies suggest that the mechanism of glutamate transport enhancement involves an interaction with the transporter that facilitates the movement of the transport domain. We identified a domain (purple star) in the glutamate transporter EAAT2 that is important for transport stimulation through a spider venom, and suggest a mechanism for enhanced transporter function through facilitated substrate translocation (arrow). Because the dysfunction of glutamate transporters is implicated in the pathogenesis of neurological disorders, understanding the mechanisms of enhanced transport could have therapeutic implications. © 2015 International Society for Neurochemistry.

Recombinant expression in E. coli of human FGFR2 with its transmembrane and extracellular domains

Directory of Open Access Journals (Sweden)

Adam Bajinting

2017-06-01

Full Text Available Fibroblast growth factor receptors (FGFRs are a family of receptor tyrosine kinases containing three domains: an extracellular receptor domain, a single transmembrane helix, and an intracellular tyrosine kinase domain. FGFRs are activated by fibroblast growth factors (FGFs as part of complex signal transduction cascades regulating angiogenesis, skeletal formation, cell differentiation, proliferation, cell survival, and cancer. We have developed the first recombinant expression system in E. coli to produce a construct of human FGFR2 containing its transmembrane and extracellular receptor domains. We demonstrate that the expressed construct is functional in binding heparin and dimerizing. Size exclusion chromatography demonstrates that the purified FGFR2 does not form a complex with FGF1 or adopts an inactive dimer conformation. Progress towards the successful recombinant production of intact FGFRs will facilitate further biochemical experiments and structure determination that will provide insight into how extracellular FGF binding activates intracellular kinase activity.

Using theory to explore facilitators and barriers to delayed prescribing in Australia: a qualitative study using the Theoretical Domains Framework and the Behaviour Change Wheel.

Science.gov (United States)

Sargent, Lucy; McCullough, Amanda; Del Mar, Chris; Lowe, John

2017-02-13

Delayed antibiotic prescribing reduces antibiotic use for acute respiratory infections in trials in general practice, but the uptake in clinical practice is low. The aim of the study was to identify facilitators and barriers to general practitioners' (GPs') use of delayed prescribing and to gain pharmacists' and the public's views about delayed prescribing in Australia. This study used the Theoretical Domains Framework and the Behaviour Change Wheel to explore facilitators and barriers to delayed prescribing in Australia. Forty-three semi-structured, face-to-face interviews with general practitioners, pharmacists and patients were conducted. Responses were coded into domains of the Theoretical Domains Framework, and specific criteria from the Behaviour Change Wheel were used to identify which domains were relevant to increasing the use of delayed prescribing by GPs. The interviews revealed nine key domains that influence GPs' use of delayed prescribing: knowledge; cognitive and interpersonal skills; memory, attention and decision-making processes; optimism; beliefs about consequences; intentions; goals; emotion; and social influences: GPs knew about delayed prescribing; however, they did not use it consistently, preferring to bring patients back for review and only using it with patients in a highly selective way. Pharmacists would support GPs and the public in delayed prescribing but would fill the prescription if people insisted. The public said they would delay taking their antibiotics if asked by their GP and given the right information on managing symptoms and when to take antibiotics. Using a theory-driven approach, we identified nine key domains that influence GPs' willingness to provide a delayed prescription to patients with an acute respiratory infection presenting to general practice. These data can be used to develop a structured intervention to change this behaviour and thus reduce antibiotic use for acute respiratory infections in general practice.

BPM-CUL3 E3 ligase modulates thermotolerance by facilitating negative regulatory domain-mediated degradation of DREB2A in Arabidopsis.

Science.gov (United States)

Morimoto, Kyoko; Ohama, Naohiko; Kidokoro, Satoshi; Mizoi, Junya; Takahashi, Fuminori; Todaka, Daisuke; Mogami, Junro; Sato, Hikaru; Qin, Feng; Kim, June-Sik; Fukao, Yoichiro; Fujiwara, Masayuki; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

2017-10-03

DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 2A (DREB2A) acts as a key transcription factor in both drought and heat stress tolerance in Arabidopsis and induces the expression of many drought- and heat stress-inducible genes. Although DREB2A expression itself is induced by stress, the posttranslational regulation of DREB2A, including protein stabilization, is required for its transcriptional activity. The deletion of a 30-aa central region of DREB2A known as the negative regulatory domain (NRD) transforms DREB2A into a stable and constitutively active form referred to as DREB2A CA. However, the molecular basis of this stabilization and activation has remained unknown for a decade. Here we identified BTB/POZ AND MATH DOMAIN proteins (BPMs), substrate adaptors of the Cullin3 (CUL3)-based E3 ligase, as DREB2A-interacting proteins. We observed that DREB2A and BPMs interact in the nuclei, and that the NRD of DREB2A is sufficient for its interaction with BPMs. BPM -knockdown plants exhibited increased DREB2A accumulation and induction of DREB2A target genes under heat and drought stress conditions. Genetic analysis indicated that the depletion of BPM expression conferred enhanced thermotolerance via DREB2A stabilization. Thus, the BPM-CUL3 E3 ligase is likely the long-sought factor responsible for NRD-dependent DREB2A degradation. Through the negative regulation of DREB2A stability, BPMs modulate the heat stress response and prevent an adverse effect of excess DREB2A on plant growth. Furthermore, we found the BPM recognition motif in various transcription factors, implying a general contribution of BPM-mediated proteolysis to divergent cellular responses via an accelerated turnover of transcription factors.

Functional analysis of the NH{sub 2}-terminal hydrophobic region and BRICHOS domain of GKN1

Energy Technology Data Exchange (ETDEWEB)

Yoon, Jung Hwan; Choi, Yoo Jin; Choi, Won Suk; Nam, Suk Woo; Lee, Jung Young; Park, Won Sang, E-mail: wonsang@catholic.ac.kr

2013-11-01

Highlights: •NH{sub 2}-terminal and BRICHOS domain of GKN1 inhibited tumor cell growth. •NH{sub 2}-terminal and BRICHOS domain of GKN1 regulated cell cycle. •NH{sub 2}-terminal and BRICHOS domain of GKN1 inhibited epigenetic regulators. -- Abstract: Gastrokine 1 (GKN1) protects the gastric antral mucosa and promotes healing by facilitating restitution and proliferation after injury. GKN1 is down-regulated in Helicobacter pylori-infected gastric epithelial cells and loss of GKN1 expression is tightly associated with gastric carcinogenesis. However, the underlying mechanisms as a tumor suppressor are largely unknown. Presently, the hydrophobic region and BRICHOS domain of GKN1, pGKN1{sup D13N}, pGKN1{sup Δ68–199}, and pGKN1{sup Δ1–67,165–199} were shown to suppress gastric cancer cell growth and recapitulate GKN1 functions. As well, the hydrophobic region and BRICHOS domain of GKN1 had a synergistic anti-cancer effect with 5-FU on tumor cell growth, implying that the NH{sub 2}-terminal hydrophobic region and BRICHOS domain of GKN1 are sufficient for tumor suppression, thereby suggesting a therapeutic intervention for gastric cancer. Also, its domain inducing endogenous miR-185 directly targeted the epigenetic effectors DNMT1 and EZH2 in gastric cancer cells. Our results suggest that the NH{sub 2}-terminal hydrophobic region and BRICHOS domain of GKN1 are sufficient for its tumor suppressor activities.

The SH2 Domain Interaction Landscape

Directory of Open Access Journals (Sweden)

Michele Tinti

2013-04-01

Full Text Available Members of the SH2 domain family modulate signal transduction by binding to short peptides containing phosphorylated tyrosines. Each domain displays a distinct preference for the sequence context of the phosphorylated residue. We have developed a high-density peptide chip technology that allows for probing of the affinity of most SH2 domains for a large fraction of the entire complement of tyrosine phosphopeptides in the human proteome. Using this technique, we have experimentally identified thousands of putative SH2-peptide interactions for more than 70 different SH2 domains. By integrating this rich data set with orthogonal context-specific information, we have assembled an SH2-mediated probabilistic interaction network, which we make available as a community resource in the PepspotDB database. A predicted dynamic interaction between the SH2 domains of the tyrosine phosphatase SHP2 and the phosphorylated tyrosine in the extracellular signal-regulated kinase activation loop was validated by experiments in living cells.

Cation diffusion facilitators transport initiation and regulation is mediated by cation induced conformational changes of the cytoplasmic domain.

Directory of Open Access Journals (Sweden)

Natalie Zeytuni

Full Text Available Cation diffusion facilitators (CDF are part of a highly conserved protein family that maintains cellular divalent cation homeostasis in all domains of life. CDF's were shown to be involved in several human diseases, such as Type-II diabetes and neurodegenerative diseases. In this work, we employed a multi-disciplinary approach to study the activation mechanism of the CDF protein family. For this we used MamM, one of the main ion transporters of magnetosomes--bacterial organelles that enable magnetotactic bacteria to orientate along geomagnetic fields. Our results reveal that the cytosolic domain of MamM forms a stable dimer that undergoes distinct conformational changes upon divalent cation binding. MamM conformational change is associated with three metal binding sites that were identified and characterized. Altogether, our results provide a novel auto-regulation mode of action model in which the cytosolic domain's conformational changes upon ligand binding allows the priming of the CDF into its transport mode.

SH2 Domain Histochemistry.

Science.gov (United States)

Buhs, Sophia; Nollau, Peter

2017-01-01

Among posttranslational modifications, the phosphorylation of tyrosine residues is a key modification in cell signaling. Because of its biological importance, characterization of the cellular state of tyrosine phosphorylation is of great interest. Based on the unique properties of endogenously expressed SH2 domains recognizing tyrosine phosphorylated signaling proteins with high specificity we have developed an alternative approach, coined SH2 profiling, enabling us to decipher complex patterns of tyrosine phosphorylation in various normal and cancerous tissues. So far, SH2 profiling has largely been applied for the analysis of protein extracts with the limitation that information on spatial distribution and intensity of tyrosine phosphorylation within a tissue is lost. Here, we describe a novel SH2 domain based strategy for differential characterization of the state of tyrosine phosphorylation in formaldehyde-fixed and paraffin-embedded tissues. This approach demonstrates that SH2 domains may serve as very valuable tools for the analysis of the differential state of tyrosine phosphorylation in primary tissues fixed and processed under conditions frequently applied by routine pathology laboratories.

Facilitating Inter-Domain Synergies in Ambient Assisted Living Environments.

Science.gov (United States)

Schwartze, Jonas; Schrom, Harald; Wolf, Klaus-Hendrik; Marschollek, Michael

2016-01-01

Current Ambient Assisted Living (AAL) environments lack integration of sensors and actuators of other sub-domains. Creating technical and organizational integration is addressed by the BASIS project (Build Automation by a Scalable and Intelligent System), which aims to build a cross-domain home bus system. The main objective of this paper is to present an overview of design, architecture and state of realization of BASIS by describing the requirements development process, underlying hardware design and software architecture. We built a distributed system of one independent building manager with several redundantly meshed segment controllers, each controlling a bus segment with any number of bus nodes. The software system layer is divided into logical partitions representing each sub-domain. Structured data storage is possible with a special FHIR based home centered data warehouse. The system has been implemented in six apartments running under daily living conditions. BASIS integrates a broad range of sub-domains, which poses challenges to all project partners in terms of a common terminology, and project management methods, but enables development of inter-domain synergies like using the same sensor and actuator hardware for a broad range of services and use cases.

The SH2 domain interaction landscape.

Science.gov (United States)

Tinti, Michele; Kiemer, Lars; Costa, Stefano; Miller, Martin L; Sacco, Francesca; Olsen, Jesper V; Carducci, Martina; Paoluzi, Serena; Langone, Francesca; Workman, Christopher T; Blom, Nikolaj; Machida, Kazuya; Thompson, Christopher M; Schutkowski, Mike; Brunak, Søren; Mann, Matthias; Mayer, Bruce J; Castagnoli, Luisa; Cesareni, Gianni

2013-04-25

Members of the SH2 domain family modulate signal transduction by binding to short peptides containing phosphorylated tyrosines. Each domain displays a distinct preference for the sequence context of the phosphorylated residue. We have developed a high-density peptide chip technology that allows for probing of the affinity of most SH2 domains for a large fraction of the entire complement of tyrosine phosphopeptides in the human proteome. Using this technique, we have experimentally identified thousands of putative SH2-peptide interactions for more than 70 different SH2 domains. By integrating this rich data set with orthogonal context-specific information, we have assembled an SH2-mediated probabilistic interaction network, which we make available as a community resource in the PepspotDB database. A predicted dynamic interaction between the SH2 domains of the tyrosine phosphatase SHP2 and the phosphorylated tyrosine in the extracellular signal-regulated kinase activation loop was validated by experiments in living cells. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Solution structure of the Grb2 SH2 domain complexed with a high-affinity inhibitor

International Nuclear Information System (INIS)

Ogura, Kenji; Shiga, Takanori; Yokochi, Masashi; Yuzawa, Satoru; Burke, Terrence R.; Inagaki, Fuyuhiko

2008-01-01

The solution structure of the growth factor receptor-bound protein 2 (Grb2) SH2 domain complexed with a high-affinity inhibitor containing a non-phosphorus phosphate mimetic within a macrocyclic platform was determined by nuclear magnetic resonance (NMR) spectroscopy. Unambiguous assignments of the bound inhibitor and intermolecular NOEs between the Grb2 SH2 domain and the inhibitor was accomplished using perdeuterated Grb2 SH2 protein. The well-defined solution structure of the complex was obtained and compared to those by X-ray crystallography. Since the crystal structure of the Grb2 SH2 domain formed a domain-swapped dimer and several inhibitors were bound to a hinge region, there were appreciable differences between the solution and crystal structures. Based on the binding interactions between the inhibitor and the Grb2 SH2 domain in solution, we proposed a design of second-generation inhibitors that could be expected to have higher affinity

Thickness-dependent a_1/a_2 domain evolution in ferroelectric PbTiO_3 films

International Nuclear Information System (INIS)

Li, S.; Zhu, Y.L.; Tang, Y.L.; Liu, Y.; Zhang, S.R.; Wang, Y.J.; Ma, X.L.

2017-01-01

Ferroelectric a_1/a_2 domain structure has great potentials in high dielectric capacitors and tunable microwave devices. Understanding its structure is crucial to better control the domain configurations for future applications. In this paper, PbTiO_3 thin films with variant thicknesses are deposited on (110)-oriented GdScO_3 substrates by Pulsed Laser Deposition (PLD) and investigated by using conventional transmission electron microscopy (TEM) and Cs-corrected Scanning TEM. Contrast analysis and electron diffractions reveal that PbTiO_3 films are domain oriented consisting of a_1/a_2 and a/c domain structure. The a_1/a_2 domains are found to distribute periodically and its width increases with increasing film thickness following square root rule. Cs-corrected STEM imaging demonstrates that the domain walls of a_1/a_2 domain structure have the rotation characteristic of 90° ferroelastic domain wall. The interchange of a_1/a_2 domains induces the formation of vertex domains composed of two 90° and one 180° domain walls. Strains are mainly concentrated on the domain walls. The formation of this complex domain configuration is discussed in terms of the effect of the misfit strain, film thickness and cooling rate. These results provide novel information about a_1/a_2 domain structures and are expected to shed some light on modulating a_1/a_2 ferroelectric domain patterns in the design of ferroelectric-based devices.

What factors influence uptake of retinal screening among young adults with type 2 diabetes? A qualitative study informed by the theoretical domains framework.

Science.gov (United States)

Lake, Amelia J; Browne, Jessica L; Rees, Gwyneth; Speight, Jane

2017-06-01

Young adults with type 2 diabetes (T2D, 18-39years) face increased risk of vision loss from diabetic retinopathy (DR). Retinal screening is essential to detect DR, yet screening rates for this group are low and little is known about the underlying factors influencing this important behavior. Using the theoretical domains framework (TDF) to guide data collection and analysis, we explored screening barriers and facilitator, contrasting them with a comparator group of older adults with T2D (40+ years). Thirty semi-structured telephone interviews (10 younger, 20 older adults) were conducted. Data were coded into TDF domains with salience identified by "frequency" of reference. Screening facilitators and barriers were systematically compared between groups. Although many screening facilitators and barriers were shared by younger and older adults, additional factors highly relevant to the former included: social comparison with others ('social influences'); concern for the impact on the family unit, unrealistic optimism and perceived invulnerability ('beliefs about consequences'); lack of time and financial resources ('environmental context and resources'), and DR misconceptions ('knowledge'). This study demonstrated that young adult retinal screening behavior was influenced by additional social cognitive factors compared to older adults, providing a first-step evidence base for clinicians and other health professionals, and potential targets for future eye health and retinal screening interventions. Copyright © 2017 Elsevier Inc. All rights reserved.

RING finger and WD repeat domain 3 (RFWD3) associates with replication protein A (RPA) and facilitates RPA-mediated DNA damage response.

Science.gov (United States)

Liu, Shangfeng; Chu, Jessica; Yucer, Nur; Leng, Mei; Wang, Shih-Ya; Chen, Benjamin P C; Hittelman, Walter N; Wang, Yi

2011-06-24

DNA damage response is crucial for maintaining genomic integrity and preventing cancer by coordinating the activation of checkpoints and the repair of damaged DNA. Central to DNA damage response are the two checkpoint kinases ATM and ATR that phosphorylate a wide range of substrates. RING finger and WD repeat domain 3 (RFWD3) was initially identified as a substrate of ATM/ATR from a proteomic screen. Subsequent studies showed that RFWD3 is an E3 ubiquitin ligase that ubiquitinates p53 in vitro and positively regulates p53 levels in response to DNA damage. We report here that RFWD3 associates with replication protein A (RPA), a single-stranded DNA-binding protein that plays essential roles in DNA replication, recombination, and repair. Binding of RPA to single-stranded DNA (ssDNA), which is generated by DNA damage and repair, is essential for the recruitment of DNA repair factors to damaged sites and the activation of checkpoint signaling. We show that RFWD3 is physically associated with RPA and rapidly localizes to sites of DNA damage in a RPA-dependent manner. In vitro experiments suggest that the C terminus of RFWD3, which encompass the coiled-coil domain and the WD40 domain, is necessary for binding to RPA. Furthermore, DNA damage-induced phosphorylation of RPA and RFWD3 is dependent upon each other. Consequently, loss of RFWD3 results in the persistent foci of DNA damage marker γH2AX and the repair protein Rad51 in damaged cells. These findings suggest that RFWD3 is recruited to sites of DNA damage and facilitates RPA-mediated DNA damage signaling and repair.

Phosphopeptide occupancy and photoaffinity cross-linking of the v-Src SH2 domain attenuates tyrosine kinase activity.

Science.gov (United States)

Garcia, P; Shoelson, S E; Drew, J S; Miller, W T

1994-12-02

Phosphorylation of c-Src at carboxyl-terminal Tyr-527 suppresses tyrosine kinase activity and transforming potential, presumably by facilitating the intramolecular interaction of the C terminus of Src with its SH2 domain. In addition, it has been shown previously that occupancy of the c-Src SH2 domain with a phosphopeptide stimulates c-Src kinase catalytic activity. We have performed analogous studies with v-Src, the transforming protein from Rous sarcoma virus, which has extensive homology with c-Src. v-Src lacks an autoregulatory phosphorylation site, and its kinase domain is constitutively active. Phosphopeptides corresponding to the sequences surrounding c-Src Tyr-527 and a Tyr-Glu-Glu-Ile motif from the hamster polyoma virus middle T antigen inhibit tyrosine kinase activity of baculovirus-expressed v-Src 2- and 4-fold, respectively. To determine the mechanism of this regulation, the Tyr-527 phosphopeptide was substituted with the photoactive amino acid p-benzoylphenylalanine at the adjacent positions (N- and C-terminal) to phosphotyrosine. These peptides photoinactivate the v-Src tyrosine kinase 5-fold in a time- and concentration-dependent manner. Furthermore, the peptides cross-link an isolated Src SH2 domain with similar rates and specificity. These data indicate that occupancy of the v-Src SH2 domain induces a conformational change that is transmitted to the kinase domain and attenuates tyrosine kinase activity.

Interactions of polyomavirus middle T with the SH2 domains of the pp85 subunit of phosphatidylinositol-3-kinase.

OpenAIRE

Yoakim, M; Hou, W; Liu, Y; Carpenter, C L; Kapeller, R; Schaffhausen, B S

1992-01-01

The binding of phosphatidylinositol-3-kinase to the polyomavirus middle T antigen is facilitated by tyrosine phosphorylation of middle T on residue 315. The pp85 subunit of phosphatidylinositol-3-kinase contains two SH2 domains, one in the middle of the molecule and one at the C terminus. When assayed by blotting with phosphorylated middle T, the more N-terminal SH2 domain is responsible for binding to middle T. When assayed in solution with glutathione S transferase fusions, both SH2s are ca...

Magnetic domain structure and domain-wall energy in UFe8Ni2Si2 and UFe6Ni4Si2 intermetallic compounds

International Nuclear Information System (INIS)

Wyslocki, J.J.; Suski, W.; Wochowski, K.

1994-01-01

Magnetic domain structures in the UFe 8 Ni 2 Si 2 and UFe 6 Ni 4 Si 2 compounds were studied using the powder pattern method. The domain structure observed is typical for uniaxial materials. The domain-wall energy density γ was determined from the average surface domain width D s observed on surfaces perpendicular to the easy axis as equal to 16 erg/cm 2 for UFe 8 Ni 2 Si 2 and 10 erg/cm 2 for UFe 6 Ni 4 Si 2 . Moreover, the critical diameter for single domain particle D c was calculated for the studied compounds

SH2 domains: modulators of nonreceptor tyrosine kinase activity.

Science.gov (United States)

Filippakopoulos, Panagis; Müller, Susanne; Knapp, Stefan

2009-12-01

The Src homology 2 (SH2) domain is a sequence-specific phosphotyrosine-binding module present in many signaling molecules. In cytoplasmic tyrosine kinases, the SH2 domain is located N-terminally to the catalytic kinase domain (SH1) where it mediates cellular localization, substrate recruitment, and regulation of kinase activity. Initially, structural studies established a role of the SH2 domain stabilizing the inactive state of Src family members. However, biochemical characterization showed that the presence of the SH2 domain is frequently required for catalytic activity, suggesting a crucial function stabilizing the active state of many nonreceptor tyrosine kinases. Recently, the structure of the SH2-kinase domain of Fes revealed that the SH2 domain stabilizes the active kinase conformation by direct interactions with the regulatory helix alphaC. Stabilizing interactions between the SH2 and the kinase domains have also been observed in the structures of active Csk and Abl. Interestingly, mutations in the SH2 domain found in human disease can be explained by SH2 domain destabilization or incorrect positioning of the SH2. Here we summarize our understanding of mechanisms that lead to tyrosine kinase activation by direct interactions mediated by the SH2 domain and discuss how mutations in the SH2 domain trigger kinase inactivation.

Expression and Production of SH2 Domain Proteins.

Science.gov (United States)

Liu, Bernard A; Ogiue-Ikeda, Mari; Machida, Kazuya

2017-01-01

The Src Homology 2 (SH2) domain lies at the heart of phosphotyrosine signaling, coordinating signaling events downstream of receptor tyrosine kinases (RTKs), adaptors, and scaffolds. Over a hundred SH2 domains are present in mammals, each having a unique specificity which determines its interactions with multiple binding partners. One of the essential tools necessary for studying and determining the role of SH2 domains in phosphotyrosine signaling is a set of soluble recombinant SH2 proteins. Here we describe methods, based on a broad experience with purification of all SH2 domains, for the production of SH2 domain proteins needed for proteomic and biochemical-based studies such as peptide arrays, mass-spectrometry, protein microarrays, reverse-phase microarrays, and high-throughput fluorescence polarization (HTP-FP). We describe stepwise protocols for expression and purification of SH2 domains using GST or poly His-tags, two widely adopted affinity tags. In addition, we address alternative approaches, challenges, and validation studies for assessing protein quality and provide general characteristics of purified human SH2 domains.

Blue Light-excited Light-Oxygen-Voltage-sensing Domain 2 (LOV2) Triggers a Rearrangement of the Kinase Domain to Induce Phosphorylation Activity in Arabidopsis Phototropin1.

Science.gov (United States)

Oide, Mao; Okajima, Koji; Kashojiya, Sachiko; Takayama, Yuki; Oroguchi, Tomotaka; Hikima, Takaaki; Yamamoto, Masaki; Nakasako, Masayoshi

2016-09-16

Phototropin1 is a blue light (BL) receptor in plants and shows BL-dependent kinase activation. The BL-excited light-oxygen-voltage-sensing domain 2 (LOV2) is primarily responsible for the activation of the kinase domain; however, the molecular mechanism by which conformational changes in LOV2 are transmitted to the kinase domain remains unclear. Here, we investigated BL-induced structural changes of a minimum functional fragment of Arabidopsis phototropin1 composed of LOV2, the kinase domain, and a linker connecting the two domains using small-angle x-ray scattering (SAXS). The fragment existed as a dimer and displayed photoreversible SAXS changes reflected in the radii of gyration of 42.9 Å in the dark and 48.8 Å under BL irradiation. In the dark, the molecular shape reconstructed from the SAXS profiles appeared as two bean-shaped lobes in a twisted arrangement that was 170 Å long, 80 Å wide, and 50 Å thick. The molecular shape under BL became slightly elongated from that in the dark. By fitting the crystal structure of the LOV2 dimer and a homology model of the kinase domain to their inferred shapes, the BL-dependent change could be interpreted as the positional shift in the kinase domain relative to that of the LOV2 dimer. In addition, we found that lysine 475, a functionally important residue, in the N-terminal region of LOV2 plays a critical role in transmitting the structural changes in LOV2 to the kinase domain. The interface between the domains is critical for signaling, suitably changing the structure to activate the kinase in response to conformational changes in the adjoining LOV2. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The linker domain of poly(rC) binding protein 2 is a major determinant in poliovirus cap-independent translation.

Science.gov (United States)

Sean, Polen; Nguyen, Joseph H C; Semler, Bert L

2008-09-01

Poliovirus, a member of the enterovirus genus in the family Picornaviridae, is the causative agent of poliomyelitis. Translation of the viral genome is mediated through an internal ribosomal entry site (IRES) encoded within the 5' noncoding region (5' NCR). IRES elements are highly structured RNA sequences that facilitate the recruitment of ribosomes for translation. Previous studies have shown that binding of a cellular protein, poly(rC) binding protein 2 (PCBP2), to a major stem-loop structure in the genomic 5' NCR is necessary for the translation of picornaviruses containing type I IRES elements, including poliovirus, coxsackievirus, and human rhinovirus. PCBP1, an isoform that shares approximately 90% amino acid identity to PCBP2, cannot efficiently stimulate poliovirus IRES-mediated translation, most likely due to its reduced binding affinity to stem-loop IV within the poliovirus IRES. The primary differences between PCBP1 and PCBP2 are found in the so-called linker domain between the second and third K-homology (KH) domains of these proteins. We hypothesize that the linker region of PCBP2 augments binding to poliovirus stem-loop IV RNA. To test this hypothesis, we generated six PCBP1/PCBP2 chimeric proteins. The recombinant PCBP1/PCBP2 chimeric proteins were able to interact with poliovirus stem-loop I RNA and participate in protein-protein interactions. We demonstrated that the PCBP1/PCBP2 chimeric proteins with the PCBP2 linker, but not with the PCBP1 linker, were able to interact with poliovirus stem-loop IV RNA, and could subsequently stimulate poliovirus IRES-mediated translation. In addition, using a monoclonal anti-PCBP2 antibody (directed against the PCBP2 linker domain) in mobility shift assays, we showed that the PCBP2 linker domain modulates binding to poliovirus stem-loop IV RNA via a mechanism that is not inhibited by the antibody.

Domain engineering of the metastable domains in the 4f-uniaxial-ferromagnet CeRu2Ga2B

Science.gov (United States)

Wulferding, D.; Kim, H.; Yang, I.; Jeong, J.; Barros, K.; Kato, Y.; Martin, I.; Ayala-Valenzuela, O. E.; Lee, M.; Choi, H. C.; Ronning, F.; Civale, L.; Baumbach, R. E.; Bauer, E. D.; Thompson, J. D.; Movshovich, R.; Kim, Jeehoon

2017-04-01

In search of novel, improved materials for magnetic data storage and spintronic devices, compounds that allow a tailoring of magnetic domain shapes and sizes are essential. Good candidates are materials with intrinsic anisotropies or competing interactions, as they are prone to host various domain phases that can be easily and precisely selected by external tuning parameters such as temperature and magnetic field. Here, we utilize vector magnetic fields to visualize directly the magnetic anisotropy in the uniaxial ferromagnet CeRu2Ga2B. We demonstrate a feasible control both globally and locally of domain shapes and sizes by the external field as well as a smooth transition from single stripe to bubble domains, which opens the door to future applications based on magnetic domain tailoring.

Progress towards the development of SH2 domain inhibitors.

Science.gov (United States)

Kraskouskaya, Dziyana; Duodu, Eugenia; Arpin, Carolynn C; Gunning, Patrick T

2013-04-21

Src homology 2 (SH2) domains are 100 amino acid modular units, which recognize and bind to tyrosyl-phosphorylated peptide sequences on their target proteins, and thereby mediate intracellular protein-protein interactions. This review summarizes the progress towards the development of synthetic agents that disrupt the function of the SH2 domains in different proteins as well as the clinical relevance of targeting a specific SH2 domain. Since 1986, SH2 domains have been identified in over 110 human proteins, including kinases, transcription factors, and adaptor proteins. A number of these proteins are over-activated in many diseases, including cancer, and their function is highly dependent on their SH2 domain. Thus, inhibition of a protein's function through disrupting that of its SH2 domain has emerged as a promising approach towards the development of novel therapeutic modalities. Although targeting the SH2 domain is a challenging task in molecular recognition, the progress reported here demonstrates the feasibility of such an approach.

Barriers and facilitators towards implementing the Sepsis Six care bundle (BLISS-1): a mixed methods investigation using the theoretical domains framework.

Science.gov (United States)

Roberts, Neil; Hooper, Guy; Lorencatto, Fabiana; Storr, Wendell; Spivey, Michael

2017-09-19

The 'Sepsis 6', a care bundle of basic, but vital, measures (e.g. intravenous fluid, antibiotics) has been implemented to improve sepsis treatment. However, uptake has been variable. Tools from behavioral sciences, such as the Theoretical Domains Framework (TDF) may be used to understand and address such implementation issues. This study used a behavioral science approach to identify barriers and facilitators towards Sepsis Six implementation at a case study hospital. Semi-structured interviews based on the TDF were conducted with a sample group of consultants, junior doctors and nurses from Emergency Department, Medical and Surgical Admissions, to explore barriers/facilitators to Sepsis Six performance. Transcripts were analyzed following the combined principles of content and framework analysis. Emerging themes informed a questionnaire to explore generalizability and importance across a sample of 261 stakeholders. Median importance and agreement ratings for each theme were calculated overall and for each role and clinical area. These were used to identify important barriers and important facilitators as targets for performance improvement. No new belief statements were discovered and data saturation was deemed achieved after 10 interviews. 1699 utterances were coded into 64 belief statements, then collated into a 51-item questionnaire. 113 questionnaire responses were obtained (44.3% response rate). Important barriers included insufficient audit and feedback, poor teamwork and communication, concerns about using the Sepsis Six in certain patients, insufficient training, and resource concerns. Facilitators included confidence in knowledge and skills, beliefs in overall benefits of the bundle, beliefs that identification and management of septic patients fell within everyone's role, and that regular use of the bundle made it easier to remember. Some beliefs were applicable for the entire group, others were specific to particular staff groups. A range of barriers

Interactions of polyomavirus middle T with the SH2 domains of the pp85 subunit of phosphatidylinositol-3-kinase.

Science.gov (United States)

Yoakim, M; Hou, W; Liu, Y; Carpenter, C L; Kapeller, R; Schaffhausen, B S

1992-01-01

The binding of phosphatidylinositol-3-kinase to the polyomavirus middle T antigen is facilitated by tyrosine phosphorylation of middle T on residue 315. The pp85 subunit of phosphatidylinositol-3-kinase contains two SH2 domains, one in the middle of the molecule and one at the C terminus. When assayed by blotting with phosphorylated middle T, the more N-terminal SH2 domain is responsible for binding to middle T. When assayed in solution with glutathione S transferase fusions, both SH2s are capable of binding phosphorylated middle T. While both SH2 fusions can compete with intact pp85 for binding to middle T, the C-terminal SH2 is the more efficient of the two. Interaction between pp85 or its SH2 domains and middle T can be blocked by a synthetic peptide comprising the tyrosine phosphorylation sequence around middle T residue 315. Despite the fact that middle T can interact with both SH2s, these domains are not equivalent. Only the C-terminal SH2-middle T interaction was blocked by anti-SH2 antibody; the two SH2 fusions also interact with different cellular proteins. Images PMID:1380095

Introduction: History of SH2 Domains and Their Applications.

Science.gov (United States)

Liu, Bernard A; Machida, Kazuya

2017-01-01

The Src Homology 2 (SH2) domain is the prototypical protein interaction module that lies at the heart of phosphotyrosine signaling. Since its serendipitous discovery, there has been a tremendous advancement in technologies and an array of techniques available for studying SH2 domains and phosphotyrosine signaling. In this chapter, we provide a glimpse of the history of SH2 domains and describe many of the tools and techniques that have been developed along the way and discuss future directions for SH2 domain studies. We highlight the gist of each chapter in this volume in the context of: the structural biology and phosphotyrosine binding; characterizing SH2 specificity and generating prediction models; systems biology and proteomics; SH2 domains in signal transduction; and SH2 domains in disease, diagnostics, and therapeutics. Many of the individual chapters provide an in-depth approach that will allow scientists to interrogate the function and role of SH2 domains.

Jahn-teller domains and magnetic domains in Mn2FeO4

NARCIS (Netherlands)

Kub, J.; Brabers, V.A.M.; Novák, P.; Gemperle, R.; Simsova, J.

2000-01-01

Elastic (Jahn–Teller) domains and magnetic domains in the tetragonal spinel Mn2FeO4 were studied using X-ray double-crystal topography, X-ray diffractometry and the colloid-SEM method. The Jahn–Teller domains of the measured samples are tetragonal with the [0 0 1] c-axis alternating perpendicularly

Dynamically Coupled Residues within the SH2 Domain of FYN Are Key to Unlocking Its Activity.

Science.gov (United States)

Huculeci, Radu; Cilia, Elisa; Lyczek, Agatha; Buts, Lieven; Houben, Klaartje; Seeliger, Markus A; van Nuland, Nico; Lenaerts, Tom

2016-11-01

Src kinase activity is controlled by various mechanisms involving a coordinated movement of kinase and regulatory domains. Notwithstanding the extensive knowledge related to the backbone dynamics, little is known about the more subtle side-chain dynamics within the regulatory domains and their role in the activation process. Here, we show through experimental methyl dynamic results and predicted changes in side-chain conformational couplings that the SH2 structure of Fyn contains a dynamic network capable of propagating binding information. We reveal that binding the phosphorylated tail of Fyn perturbs a residue cluster near the linker connecting the SH2 and SH3 domains of Fyn, which is known to be relevant in the regulation of the activity of Fyn. Biochemical perturbation experiments validate that those residues are essential for inhibition of Fyn, leading to a gain of function upon mutation. These findings reveal how side-chain dynamics may facilitate the allosteric regulation of the different members of the Src kinase family. Copyright © 2016 Elsevier Ltd. All rights reserved.

Nucleic acid sequences encoding D1 and D1/D2 domains of human coxsackievirus and adenovirus receptor (CAR)

Science.gov (United States)

Freimuth, Paul I.

2010-04-06

The invention provides recombinant human CAR (coxsackievirus and adenovirus receptor) polypeptides which bind adenovirus. Specifically, polypeptides corresponding to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2 are provided. In another aspect, the invention provides nucleic acid sequences encoding these domains and expression vectors for producing the domains and bacterial cells containing such vectors. The invention also includes an isolated fusion protein comprised of the D1 polypeptide fused to a polypeptide which facilitates folding of D1 when expressed in bacteria. The functional D1 domain finds application in a therapeutic method for treating a patient infected with a CAR D1-binding virus, and also in a method for identifying an antiviral compound which interferes with viral attachment. The invention also provides a method for specifically targeting a cell for infection by a virus which binds to D1.

SH2 domains: modulators of nonreceptor tyrosine kinase activity

OpenAIRE

Filippakopoulos, Panagis; Müller, Susanne; Knapp, Stefan

2009-01-01

The Src homology 2 (SH2) domain is a sequence-specific phosphotyrosine-binding module present in many signaling molecules. In cytoplasmic tyrosine kinases, the SH2 domain is located N-terminally to the catalytic kinase domain (SH1) where it mediates cellular localization, substrate recruitment, and regulation of kinase activity. Initially, structural studies established a role of the SH2 domain stabilizing the inactive state of Src family members. However, biochemical characterization showed ...

Distinct mechanisms of a phosphotyrosyl peptide binding to two SH2 domains.

Science.gov (United States)

Pang, Xiaodong; Zhou, Huan-Xiang

2014-05-01

Protein phosphorylation is very common post-translational modification, catalyzed by kinases, for signaling and regulation. Phosphotyrosines frequently target SH2 domains. The spleen tyrosine kinase (Syk) is critical for tyrosine phosphorylation of multiple proteins and for regulation of important pathways. Phosphorylation of both Y342 and Y346 in Syk linker B is required for optimal signaling. The SH2 domains of Vav1 and PLC-γ both bind this doubly phosphorylated motif. Here we used a recently developed method to calculate the effects of Y342 and Y346 phosphorylation on the rate constants of a peptide from Syk linker B binding to the SH2 domains of Vav1 and PLC-γ. The predicted effects agree well with experimental observations. Moreover, we found that the same doubly phosphorylated peptide binds the two SH2 domains via distinct mechanisms, with apparent rigid docking for Vav1 SH2 and dock-and-coalesce for PLC-γ SH2.

Quantifying information transfer by protein domains: Analysis of the Fyn SH2 domain structure

Directory of Open Access Journals (Sweden)

Serrano Luis

2008-10-01

Full Text Available Abstract Background Efficient communication between distant sites within a protein is essential for cooperative biological response. Although often associated with large allosteric movements, more subtle changes in protein dynamics can also induce long-range correlations. However, an appropriate formalism that directly relates protein structural dynamics to information exchange between functional sites is still lacking. Results Here we introduce a method to analyze protein dynamics within the framework of information theory and show that signal transduction within proteins can be considered as a particular instance of communication over a noisy channel. In particular, we analyze the conformational correlations between protein residues and apply the concept of mutual information to quantify information exchange. Mapping out changes of mutual information on the protein structure then allows visualizing how distal communication is achieved. We illustrate the approach by analyzing information transfer by the SH2 domain of Fyn tyrosine kinase, obtained from Monte Carlo dynamics simulations. Our analysis reveals that the Fyn SH2 domain forms a noisy communication channel that couples residues located in the phosphopeptide and specificity binding sites and a number of residues at the other side of the domain near the linkers that connect the SH2 domain to the SH3 and kinase domains. We find that for this particular domain, communication is affected by a series of contiguous residues that connect distal sites by crossing the core of the SH2 domain. Conclusion As a result, our method provides a means to directly map the exchange of biological information on the structure of protein domains, making it clear how binding triggers conformational changes in the protein structure. As such it provides a structural road, next to the existing attempts at sequence level, to predict long-range interactions within protein structures.

Domain 2: Sport Safety and Injury Prevention

Science.gov (United States)

Gurchiek, Larry; Mokha, Monique Butcher

2004-01-01

Most coaches recognize the importance of creating a safe environment and preventing injuries of their athletes. Domain 2 is dedicated to this important aspect of coaching, and outlines specific areas within safety and injury prevention that coaches should address. Domain 2 sets the standards for facility, equipment, and environmental safety…

Structure of an isolated unglycosylated antibody CH2 domain

International Nuclear Information System (INIS)

Prabakaran, Ponraj; Vu, Bang K.; Gan, Jianhua; Feng, Yang; Dimitrov, Dimiter S.; Ji, Xinhua

2008-01-01

The crystal structure of an isolated unglycosylated antibody C H 2 domain has been determined at 1.7 Å resolution. The C H 2 (C H 3 for IgM and IgE) domain of an antibody plays an important role in mediating effector functions and preserving antibody stability. It is the only domain in human immunoglobulins (Igs) which is involved in weak interchain protein–protein interactions with another C H 2 domain solely through sugar moieties. The N-linked glycosylation at Asn297 is conserved in mammalian IgGs as well as in homologous regions of other antibody isotypes. To examine the structural details of the C H 2 domain in the absence of glycosylation and other antibody domains, the crystal structure of an isolated unglycosylated antibody γ1 C H 2 domain was determined at 1.7 Å resolution and compared with corresponding C H 2 structures from intact Fc, IgG and Fc receptor complexes. Furthermore, the oligomeric state of the protein in solution was studied using size-exclusion chromatography. The results suggested that the unglycosylated human antibody C H 2 domain is a monomer and that its structure is similar to that found in the intact Fc, IgG and Fc receptor complex structures. However, certain structural variations were observed in the Fc receptor-binding sites. Owing to its small size, stability and non-immunogenic Ig template, the C H 2-domain structure could be useful for the development by protein design of antibody domains exerting effector functions and/or antigen specificity and as a robust scaffold in protein-engineering applications

High resolution crystal structure of the Grb2 SH2 domain with a phosphopeptide derived from CD28.

Directory of Open Access Journals (Sweden)

Kunitake Higo

Full Text Available Src homology 2 (SH2 domains play a critical role in cellular signal transduction. They bind to peptides containing phosphotyrosine (pY with various specificities that depend on the flanking amino-acid residues. The SH2 domain of growth-factor receptor-bound protein 2 (Grb2 specifically recognizes pY-X-N-X, whereas the SH2 domains in phosphatidylinositol 3-kinase (PI3K recognize pY-X-X-M. Binding of the pY site in CD28 (pY-M-N-M by PI3K and Grb2 through their SH2 domains is a key step that triggers the CD28 signal transduction for T cell activation and differentiation. In this study, we determined the crystal structure of the Grb2 SH2 domain in complex with a pY-containing peptide derived from CD28 at 1.35 Å resolution. The peptide was found to adopt a twisted U-type conformation, similar to, but distinct from type-I β-turn. In all previously reported crystal structures, the peptide bound to the Grb2 SH2 domains adopts a type-I β-turn conformation, except those with a proline residue at the pY+3 position. Molecular modeling also suggests that the same peptide bound to PI3K might adopt a very different conformation.

High resolution crystal structure of the Grb2 SH2 domain with a phosphopeptide derived from CD28.

Science.gov (United States)

Higo, Kunitake; Ikura, Teikichi; Oda, Masayuki; Morii, Hisayuki; Takahashi, Jun; Abe, Ryo; Ito, Nobutoshi

2013-01-01

Src homology 2 (SH2) domains play a critical role in cellular signal transduction. They bind to peptides containing phosphotyrosine (pY) with various specificities that depend on the flanking amino-acid residues. The SH2 domain of growth-factor receptor-bound protein 2 (Grb2) specifically recognizes pY-X-N-X, whereas the SH2 domains in phosphatidylinositol 3-kinase (PI3K) recognize pY-X-X-M. Binding of the pY site in CD28 (pY-M-N-M) by PI3K and Grb2 through their SH2 domains is a key step that triggers the CD28 signal transduction for T cell activation and differentiation. In this study, we determined the crystal structure of the Grb2 SH2 domain in complex with a pY-containing peptide derived from CD28 at 1.35 Å resolution. The peptide was found to adopt a twisted U-type conformation, similar to, but distinct from type-I β-turn. In all previously reported crystal structures, the peptide bound to the Grb2 SH2 domains adopts a type-I β-turn conformation, except those with a proline residue at the pY+3 position. Molecular modeling also suggests that the same peptide bound to PI3K might adopt a very different conformation.

ViSlang: A system for interpreted domain-specific languages for scientific visualization

KAUST Repository

Rautek, Peter; Bruckner, Stefan; Grö ller, Meister Eduard; Hadwiger, Markus

2014-01-01

-level interface). In this paper we present a system that integrates domain-specific languages (DSLs) and facilitates the creation of new DSLs. DSLs provide an effective interface for domain scientists avoiding the difficulties involved with low-level interfaces

Superbinder SH2 domains act as antagonists of cell signaling.

Science.gov (United States)

Kaneko, Tomonori; Huang, Haiming; Cao, Xuan; Li, Xing; Li, Chengjun; Voss, Courtney; Sidhu, Sachdev S; Li, Shawn S C

2012-09-25

Protein-ligand interactions mediated by modular domains, which often play important roles in regulating cellular functions, are generally of moderate affinities. We examined the Src homology 2 (SH2) domain, a modular domain that recognizes phosphorylated tyrosine (pTyr) residues, to investigate how the binding affinity of a modular domain for its ligand influences the structure and cellular function of the protein. We used the phage display method to perform directed evolution of the pTyr-binding residues in the SH2 domain of the tyrosine kinase Fyn and identified three amino acid substitutions that critically affected binding. We generated three SH2 domain triple-point mutants that were "superbinders" with much higher affinities for pTyr-containing peptides than the natural domain. Crystallographic analysis of one of these superbinders revealed that the superbinder SH2 domain recognized the pTyr moiety in a bipartite binding mode: A hydrophobic surface encompassed the phenyl ring, and a positively charged site engaged the phosphate. When expressed in mammalian cells, the superbinder SH2 domains blocked epidermal growth factor receptor signaling and inhibited anchorage-independent cell proliferation, suggesting that pTyr superbinders might be explored for therapeutic applications and useful as biological research tools. Although the SH2 domain fold can support much higher affinity for its ligand than is observed in nature, our results suggest that natural SH2 domains are not optimized for ligand binding but for specificity and flexibility, which are likely properties important for their function in signaling and regulatory processes.

The facilitative effects of glucose ingestion on memory retrieval in younger and older adults: is task difficulty or task domain critical?

Science.gov (United States)

Riby, Leigh M; McMurtrie, Hazel; Smallwood, Jonathan; Ballantyne, Carrie; Meikle, Andrew; Smith, Emily

2006-02-01

The ingestion of a glucose-containing drink has been shown to improve cognitive performance, particularly memory functioning. However, it remains unclear as to the extent to which task domain and task difficulty moderate the glucose enhancement effect. The aim of this research was to determine whether boosts in performance are restricted to particular classes of memory (episodic v. semantic) or to tasks of considerable cognitive load. A repeated measures (25 g glucose v. saccharin), counterbalanced, double-blind design was used with younger and older adults. Participants performed a battery of episodic (e.g. paired associate learning) and semantic memory (e.g. category verification) tasks under low and high cognitive load. Electrophysiological measures (heart rate and galvanic skin response) of arousal and mental effort were also gathered. The results indicated that whilst glucose appeared to aid episodic remembering, cognitive load did not exaggerate the facilitative effect. For semantic memory, there was little evidence to suggest that glucose can boost semantic memory retrieval even when the load was manipulated. One exception was that glucose facilitated performance during the difficult category fluency task. Regardless, the present findings are consistent with the domain-specific account in which glucose acts primarily on the hippocampal region, which is known to support episodic memory. The possible contribution of the hippocampus in semantic memory processing is also discussed.

Hydrophobic interaction between the SH2 domain and the kinase domain is required for the activation of Csk.

Science.gov (United States)

Mikkola, Esa T; Gahmberg, Carl G

2010-06-18

The protein tyrosine kinase C-terminal Src kinase (Csk) is activated by the engagement of its Src homology (SH) 2 domain. However, the molecular mechanism required for this is not completely understood. The crystal structure of the active Csk indicates that Csk could be activated by contact between the SH2 domain and the beta3-alphaC loop in the N-terminal lobe of the kinase domain. To study the importance of this interaction for the SH2-domain-mediated activation of Csk, we mutated the amino acid residues forming the contacts between the SH2 domain and the beta3-alphaC loop. The mutation of the beta3-alphaC loop Ala228 to glycine and of the SH2 domain Tyr116, Tyr133, Leu138, and Leu149 to alanine resulted in the inability of the SH2 domain ligand to activate Csk. Furthermore, the overexpressed Csk mutants A228G, Y133A/Y116A, L138A, and L149A were unable to efficiently inactivate endogenous Src in human embryonic kidney 293 cells. The results suggest that the SH2-domain-mediated activation of Csk is dependent on the binding of the beta3-alphaC loop Ala228 to the hydrophobic pocket formed by the side chains of Tyr116, Tyr133, Leu138, and Leu149 on the surface of the SH2 domain. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Crystal structure of the Ig1 domain of the neural cell adhesion molecule NCAM2 displays domain swapping

DEFF Research Database (Denmark)

Rasmussen, Kim Krighaar; Kulahin, Nikolaj; Kristensen, Ole

2008-01-01

The crystal structure of the first immunoglobulin (Ig1) domain of neural cell adhesion molecule 2 (NCAM2/OCAM/RNCAM) is presented at a resolution of 2.7 A. NCAM2 is a member of the immunoglobulin superfamily of cell adhesion molecules (IgCAMs). In the structure, two Ig domains interact by domain...

Lyso-myristoyl phosphatidylcholine micelles sustain the activity of Dengue non-structural (NS) protein 3 protease domain fused with the full-length NS2B.

Science.gov (United States)

Huang, Qiwei; Li, Qingxin; Joy, Joma; Chen, Angela Shuyi; Ruiz-Carrillo, David; Hill, Jeffrey; Lescar, Julien; Kang, Congbao

2013-12-01

Dengue virus (DENV), a member of the flavivirus genus, affects 50-100 million people in tropical and sub-tropical regions. The DENV protease domain is located at the N-terminus of the NS3 protease and requires for its enzymatic activity a hydrophilic segment of the NS2B that acts as a cofactor. The protease is an important antiviral drug target because it plays a crucial role in virus replication by cleaving the genome-coded polypeptide into mature functional proteins. Currently, there are no drugs to inhibit DENV protease activity. Most structural and functional studies have been conducted using protein constructs containing the NS3 protease domain connected to a soluble segment of the NS2B membrane protein via a nine-residue linker. For in vitro structural and functional studies, it would be useful to produce a natural form of the DENV protease containing the NS3 protease domain and the full-length NS2B protein. Herein, we describe the expression and purification of a natural form of DENV protease (NS2BFL-NS3pro) containing the full-length NS2B protein and the protease domain of NS3 (NS3pro). The protease was expressed and purified in detergent micelles necessary for its folding. Our results show that this purified protein was active in detergent micelles such as lyso-myristoyl phosphatidylcholine (LMPC). These findings should facilitate further structural and functional studies of the protease and will facilitate drug discovery targeting DENV. Copyright © 2013 Elsevier Inc. All rights reserved.

Functional diversity of Csk, Chk, and Src SH2 domains due to a single residue variation.

Science.gov (United States)

Ayrapetov, Marina K; Nam, Nguyen Hai; Ye, Guofeng; Kumar, Anil; Parang, Keykavous; Sun, Gongqin

2005-07-08

The C-terminal Src kinase (Csk) family of protein tyrosine kinases contains two members: Csk and Csk homologous kinase (Chk). Both phosphorylate and inactivate Src family kinases. Recent reports suggest that the Src homology (SH) 2 domains of Csk and Chk may bind to different phosphoproteins, which provides a basis for different cellular functions for Csk and Chk. To verify and characterize such a functional divergence, we compared the binding properties of the Csk, Chk, and Src SH2 domains and investigated the structural basis for the functional divergence. First, the study demonstrated striking functional differences between the Csk and Chk SH2 domains and revealed functional similarities between the Chk and Src SH2 domains. Second, structural analysis and mutagenic studies revealed that the functional differences among the three SH2 domains were largely controlled by one residue, Glu127 in Csk, Ile167 in Chk, and Lys200 in Src. Mutating these residues in the Csk or Chk SH2 domain to the Src counterpart resulted in dramatic gain of function similar to Src SH2 domain, whereas mutating Lys200 in Src SH2 domain to Glu (the Csk counterpart) resulted in loss of Src SH2 function. Third, a single point mutation of E127K rendered Csk responsive to activation by a Src SH2 domain ligand. Finally, the optimal phosphopeptide sequence for the Chk SH2 domain was determined. These results provide a compelling explanation for the functional differences between two homologous protein tyrosine kinases and reveal a new structure-function relationship for the SH2 domains.

Solution structure of tensin2 SH2 domain and its phosphotyrosine-independent interaction with DLC-1.

Directory of Open Access Journals (Sweden)

Kun Dai

Full Text Available Src homology 2 (SH2 domain is a conserved module involved in various biological processes. Tensin family member was reported to be involved in tumor suppression by interacting with DLC-1 (deleted-in-liver-cancer-1 via its SH2 domain. We explore here the important questions that what the structure of tensin2 SH2 domain is, and how it binds to DLC-1, which might reveal a novel binding mode.Tensin2 SH2 domain adopts a conserved SH2 fold that mainly consists of five β-strands flanked by two α-helices. Most SH2 domains recognize phosphorylated ligands specifically. However, tensin2 SH2 domain was identified to interact with nonphosphorylated ligand (DLC-1 as well as phosphorylated ligand.We determined the solution structure of tensin2 SH2 domain using NMR spectroscopy, and revealed the interactions between tensin2 SH2 domain and its ligands in a phosphotyrosine-independent manner.

A new tagging system for production of recombinant proteins in Drosophila S2 cells using the third domain of the urokinase receptor

DEFF Research Database (Denmark)

Gårdsvoll, Henrik; Hansen, Line V; Jørgensen, Thomas J D

2007-01-01

The use of protein fusion tag technology greatly facilitates detection, expression and purification of recombinant proteins, and the demands for new and more effective systems are therefore expanding. We have used a soluble truncated form of the third domain of the urokinase receptor...... as a convenient C-terminal fusion partner for various recombinant extracellular human proteins used in basic cancer research. The stability of this cystein-rich domain, which structure adopts a three-finger fold, provides an important asset for its applicability as a fusion tag for expression of recombinant...... chromatography procedure using the immobilized anti-uPAR monoclonal antibody R2. An optional enterokinase cleavage site is included between the various recombinant proteins and the linker region of the tag, which enables generation of highly pure preparations of tag-free recombinant proteins. Using this system...

A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain

Science.gov (United States)

Harper, J. F.; Hong, B.; Hwang, I.; Guo, H. Q.; Stoddard, R.; Huang, J. F.; Palmgren, M. G.; Sze, H.; Evans, M. L. (Principal Investigator)

1998-01-01

To study transporters involved in regulating intracellular Ca2+, we isolated a full-length cDNA encoding a Ca2+-ATPase from a model plant, Arabidopsis, and named it ACA2 (Arabidopsis Ca2+-ATPase, isoform 2). ACA2p is most similar to a "plasma membrane-type" Ca2+-ATPase, but is smaller (110 kDa), contains a unique N-terminal domain, and is missing a long C-terminal calmodulin-binding regulatory domain. In addition, ACA2p is localized to an endomembrane system and not the plasma membrane, as shown by aqueous-two phase fractionation of microsomal membranes. ACA2p was expressed in yeast as both a full-length protein (ACA2-1p) and an N-terminal truncation mutant (ACA2-2p; Delta residues 2-80). Only the truncation mutant restored the growth on Ca2+-depleted medium of a yeast mutant defective in both endogenous Ca2+ pumps, PMR1 and PMC1. Although basal Ca2+-ATPase activity of the full-length protein was low, it was stimulated 5-fold by calmodulin (50% activation around 30 nM). In contrast, the truncated pump was fully active and insensitive to calmodulin. A calmodulin-binding sequence was identified within the first 36 residues of the N-terminal domain, as shown by calmodulin gel overlays on fusion proteins. Thus, ACA2 encodes a novel calmodulin-regulated Ca2+-ATPase distinguished by a unique N-terminal regulatory domain and a non-plasma membrane localization.

A domain-independent descriptive design model and its application to structured reflection on design processes

NARCIS (Netherlands)

Reymen, Isabelle; Hammer, D.K.; Kroes, P.A.; van Aken, Joan Ernst; van Aken, J.E.; Dorst, C.H.; Bax, M.F.T.; Basten, T

2006-01-01

Domain-independent models of the design process are an important means for facilitating interdisciplinary communication and for supporting multidisciplinary design. Many so-called domain-independent models are, however, not really domain independent. We state that to be domain independent, the

Ligand binding by PDZ domains

DEFF Research Database (Denmark)

Chi, Celestine N.; Bach, Anders; Strømgaard, Kristian

2012-01-01

, for example, are particularly rich in these domains. The general function of PDZ domains is to bring proteins together within the appropriate cellular compartment, thereby facilitating scaffolding, signaling, and trafficking events. The many functions of PDZ domains under normal physiological as well...... as pathological conditions have been reviewed recently. In this review, we focus on the molecular details of how PDZ domains bind their protein ligands and their potential as drug targets in this context....

The Zinc Finger of Prolyl Hydroxylase Domain Protein 2 Is Essential for Efficient Hydroxylation of Hypoxia-Inducible Factor α.

Science.gov (United States)

Arsenault, Patrick R; Song, Daisheng; Chung, Yu Jin; Khurana, Tejvir S; Lee, Frank S

2016-09-15

Prolyl hydroxylase domain protein 2 (PHD2) (also known as EGLN1) is a key oxygen sensor in mammals that posttranslationally modifies hypoxia-inducible factor α (HIF-α) and targets it for degradation. In addition to its catalytic domain, PHD2 contains an evolutionarily conserved zinc finger domain, which we have previously proposed recruits PHD2 to the HSP90 pathway to promote HIF-α hydroxylation. Here, we provide evidence that this recruitment is critical both in vitro and in vivo We show that in vitro, the zinc finger can function as an autonomous recruitment domain to facilitate interaction with HIF-α. In vivo, ablation of zinc finger function by a C36S/C42S Egln1 knock-in mutation results in upregulation of the erythropoietin gene, erythrocytosis, and augmented hypoxic ventilatory response, all hallmarks of Egln1 loss of function and HIF stabilization. Hence, the zinc finger ordinarily performs a critical positive regulatory function. Intriguingly, the function of this zinc finger is impaired in high-altitude-adapted Tibetans, suggesting that their adaptation to high altitude may, in part, be due to a loss-of-function EGLN1 allele. Thus, these findings have important implications for understanding both the molecular mechanism of the hypoxic response and human adaptation to high altitude. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Fusion protein based on Grb2-SH2 domain for cancer therapy

International Nuclear Information System (INIS)

Saito, Yuriko; Furukawa, Takako; Arano, Yasushi; Fujibayashi, Yasuhisa; Saga, Tsuneo

2010-01-01

Research highlights: → Grb2 mediates EGFR signaling through binding to phosphorylate EGFR with SH2 domain. → We generated fusion proteins containing 1 or 2 SH2 domains of Grb2 added with TAT. → The one with 2 SH2 domains (TSSF) interfered ERK phosphorylation. → TSSF significantly delayed the growth of EGFR overexpressing tumor in a mouse model. -- Abstract: Epidermal growth factor receptor (EGFR) is one of the very attractive targets for cancer therapy. In this study, we generated fusion proteins containing one or two Src-homology 2 (SH2) domains of growth factor receptor bound protein 2 (Grb2), which bind to phosphorylated EGFR, added with HIV-1 transactivating transcription for cell membrane penetration (termed TSF and TSSF, respectively). We examined if they can interfere Grb2-mediated signaling pathway and suppress tumor growth as expected from the lack of SH3 domain, which is necessary to intermediate EGFR-Grb2 cell signaling, in the fusion proteins. The transduction efficiency of TSSF was similar to that of TSF, but the binding activity of TSSF to EGFR was higher than that of TSF. Treatment of EGFR-overexpressing cells showed that TSSF decreased p42-ERK phosphorylation, while TSF did not. Both the proteins delayed cell growth but did not induce cell death in culture. TSSF also significantly suppressed tumor growth in vivo under consecutive administration. In conclusion, TSSF showed an ability to inhibit EGFR-Grb2 signaling and could have a potential to treat EGFR-activated cancer.

Mutations in the FMN domain modulate MCD spectra of the heme site in the oxygenase domain of inducible nitric oxide synthase.

Science.gov (United States)

Sempombe, Joseph; Elmore, Bradley O; Sun, Xi; Dupont, Andrea; Ghosh, Dipak K; Guillemette, J Guy; Kirk, Martin L; Feng, Changjian

2009-05-27

The nitric oxide synthase (NOS) output state for NO production is a complex of the flavin mononucleotide (FMN)-binding domain and the heme domain, and thereby it facilitates the interdomain electron transfer from the FMN to the catalytic heme site. Emerging evidence suggests that interdomain FMN-heme interactions are important in the formation of the output state because they guide the docking of the FMN domain to the heme domain. In this study, notable effects of mutations in the adjacent FMN domain on the heme structure in a human iNOS bidomain oxygenase/FMN construct have been observed by using low-temperature magnetic circular dichroism (MCD) spectroscopy. The comparative MCD study of wild-type and mutant proteins clearly indicates that a properly docked FMN domain contributes to the observed L-Arg perturbation of the heme MCD spectrum in the wild-type protein and that the conserved surface residues in the FMN domain (E546 and E603) play key roles in facilitating a productive alignment of the FMN and heme domains in iNOS.

A Hybrid Approach to Finding Relevant Social Media Content for Complex Domain Specific Information Needs.

Science.gov (United States)

Cameron, Delroy; Sheth, Amit P; Jaykumar, Nishita; Thirunarayan, Krishnaprasad; Anand, Gaurish; Smith, Gary A

2014-12-01

While contemporary semantic search systems offer to improve classical keyword-based search, they are not always adequate for complex domain specific information needs. The domain of prescription drug abuse, for example, requires knowledge of both ontological concepts and "intelligible constructs" not typically modeled in ontologies. These intelligible constructs convey essential information that include notions of intensity, frequency, interval, dosage and sentiments, which could be important to the holistic needs of the information seeker. In this paper, we present a hybrid approach to domain specific information retrieval that integrates ontology-driven query interpretation with synonym-based query expansion and domain specific rules, to facilitate search in social media on prescription drug abuse. Our framework is based on a context-free grammar (CFG) that defines the query language of constructs interpretable by the search system. The grammar provides two levels of semantic interpretation: 1) a top-level CFG that facilitates retrieval of diverse textual patterns, which belong to broad templates and 2) a low-level CFG that enables interpretation of specific expressions belonging to such textual patterns. These low-level expressions occur as concepts from four different categories of data: 1) ontological concepts, 2) concepts in lexicons (such as emotions and sentiments), 3) concepts in lexicons with only partial ontology representation, called lexico-ontology concepts (such as side effects and routes of administration (ROA)), and 4) domain specific expressions (such as date, time, interval, frequency and dosage) derived solely through rules. Our approach is embodied in a novel Semantic Web platform called PREDOSE, which provides search support for complex domain specific information needs in prescription drug abuse epidemiology. When applied to a corpus of over 1 million drug abuse-related web forum posts, our search framework proved effective in retrieving

Phrenic motor neuron adenosine 2A receptors elicit phrenic motor facilitation.

Science.gov (United States)

Seven, Yasin B; Perim, Raphael R; Hobson, Orinda R; Simon, Alec K; Tadjalli, Arash; Mitchell, Gordon S

2018-04-15

Although adenosine 2A (A 2A ) receptor activation triggers specific cell signalling cascades, the ensuing physiological outcomes depend on the specific cell type expressing these receptors. Cervical spinal adenosine 2A (A 2A ) receptor activation elicits a prolonged facilitation in phrenic nerve activity, which was nearly abolished following intrapleural A 2A receptor siRNA injections. A 2A receptor siRNA injections selectively knocked down A 2A receptors in cholera toxin B-subunit-identified phrenic motor neurons, sparing cervical non-phrenic motor neurons. Collectively, our results support the hypothesis that phrenic motor neurons express the A 2A receptors relevant to A 2A receptor-induced phrenic motor facilitation. Upregulation of A 2A receptor expression in the phrenic motor neurons per se may potentially be a useful approach to increase phrenic motor neuron excitability in conditions such as spinal cord injury. Cervical spinal adenosine 2A (A 2A ) receptor activation elicits a prolonged increase in phrenic nerve activity, an effect known as phrenic motor facilitation (pMF). The specific cervical spinal cells expressing the relevant A 2A receptors for pMF are unknown. This is an important question since the physiological outcome of A 2A receptor activation is highly cell type specific. Thus, we tested the hypothesis that the relevant A 2A receptors for pMF are expressed in phrenic motor neurons per se versus non-phrenic neurons of the cervical spinal cord. A 2A receptor immunostaining significantly colocalized with NeuN-positive neurons (89 ± 2%). Intrapleural siRNA injections were used to selectively knock down A 2A receptors in cholera toxin B-subunit-labelled phrenic motor neurons. A 2A receptor knock-down was verified by a ∼45% decrease in A 2A receptor immunoreactivity within phrenic motor neurons versus non-targeting siRNAs (siNT; P phrenic motor neurons. In rats that were anaesthetized, subjected to neuromuscular blockade and ventilated, p

Barriers and Facilitators to Implementing the HEADS-ED: A Rapid Screening Tool for Pediatric Patients in Emergency Departments.

Science.gov (United States)

MacWilliams, Kate; Curran, Janet; Racek, Jakub; Cloutier, Paula; Cappelli, Mario

2017-12-01

This study sought to identify barriers and facilitators to the implementation of the HEADS-ED, a screening tool appropriate for use in the emergency department (ED) that facilitates standardized assessments, discharge planning, charting, and linking pediatric mental health patients to appropriate community resources. A qualitative theory-based design was used to identify barriers and facilitators to implementing the HEADS-ED tool. Focus groups were conducted with participants recruited from 6 different ED settings across 2 provinces (Ontario and Nova Scotia). The Theoretical Domains Framework was used as a conceptual framework to guide data collection and to identify themes from focus group discussions. The following themes spanning 12 domains were identified as reflective of participants' beliefs about the barriers and facilitators to implementing the HEADS-ED tool: knowledge, skills, beliefs about capabilities, social professional role and identity, optimism, beliefs about consequences, reinforcement, environmental context and resources, social influences, emotion, behavioral regulation and memory, and attention and decision process. The HEADS-ED has the potential to address the need for better discharge planning, complete charting, and standardized assessments for the increasing population of pediatric mental health patients who present to EDs. This study has identified potential barriers and facilitators, which should be considered when developing an implementation plan for adopting the HEADS-ED tool into practice within EDs.

Evolution of SH2 domains and phosphotyrosine signalling networks

Science.gov (United States)

Liu, Bernard A.; Nash, Piers D.

2012-01-01

Src homology 2 (SH2) domains mediate selective protein–protein interactions with tyrosine phosphorylated proteins, and in doing so define specificity of phosphotyrosine (pTyr) signalling networks. SH2 domains and protein-tyrosine phosphatases expand alongside protein-tyrosine kinases (PTKs) to coordinate cellular and organismal complexity in the evolution of the unikont branch of the eukaryotes. Examination of conserved families of PTKs and SH2 domain proteins provides fiduciary marks that trace the evolutionary landscape for the development of complex cellular systems in the proto-metazoan and metazoan lineages. The evolutionary provenance of conserved SH2 and PTK families reveals the mechanisms by which diversity is achieved through adaptations in tissue-specific gene transcription, altered ligand binding, insertions of linear motifs and the gain or loss of domains following gene duplication. We discuss mechanisms by which pTyr-mediated signalling networks evolve through the development of novel and expanded families of SH2 domain proteins and the elaboration of connections between pTyr-signalling proteins. These changes underlie the variety of general and specific signalling networks that give rise to tissue-specific functions and increasingly complex developmental programmes. Examination of SH2 domains from an evolutionary perspective provides insight into the process by which evolutionary expansion and modification of molecular protein interaction domain proteins permits the development of novel protein-interaction networks and accommodates adaptation of signalling networks. PMID:22889907

Src binds cortactin through an SH2 domain cystine-mediated linkage

Science.gov (United States)

Evans, Jason V.; Ammer, Amanda G.; Jett, John E.; Bolcato, Chris A.; Breaux, Jason C.; Martin, Karen H.; Culp, Mark V.; Gannett, Peter M.; Weed, Scott A.

2012-01-01

Summary Tyrosine-kinase-based signal transduction mediated by modular protein domains is critical for cellular function. The Src homology (SH)2 domain is an important conductor of intracellular signaling that binds to phosphorylated tyrosines on acceptor proteins, producing molecular complexes responsible for signal relay. Cortactin is a cytoskeletal protein and tyrosine kinase substrate that regulates actin-based motility through interactions with SH2-domain-containing proteins. The Src kinase SH2 domain mediates cortactin binding and tyrosine phosphorylation, but how Src interacts with cortactin is unknown. Here we demonstrate that Src binds cortactin through cystine bonding between Src C185 in the SH2 domain within the phosphotyrosine binding pocket and cortactin C112/246 in the cortactin repeats domain, independent of tyrosine phosphorylation. Interaction studies show that the presence of reducing agents ablates Src-cortactin binding, eliminates cortactin phosphorylation by Src, and prevents Src SH2 domain binding to cortactin. Tandem MS/MS sequencing demonstrates cystine bond formation between Src C185 and cortactin C112/246. Mutational studies indicate that an intact cystine binding interface is required for Src-mediated cortactin phosphorylation, cell migration, and pre-invadopodia formation. Our results identify a novel phosphotyrosine-independent binding mode between the Src SH2 domain and cortactin. Besides Src, one quarter of all SH2 domains contain cysteines at or near the analogous Src C185 position. This provides a potential alternative mechanism to tyrosine phosphorylation for cysteine-containing SH2 domains to bind cognate ligands that may be widespread in propagating signals regulating diverse cellular functions. PMID:23097045

Src binds cortactin through an SH2 domain cystine-mediated linkage.

Science.gov (United States)

Evans, Jason V; Ammer, Amanda G; Jett, John E; Bolcato, Chris A; Breaux, Jason C; Martin, Karen H; Culp, Mark V; Gannett, Peter M; Weed, Scott A

2012-12-15

Tyrosine-kinase-based signal transduction mediated by modular protein domains is critical for cellular function. The Src homology (SH)2 domain is an important conductor of intracellular signaling that binds to phosphorylated tyrosines on acceptor proteins, producing molecular complexes responsible for signal relay. Cortactin is a cytoskeletal protein and tyrosine kinase substrate that regulates actin-based motility through interactions with SH2-domain-containing proteins. The Src kinase SH2 domain mediates cortactin binding and tyrosine phosphorylation, but how Src interacts with cortactin is unknown. Here we demonstrate that Src binds cortactin through cystine bonding between Src C185 in the SH2 domain within the phosphotyrosine binding pocket and cortactin C112/246 in the cortactin repeats domain, independent of tyrosine phosphorylation. Interaction studies show that the presence of reducing agents ablates Src-cortactin binding, eliminates cortactin phosphorylation by Src, and prevents Src SH2 domain binding to cortactin. Tandem MS/MS sequencing demonstrates cystine bond formation between Src C185 and cortactin C112/246. Mutational studies indicate that an intact cystine binding interface is required for Src-mediated cortactin phosphorylation, cell migration, and pre-invadopodia formation. Our results identify a novel phosphotyrosine-independent binding mode between the Src SH2 domain and cortactin. Besides Src, one quarter of all SH2 domains contain cysteines at or near the analogous Src C185 position. This provides a potential alternative mechanism to tyrosine phosphorylation for cysteine-containing SH2 domains to bind cognate ligands that may be widespread in propagating signals regulating diverse cellular functions.

A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression

Science.gov (United States)

Syed, Salahuddin; Desler, Claus; Rasmussen, Lene J.; Schmidt, Kristina H.

2016-01-01

In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N-terminal domain required for control of DNA synthesis maps to residues 186–212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each Rrm3 function are distinct. Although both lesion types activate the DNA-damage checkpoint, we find that the resultant increase in nucleotide levels is not sufficient for continued DNA synthesis under replication stress. Together, our findings suggest a role of Rrm3, via its Orc5-binding domain, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks. PMID:27923055

A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression.

Science.gov (United States)

Syed, Salahuddin; Desler, Claus; Rasmussen, Lene J; Schmidt, Kristina H

2016-12-01

In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N-terminal domain required for control of DNA synthesis maps to residues 186-212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each Rrm3 function are distinct. Although both lesion types activate the DNA-damage checkpoint, we find that the resultant increase in nucleotide levels is not sufficient for continued DNA synthesis under replication stress. Together, our findings suggest a role of Rrm3, via its Orc5-binding domain, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks.

SH2 Binding Site Protection Assay: A Method for Identification of SH2 Domain Interaction Partners by Exploiting SH2 Mediated Phosphosite Protection.

Science.gov (United States)

Jadwin, Joshua A

2017-01-01

Over the last two decades there has been a significant effort in the field to characterize the phosphosite binding specificities of SH2 domains with the goal of deciphering the pY signaling code. Although high throughput studies in various formats using most SH2 domains have collectively provided a rich resource of in vitro SH2-pTyr site specificity maps, this data can only be used approximate what is happening in the cell where protein concentrations and localization are not homogenous, as they are for in vitro experiments. Here we describe an in vivo approach, SH2 site protection assay, which can capture the pTyr binding specificity of SH2 domains in the cell. The basis of this approach is SH2-pY site protection, the ability of SH2 domains to prevent the PTP-dependent dephosphorylation of their pY site binding partners. We overexpress a tracer SH2 domain in cells and quantify the change in abundance of tyrosine phosphorylated sites using MS. Since the method is performed in vivo, it has the advantage of identifying SH2-pY interactions as they occur within in the cell.

Domain-orientation dependence of levitation force in seeded melt grown single-domain YBa2Cu3Ox

International Nuclear Information System (INIS)

Shi, D.; Qu, D.; Sagar, S.; Lahiri, K.

1997-01-01

Domain-orientation dependence of levitation force has been determined for single-domain YBa 2 Cu 3 O x . The single-domain material is obtained from a seeded melt growth process. The levitation force has been found to reach a maximum as the c axis of the domain is parallel to the direction of the force. The levitation force decreases in a cosine law fashion as the angle θ (the angle between the direction of the force and the c axis) increases from 0 degree to 60 degree. A maximum anisotropy of levitation force of 2.29 has been found. A physical model is proposed to explain the observed orientation dependence. copyright 1997 American Institute of Physics

The BRCT domain is a phospho-protein binding domain.

Science.gov (United States)

Yu, Xiaochun; Chini, Claudia Christiano Silva; He, Miao; Mer, Georges; Chen, Junjie

2003-10-24

The carboxyl-terminal domain (BRCT) of the Breast Cancer Gene 1 (BRCA1) protein is an evolutionarily conserved module that exists in a large number of proteins from prokaryotes to eukaryotes. Although most BRCT domain-containing proteins participate in DNA-damage checkpoint or DNA-repair pathways, or both, the function of the BRCT domain is not fully understood. We show that the BRCA1 BRCT domain directly interacts with phosphorylated BRCA1-Associated Carboxyl-terminal Helicase (BACH1). This specific interaction between BRCA1 and phosphorylated BACH1 is cell cycle regulated and is required for DNA damage-induced checkpoint control during the transition from G2 to M phase of the cell cycle. Further, we show that two other BRCT domains interact with their respective physiological partners in a phosphorylation-dependent manner. Thirteen additional BRCT domains also preferentially bind phospho-peptides rather than nonphosphorylated control peptides. These data imply that the BRCT domain is a phospho-protein binding domain involved in cell cycle control.

In-Solution SH2 Domain Binding Assay Based on Proximity Ligation.

Science.gov (United States)

Machida, Kazuya

2017-01-01

Protein-protein interactions mediated by SH2 domains confer specificity in tyrosine kinase pathways. Traditional assays for assessing interactions between an SH2 domain and its interacting protein such as far-Western and pull-down are inherently low throughput. We developed SH2-PLA, an in-solution SH2 domain binding assay, that takes advantage of the speed and sensitivity of proximity ligation and real-time PCR. SH2-PLA allows for rapid assessment of SH2 domain binding to a target protein using only a few microliters of cell lysate, thereby making it an attractive new tool to study tyrosine kinase signaling.

Structural and biophysical investigation of the interaction of a mutant Grb2 SH2 domain (W121G) with its cognate phosphopeptide.

Science.gov (United States)

Papaioannou, Danai; Geibel, Sebastian; Kunze, Micha B A; Kay, Christopher W M; Waksman, Gabriel

2016-03-01

The adaptor protein Grb2 is a key element of mitogenetically important signaling pathways. With its SH2 domain it binds to upstream targets while its SH3 domains bind to downstream proteins thereby relaying signals from the cell membranes to the nucleus. The Grb2 SH2 domain binds to its targets by recognizing a phosphotyrosine (pY) in a pYxNx peptide motif, requiring an Asn at the +2 position C-terminal to the pY with the residue either side of this Asn being hydrophobic. Structural analysis of the Grb2 SH2 domain in complex with its cognate peptide has shown that the peptide adopts a unique β-turn conformation, unlike the extended conformation that phosphopeptides adopt when bound to other SH2 domains. TrpEF1 (W121) is believed to force the peptide into this unusual conformation conferring this unique specificity to the Grb2 SH2 domain. Using X-ray crystallography, electron paramagnetic resonance (EPR) spectroscopy, and isothermal titration calorimetry (ITC), we describe here a series of experiments that explore the role of TrpEF1 in determining the specificity of the Grb2 SH2 domain. Our results demonstrate that the ligand does not adopt a pre-organized structure before binding to the SH2 domain, rather it is the interaction between the two that imposes the hairpin loop to the peptide. Furthermore, we find that the peptide adopts a similar structure when bound to both the wild-type Grb2 SH2 domain and a TrpEF1Gly mutant. This suggests that TrpEF1 is not the determining factor for the conformation of the phosphopeptide. © 2015 The Protein Society.

Facilitators and barriers to quality of care in maternal, newborn and child health: a global situational analysis through metareview.

Science.gov (United States)

Nair, Manisha; Yoshida, Sachiyo; Lambrechts, Thierry; Boschi-Pinto, Cynthia; Bose, Krishna; Mason, Elizabeth Mary; Mathai, Matthews

2014-05-22

Conduct a global situational analysis to identify the current facilitators and barriers to improving quality of care (QoC) for pregnant women, newborns and children. Metareview of published and unpublished systematic reviews and meta-analyses conducted between January 2000 and March 2013 in any language. Assessment of Multiple Systematic Reviews (AMSTAR) is used to assess the methodological quality of systematic reviews. Health systems of all countries. Study outcome: QoC measured using surrogate indicators--effective, efficient, accessible, acceptable/patient centred, equitable and safe. Conducted in two phases (1) qualitative synthesis of extracted data to identify and group the facilitators and barriers to improving QoC, for each of the three population groups, into the six domains of WHO's framework and explore new domains and (2) an analysis grid to map the common facilitators and barriers. We included 98 systematic reviews with 110 interventions to improve QoC from countries globally. The facilitators and barriers identified fitted the six domains of WHO's framework--information, patient-population engagement, leadership, regulations and standards, organisational capacity and models of care. Two new domains, 'communication' and 'satisfaction', were generated. Facilitators included active and regular interpersonal communication between users and providers; respect, confidentiality, comfort and support during care provision; engaging users in decision-making; continuity of care and effective audit and feedback mechanisms. Key barriers identified were language barriers in information and communication; power difference between users and providers; health systems not accounting for user satisfaction; variable standards of implementation of standard guidelines; shortage of resources in health facilities and lack of studies assessing the role of leadership in improving QoC. These were common across the three population groups. The barriers to good

The selectivity of receptor tyrosine kinase signaling is controlled by a secondary SH2 domain binding site.

Science.gov (United States)

Bae, Jae Hyun; Lew, Erin Denise; Yuzawa, Satoru; Tomé, Francisco; Lax, Irit; Schlessinger, Joseph

2009-08-07

SH2 domain-mediated interactions represent a crucial step in transmembrane signaling by receptor tyrosine kinases. SH2 domains recognize phosphotyrosine (pY) in the context of particular sequence motifs in receptor phosphorylation sites. However, the modest binding affinity of SH2 domains to pY containing peptides may not account for and likely represents an oversimplified mechanism for regulation of selectivity of signaling pathways in living cells. Here we describe the crystal structure of the activated tyrosine kinase domain of FGFR1 in complex with a phospholipase Cgamma fragment. The structural and biochemical data and experiments with cultured cells show that the selectivity of phospholipase Cgamma binding and signaling via activated FGFR1 are determined by interactions between a secondary binding site on an SH2 domain and a region in FGFR1 kinase domain in a phosphorylation independent manner. These experiments reveal a mechanism for how SH2 domain selectivity is regulated in vivo to mediate a specific cellular process.

Barriers and Facilitators to Self-Directed Learning in Continuing Professional Development for Physicians in Canada: A Scoping Review.

Science.gov (United States)

Jeong, Dahn; Presseau, Justin; ElChamaa, Rima; Naumann, Danielle N; Mascaro, Colin; Luconi, Francesca; Smith, Karen M; Kitto, Simon

2018-04-10

This scoping review explored the barriers and facilitators that influence engagement in and implementation of self-directed learning (SDL) in continuing professional development (CPD) for physicians in Canada. This review followed the six-stage scoping review framework of Arksey and O'Malley and of Daudt et al. In 2015, the authors searched eight online databases for English-language Canadian articles published January 2005-December 2015. To chart and analyze the data from the 17 included studies, they employed two-step analysis process of conventional content analysis followed by directed coding guided by the Theoretical Domains Framework (TDF). Conventional content analysis generated five categories of barriers and facilitators: individual, program, technological, environmental, and workplace/organizational. Directed coding guided by the TDF allowed analysis of barriers and facilitators to behavior change according to two key groups: physicians engaging in SDL and SDL developers designing and implementing SDL programs. Of the 318 total barriers and facilitators coded, 290 (91.2%) were coded for physicians and 28 (8.8%) for SDL developers. The majority (209; 65.7%) were coded in four key TDF domains: environmental context and resources, social influences, beliefs about consequences, and behavioral regulation. This scoping review identified five categories of barriers and facilitators in the literature and four key TDF domains where most factors related to behavior change of physicians and SDL developers regarding SDL programs in CPD were coded. There was a significant gap in the literature about factors that may contribute to SDL developers' capacity to design and implement SDL programs in CPD.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way

Probing SH2-domains using Inhibitor Affinity Purification (IAP).

Science.gov (United States)

Höfener, Michael; Heinzlmeir, Stephanie; Kuster, Bernhard; Sewald, Norbert

2014-01-01

Many human diseases are correlated with the dysregulation of signal transduction processes. One of the most important protein interaction domains in the context of signal transduction is the Src homology 2 (SH2) domain that binds phosphotyrosine residues. Hence, appropriate methods for the investigation of SH2 proteins are indispensable in diagnostics and medicinal chemistry. Therefore, an affinity resin for the enrichment of all SH2 proteins in one experiment would be desirable. However, current methods are unable to address all SH2 proteins simultaneously with a single compound or a small array of compounds. In order to overcome these limitations for the investigation of this particular protein family in future experiments, a dipeptide-derived probe has been designed, synthesized and evaluated. This probe successfully enriched 22 SH2 proteins from mixed cell lysates which contained 50 SH2 proteins. Further characterization of the SH2 binding properties of the probe using depletion and competition experiments indicated its ability to enrich complexes consisting of SH2 domain bearing regulatory PI3K subunits and catalytic phosphoinositide 3-kinase (PI3K) subunits that have no SH2 domain. The results make this probe a promising starting point for the development of a mixed affinity resin with complete SH2 protein coverage. Moreover, the additional findings render it a valuable tool for the evaluation of PI3K complex interrupting inhibitors.

Rosette Assay: Highly Customizable Dot-Blot for SH2 Domain Screening.

Science.gov (United States)

Ng, Khong Y; Machida, Kazuya

2017-01-01

With a growing number of high-throughput studies, structural analyses, and availability of protein-protein interaction databases, it is now possible to apply web-based prediction tools to SH2 domain-interactions. However, in silico prediction is not always reliable and requires experimental validation. Rosette assay is a dot blot-based reverse-phase assay developed for the assessment of binding between SH2 domains and their ligands. It is conveniently customizable, allowing for low- to high-throughput analysis of interactions between various numbers of SH2 domains and their ligands, e.g., short peptides, purified proteins, and cell lysates. The binding assay is performed in a 96-well plate (MBA or MWA apparatus) in which a sample spotted membrane is incubated with up to 96 labeled SH2 domains. Bound domains are detected and quantified using a chemiluminescence or near-infrared fluorescence (IR) imaging system. In this chapter, we describe a practical protocol for rosette assay to assess interactions between synthesized tyrosine phosphorylated peptides and a library of GST-tagged SH2 domains. Since the methodology is not confined to assessment of SH2-pTyr interactions, rosette assay can be broadly utilized for ligand and drug screening using different protein interaction domains or antibodies.

Analysis of Domain Architecture and Phylogenetics of Family 2 Glycoside Hydrolases (GH2.

Directory of Open Access Journals (Sweden)

David Talens-Perales

Full Text Available In this work we report a detailed analysis of the topology and phylogenetics of family 2 glycoside hydrolases (GH2. We distinguish five topologies or domain architectures based on the presence and distribution of protein domains defined in Pfam and Interpro databases. All of them share a central TIM barrel (catalytic module with two β-sandwich domains (non-catalytic at the N-terminal end, but differ in the occurrence and nature of additional non-catalytic modules at the C-terminal region. Phylogenetic analysis was based on the sequence of the Pfam Glyco_hydro_2_C catalytic module present in most GH2 proteins. Our results led us to propose a model in which evolutionary diversity of GH2 enzymes is driven by the addition of different non-catalytic domains at the C-terminal region. This model accounts for the divergence of β-galactosidases from β-glucuronidases, the diversification of β-galactosidases with different transglycosylation specificities, and the emergence of bicistronic β-galactosidases. This study also allows the identification of groups of functionally uncharacterized protein sequences with potential biotechnological interest.

The development and application of a quantitative peptide microarray platform to SH2 domain specificity space

Science.gov (United States)

Engelmann, Brett Warren

The Src homology 2 (SH2) domains evolved alongside protein tyrosine kinases (PTKs) and phosphatases (PTPs) in metazoans to recognize the phosphotyrosine (pY) post-translational modification. The human genome encodes 121 SH2 domains within 111 SH2 domain containing proteins that represent the primary mechanism for cellular signal transduction immediately downstream of PTKs. Despite pY recognition contributing to roughly half of the binding energy, SH2 domains possess substantial binding specificity, or affinity discrimination between phosphopeptide ligands. This specificity is largely imparted by amino acids (AAs) adjacent to the pY, typically from positions +1 to +4 C-terminal to the pY. Much experimental effort has been undertaken to construct preferred binding motifs for many SH2 domains. However, due to limitations in previous experimental methodologies these motifs do not account for the interplay between AAs. It was therefore not known how AAs within the context of individual peptides function to impart SH2 domain specificity. In this work we identified the critical role context plays in defining SH2 domain specificity for physiological ligands. We also constructed a high quality interactome using 50 SH2 domains and 192 physiological ligands. We next developed a quantitative high-throughput (Q-HTP) peptide microarray platform to assess the affinities four SH2 domains have for 124 physiological ligands. We demonstrated the superior characteristics of our platform relative to preceding approaches and validated our results using established biophysical techniques, literature corroboration, and predictive algorithms. The quantitative information provided by the arrays was leveraged to investigate SH2 domain binding distributions and identify points of binding overlap. Our microarray derived affinity estimates were integrated to produce quantitative interaction motifs capable of predicting interactions. Furthermore, our microarrays proved capable of resolving

Structure of the thioredoxin-fold domain of human phosducin-like protein 2

International Nuclear Information System (INIS)

Lou, Xiaochu; Bao, Rui; Zhou, Cong-Zhao; Chen, Yuxing

2009-01-01

The X-ray crystal structure of the Trx-fold domain of hPDCL2 was solved at 2.70 Å resolution and resembled the Trx-fold domain of rat phosducin. Human phosducin-like protein 2 (hPDCL2) has been identified as belonging to subgroup II of the phosducin (Pdc) family. The members of this family share an N-terminal helix domain and a C-terminal thioredoxin-fold (Trx-fold) domain. The X-ray crystal structure of the Trx-fold domain of hPDCL2 was solved at 2.70 Å resolution and resembled the Trx-fold domain of rat phosducin. Comparative structural analysis revealed the structural basis of their putative functional divergence

Facilitation as a teaching strategy : experiences of facilitators

Directory of Open Access Journals (Sweden)

E Lekalakala-Mokgele

2006-09-01

Full Text Available Changes in nursing education involve the move from traditional teaching approaches that are teacher-centred to facilitation, a student centred approach. The studentcentred approach is based on a philosophy of teaching and learning that puts the learner on centre-stage. The aim of this study was to identify the challenges of facilitators of learning using facilitation as a teaching method and recommend strategies for their (facilitators development and support. A qualitative, explorative and contextual design was used. Four (4 universities in South Africa which utilize facilitation as a teaching/ learning process were identified and the facilitators were selected to be the sample of the study. The main question posed during in-depth group interviews was: How do you experience facilitation as a teaching/learning method?. Facilitators indicated different experiences and emotions when they first had to facilitate learning. All of them indicated that it was difficult to facilitate at the beginning as they were trained to lecture and that no format for facilitation was available. They experienced frustrations and anxieties as a result. The lack of knowledge of facilitation instilled fear in them. However they indicated that facilitation had many benefits for them and for the students. Amongst the ones mentioned were personal and professional growth. Challenges mentioned were the fear that they waste time and that they do not cover the content. It is therefore important that facilitation be included in the training of nurse educators.

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.

Facilitators for practice change in Spanish community pharmacy.

Science.gov (United States)

Gastelurrutia, Miguel A; Benrimoj, S I Charlie; Castrillon, Carla C; de Amezua, María J Casado; Fernandez-Llimos, Fernando; Faus, Maria J

2009-02-01

To identify and prioritise facilitators for practice change in Spanish community pharmacy. Spanish community pharmacies. Qualitative study. Thirty-three semi-structured interviews were conducted with community pharmacists (n = 15) and pharmacy strategists (n = 18), and the results were examined using the content analysis method. In addition, two nominal groups (seven community pharmacists and seven strategists) were formed to identify and prioritise facilitators. Results of both techniques were then triangulated. Facilitators for practice change. Twelve facilitators were identified and grouped into four domains (D1: Pharmacist; D2: Pharmacy as an organisation; D3: Pharmaceutical profession; D4: Miscellaneous). Facilitators identified in D1 include: the need for more clinical education at both pre- and post-graduate levels; the need for clearer and unequivocal messages from professional leaders about the future of the professional practice; and the need for a change in pharmacists' attitudes. Facilitators in D2 are: the need to change the reimbursement system to accommodate cognitive service delivery as well as dispensing; and the need to change the front office of pharmacies. Facilitators identified in D3 are: the need for the Spanish National Professional Association to take a leadership role in the implementation of cognitive services; the need to reduce administrative workload; and the need for universities to reduce the gap between education and research. Other facilitators identified in this study include: the need to increase patients' demand for cognitive services at pharmacies; the need to improve pharmacist-physician relationships; the need for support from health care authorities; and the need for improved marketing of cognitive services and their benefits to society, including physicians and health care authorities. Twelve facilitators were identified. Strategists considered clinical education and pharmacists' attitude as the most important, and

Facilitation of learning: part 1.

Science.gov (United States)

Warburton, Tyler; Trish, Houghton; Barry, Debbie

2016-04-06

This article, the fourth in a series of 11, discusses the context for the facilitation of learning. It outlines the main principles and theories for understanding the process of learning, including examples which link these concepts to practice. The practical aspects of using these theories in a practice setting will be discussed in the fifth article of this series. Together, these two articles will provide mentors and practice teachers with knowledge of the learning process, which will enable them to meet the second domain of the Nursing and Midwifery Council's Standards to Support Learning and Assessment in Practice on facilitation of learning.

PDDL4J: a planning domain description library for java

Science.gov (United States)

Pellier, D.; Fiorino, H.

2018-01-01

PDDL4J (Planning Domain Description Library for Java) is an open source toolkit for Java cross-platform developers meant (1) to provide state-of-the-art planners based on the Pddl language, and (2) to facilitate research works on new planners. In this article, we present an overview of the Automated Planning concepts and languages. We present some planning systems and their most significant applications. Then, we detail the Pddl4j toolkit with an emphasis on the available informative structures, heuristics and search algorithms.

Modeling CO2-facilitated transport across a diethanolamine liquid membrane

Energy Technology Data Exchange (ETDEWEB)

Lihong Bao; Michael C. Trachtenberg [Carbozyme Inc., Monmouth Junction, NJ (United States)

2005-12-15

We compared experimental and model data for the facilitated transport of CO2 from a CO2-air mixture across an aqueous solution of diethanolamine (DEA) via a hollow fiber, contained liquid membrane (HFCLM) permeator. A two-step carbamate formation model was devised to analyze the data instead of the one-step mechanism used by previous investigators. The effects of DEA concentration, liquid membrane thickness and feed CO2 concentration were also studied. With a 20% (wt) DEA liquid membrane and feed of 15% CO2 in CO2-air mixture at atmosphere pressure, the permeance reached 1.51E-8 mol/m{sup 2} s Pa with a CO2/N2 selectivity of 115. Model predictions compared well with the experimental results at CO2 concentrations of industrial importance. Short-term stability of the HFCLM permeator performance was examined. The system was stable during 5-days of testing.

Transcriptional machinery of TNF-α-inducible YTH domain containing 2 (YTHDC2) gene.

Science.gov (United States)

Tanabe, Atsushi; Konno, Junpei; Tanikawa, Kenya; Sahara, Hiroeki

2014-02-01

We previously demonstrated that a cellular factor, cyclosporin A (CsA) associated helicase-like protein (CAHL) that is identical to YTH domain containing 2 (YTHDC2), forms trimer complex with cyclophilin B and NS5B of hepatitis C virus (HCV) and facilitates HCV genome replication. Gene expression of YTHDC2 was shown in tumor cell lines and tumor necrosis factor (TNF)-α-treated hepatocytes, but not in untreated. However, the function of YTHDC2 in the tumor cells and the mechanism by which the YTHDC2 gene is transcribed in these cells is largely unknown. We first evaluated that the role of YTHDC2 in the proliferation of hepatocellular carcinoma (HCC) cell line Huh7 using RNA interference and found that YTHDC2-downregulated Huh7 were significantly decreased cell growth as compared to control. We next demonstrated that the cAMP response element (CRE) site in the promoter region of the YTHDC2 gene is critical for YTHDC2 transcription. To further investigate the transcription factors bound to the CRE site, we performed chromatin immunoprecipitation assays. Our findings demonstrate that c-Jun and ATF-2 bind to the CRE site in Huh7, and that TNF-α induces the biological activity of these transcription factors in hepatocytes as well as Huh7. Moreover, treatment with the HDAC inhibitor, trichostatin A (TSA), reduces YTHDC2 expression in Huh7 and in TNF-α-stimulated hepatocytes. Collectively, these data show that YTHDC2 plays an important role in tumor cells growth and activation/recruitment of c-Jun and ATF-2 to the YTHDC2 promoter is necessary for the transcription of YTHDC2, and that HDAC activity is required for the efficient expression of YTHDC2 in both of hepatocyte and HCC cells. Copyright © 2013 Elsevier B.V. All rights reserved.

Polar Domain Discovery with Sparkler

Science.gov (United States)

Duerr, R.; Khalsa, S. J. S.; Mattmann, C. A.; Ottilingam, N. K.; Singh, K.; Lopez, L. A.

2017-12-01

The scientific web is vast and ever growing. It encompasses millions of textual, scientific and multimedia documents describing research in a multitude of scientific streams. Most of these documents are hidden behind forms which require user action to retrieve and thus can't be directly accessed by content crawlers. These documents are hosted on web servers across the world, most often on outdated hardware and network infrastructure. Hence it is difficult and time-consuming to aggregate documents from the scientific web, especially those relevant to a specific domain. Thus generating meaningful domain-specific insights is currently difficult. We present an automated discovery system (Figure 1) using Sparkler, an open-source, extensible, horizontally scalable crawler which facilitates high throughput and focused crawling of documents pertinent to a particular domain such as information about polar regions. With this set of highly domain relevant documents, we show that it is possible to answer analytical questions about that domain. Our domain discovery algorithm leverages prior domain knowledge to reach out to commercial/scientific search engines to generate seed URLs. Subject matter experts then annotate these seed URLs manually on a scale from highly relevant to irrelevant. We leverage this annotated dataset to train a machine learning model which predicts the `domain relevance' of a given document. We extend Sparkler with this model to focus crawling on documents relevant to that domain. Sparkler avoids disruption of service by 1) partitioning URLs by hostname such that every node gets a different host to crawl and by 2) inserting delays between subsequent requests. With an NSF-funded supercomputer Wrangler, we scaled our domain discovery pipeline to crawl about 200k polar specific documents from the scientific web, within a day.

The N-methyl-D-aspartate receptor subunits NR2A and NR2B bind to the SH2 domains of phospholipase C-gamma.

Science.gov (United States)

Gurd, J W; Bissoon, N

1997-08-01

The NMDA receptor has recently been found to be phosphorylated on tyrosine. To assess the possible connection between tyrosine phosphorylation of the NMDA receptor and signaling pathways in the postsynaptic cell, we have investigated the relationship between tyrosine phosphorylation and the binding of NMDA receptor subunits to the SH2 domains of phospholipase C-gamma (PLC-gamma). A glutathione S-transferase (GST) fusion protein containing both the N- and the C-proximal SH2 domains of PLC-gamma was bound to glutathione-agarose and reacted with synaptic junctional proteins and glycoproteins. Tyrosine-phosphorylated PSD-GP180, which has been identified as the NR2B subunit of the NMDA receptor, bound to the SH2-agarose beads in a phosphorylation-dependent fashion. Immunoblot analysis with antibodies specific for individual NMDA receptor subunits showed that both NR2A and NR2B subunits bound to the SH2-agarose. No binding occurred to GST-agarose lacking an associated SH2 domain, indicating that binding was specific for the SH2 domains. The binding of receptor subunits increased after the incubation of synaptic junctions with ATP and decreased after treatment of synaptic junctions with exogenous protein tyrosine phosphatase. Immunoprecipitation experiments confirmed that NR2A and NR2B were phosphorylated on tyrosine and further that tyrosine phosphorylation of each of the subunits was increased after incubation with ATP. The results demonstrate that NMDA receptor subunits NR2A and NR2B will bind to the SH2 domains of PLC-gamma and that isolated synaptic junctions contain endogenous protein tyrosine kinase(s) that can phosphorylate both NR2A and NR2B receptor subunits, and suggest that interaction of the tyrosine-phosphorylated NMDA receptor with proteins that contain SH2 domains may serve to link it to signaling pathways in the postsynaptic cell.

The BID Domain of Type IV Secretion Substrates Forms a Conserved Four-Helix Bundle Topped with a Hook.

Science.gov (United States)

Stanger, Frédéric V; de Beer, Tjaart A P; Dranow, David M; Schirmer, Tilman; Phan, Isabelle; Dehio, Christoph

2017-01-03

The BID (Bep intracellular delivery) domain functions as secretion signal in a subfamily of protein substrates of bacterial type IV secretion (T4S) systems. It mediates transfer of (1) relaxases and the attached DNA during bacterial conjugation, and (2) numerous Bartonella effector proteins (Beps) during protein transfer into host cells infected by pathogenic Bartonella species. Furthermore, BID domains of Beps have often evolved secondary effector functions within host cells. Here, we provide crystal structures for three representative BID domains and describe a novel conserved fold characterized by a compact, antiparallel four-helix bundle topped with a hook. The conserved hydrophobic core provides a rigid scaffold to a surface that, despite a few conserved exposed residues and similarities in charge distribution, displays significant variability. We propose that the genuine function of BID domains as T4S signal may primarily depend on their rigid structure, while the plasticity of their surface may facilitate adaptation to secondary effector functions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Tyrosine Phosphorylation of the Lyn Src Homology 2 (SH2) Domain Modulates Its Binding Affinity and Specificity*

Science.gov (United States)

Jin, Lily L.; Wybenga-Groot, Leanne E.; Tong, Jiefei; Taylor, Paul; Minden, Mark D.; Trudel, Suzanne; McGlade, C. Jane; Moran, Michael F.

2015-01-01

Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y194 impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y194 on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases. PMID:25587033

The Abl SH2-kinase linker naturally adopts a conformation competent for SH3 domain binding.

Science.gov (United States)

Chen, Shugui; Brier, Sébastien; Smithgall, Thomas E; Engen, John R

2007-04-01

The core of the Abelson tyrosine kinase (c-Abl) is structurally similar to Src-family kinases where SH3 and SH2 domains pack against the backside of the kinase domain in the down-regulated conformation. Both kinase families depend upon intramolecular association of SH3 with the linker joining the SH2 and kinase domains for suppression of kinase activity. Hydrogen deuterium exchange (HX) and mass spectrometry (MS) were used to probe intramolecular interaction of the c-Abl SH3 domain with the linker in recombinant constructs lacking the kinase domain. Under physiological conditions, the c-Abl SH3 domain undergoes partial unfolding, which is stabilized by ligand binding, providing a unique assay for SH3:linker interaction in solution. Using this approach, we observed dynamic association of the SH3 domain with the linker in the absence of the kinase domain. Truncation of the linker before W254 completely prevented cis-interaction with SH3, while constructs containing amino acids past this point showed SH3:linker interactions. The observation that the Abl linker sequence exhibits SH3-binding activity in the absence of the kinase domain is unique to Abl and was not observed with Src-family kinases. These results suggest that SH3:linker interactions may have a more prominent role in Abl regulation than in Src kinases, where the down-regulated conformation is further stabilized by a second intramolecular interaction between the C-terminal tail and the SH2 domain.

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.

A novel disulfide bond in the SH2 Domain of the C-terminal Src kinase controls catalytic activity.

Science.gov (United States)

Mills, Jamie E; Whitford, Paul C; Shaffer, Jennifer; Onuchic, Jose N; Adams, Joseph A; Jennings, Patricia A

2007-02-02

The SH2 domain of the C-terminal Src kinase [Csk] contains a unique disulfide bond that is not present in other known SH2 domains. To investigate whether this unusual disulfide bond serves a novel function, the effects of disulfide bond formation on catalytic activity of the full-length protein and on the structure of the SH2 domain were investigated. The kinase activity of full-length Csk decreases by an order of magnitude upon formation of the disulfide bond in the distal SH2 domain. NMR spectra of the fully oxidized and fully reduced SH2 domains exhibit similar chemical shift patterns and are indicative of similar, well-defined tertiary structures. The solvent-accessible disulfide bond in the isolated SH2 domain is highly stable and far from the small lobe of the kinase domain. However, reduction of this bond results in chemical shift changes of resonances that map to a cluster of residues that extend from the disulfide bond across the molecule to a surface that is in direct contact with the small lobe of the kinase domain in the intact molecule. Normal mode analyses and molecular dynamics calculations suggest that disulfide bond formation has large effects on residues within the kinase domain, most notably within the active-site cleft. Overall, the data indicate that reversible cross-linking of two cysteine residues in the SH2 domain greatly impacts catalytic function and interdomain communication in Csk.

In Vitro Maturation of a Humanized Shark VNAR Domain to Improve Its Biophysical Properties to Facilitate Clinical Development

Directory of Open Access Journals (Sweden)

John Steven

2017-10-01

Full Text Available Molecular engineering to increase the percentage identity to common human immunoglobulin sequences of non-human therapeutic antibodies and scaffolds has become standard practice. This strategy is often used to reduce undesirable immunogenic responses, accelerating the clinical development of candidate domains. The first humanized shark variable domain (VNAR was reported by Kovalenko and colleagues and used the anti-human serum albumin (HSA domain, clone E06, as a model to construct a number of humanized versions including huE06v1.10. This study extends this work by using huE06v1.10 as a template to isolate domains with improved biophysical properties and reduced antigenicity. Random mutagenesis was conducted on huE06v1.10 followed by refinement of clones through an off-rate ranking-based selection on target antigen. Many of these next-generation binders retained high affinity for target, together with good species cross-reactivity. Lead domains were assessed for any tendency to dimerize, tolerance to N- and C-terminal fusions, affinity, stability, and relative antigenicity in human dendritic cell assays. Functionality of candidate clones was verified in vivo through the extension of serum half-life in a typical drug format. From these analyses the domain, BA11, exhibited negligible antigenicity, high stability and high affinity for mouse, rat, and HSA. When these attributes were combined with demonstrable functionality in a rat model of PK, the BA11 clone was established as our clinical candidate.

Design of potentially active ligands for SH2 domains by molecular modeling methods

Directory of Open Access Journals (Sweden)

Hurmach V. V.

2014-07-01

Full Text Available Search for new chemical structures possessing specific biological activity is a complex problem that needs the use of the latest achievements of molecular modeling technologies. It is well known that SH2 domains play a major role in ontogenesis as intermediaries of specific protein-protein interactions. Aim. Developing an algorithm to investigate the properties of SH2 domain binding, search for new potential active compounds for the whole SH2 domains class. Methods. In this paper, we utilize a complex of computer modeling methods to create a generic set of potentially active compounds targeting universally at the whole class of SH2 domains. A cluster analysis of all available three-dimensional structures of SH2 domains was performed and general pharmacophore models were formulated. The models were used for virtual screening of collection of drug-like compounds provided by Enamine Ltd. Results. The design technique for library of potentially active compounds for SH2 domains class was proposed. Conclusions. The original algorithm of SH2 domains research with molecular docking method was developed. Using our algorithm, the active compounds for SH2 domains were found.

The LIM domain protein FHL2 interacts with the NR5A family of nuclear receptors and CREB to activate the inhibin-α subunit gene in ovarian granulosa cells.

Science.gov (United States)

Matulis, Christina K; Mayo, Kelly E

2012-08-01

Nuclear receptor transcriptional activity is enhanced by interaction with coactivators. The highly related nuclear receptor 5A (NR5A) subfamily members liver receptor homolog 1 and steroidogenic factor 1 bind to and activate several of the same genes, many of which are important for reproductive function. To better understand transcriptional activation by these nuclear receptors, we sought to identify interacting proteins that might function as coactivators. The LIM domain protein four and a half LIM domain 2 (FHL2) was identified as interacting with the NR5A receptors in a yeast two-hybrid screen of a human ovary cDNA library. FHL2, and the closely related FHL1, are both expressed in the rodent ovary and in granulosa cells. Small interfering RNA-mediated knockdown of FHL1 and FHL2 in primary mouse granulosa cells reduced expression of the NR5A target genes encoding inhibin-α and P450scc. In vitro assays confirmed the interaction between the FHL and NR5A proteins and revealed that a single LIM domain of FHL2 is sufficient for this interaction, whereas determinants in both the ligand binding domain and DNA binding domain of NR5A proteins are important. FHL2 enhances the ability of both liver receptor homolog 1 and steroidogenic factor 1 to activate the inhibin-α subunit gene promoter in granulosa cells and thus functions as a transcriptional coactivator. FHL2 also interacts with cAMP response element-binding protein and substantially augments activation of inhibin gene expression by the combination of NR5A receptors and forskolin, suggesting that FHL2 may facilitate integration of these two signals. Collectively these results identify FHL2 as a novel coactivator of NR5A nuclear receptors in ovarian granulosa cells and suggest its involvement in regulating target genes important for mammalian reproduction.

Tyrosine phosphorylation of the Lyn Src homology 2 (SH2) domain modulates its binding affinity and specificity.

Science.gov (United States)

Jin, Lily L; Wybenga-Groot, Leanne E; Tong, Jiefei; Taylor, Paul; Minden, Mark D; Trudel, Suzanne; McGlade, C Jane; Moran, Michael F

2015-03-01

Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y(194) impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y(194) on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Preliminary crystallographic characterization of the Grb2 SH2 domain in complex with a FAK-derived phosphotyrosyl peptide

International Nuclear Information System (INIS)

Chen, Hsiao-Hsin; Chen, Cuei-Wen; Chang, Yu-Yung; Shen, Tang-Long; Hsu, Chun-Hua

2010-01-01

Crystals of the Grb2 SH2 domain in complex with a phosphotyrosyl peptide corresponding to residues 921–930 of focal adhesion kinase (FAK) have been obtained using the sitting-drop vapour-diffusion technique. Data have been collected to 2.49 Å resolution. Growth factor receptor-bound protein 2 (Grb2) is an adaptor protein with a single SH2 domain that specifically binds to focal adhesion kinase (FAK) when residue Tyr925 of FAK is phosphorylated. The Grb2–FAK interaction is associated with cellular integrin-activated signal transduction events leading to the activation of the Ras-MAPK pathway. Crystals of the Grb2 SH2 domain in complex with a phosphopeptide corresponding to residues 921–930 of FAK have been obtained using the sitting-drop vapour-diffusion technique. The crystals belonged to space group P3 1 21, with unit-cell parameters a = b = 102.7, c = 127.6 Å, α = β = 90.0, γ = 120.0°. A diffraction data set was collected from a flash-cooled crystal at 100 K to 2.49 Å resolution using synchrotron radiation. Structure determination by molecular replacement and analysis of the detailed structure of the complex are currently in progress

Crystallization and preliminary crystallographic studies of the W2 domain of Drosophila melanogaster eukaryotic translation initiation factor 5C domain-containing protein

International Nuclear Information System (INIS)

Zhao, Hui; Wang, Hong; Liu, Huihui; Teng, Maikun; Li, Xu

2012-01-01

The crystallization and preliminary crystallographic studies of the carboxy-terminal domain of D. melanogaster eukaryotic translation initiation factor 5C domain-containing protein are reported. The Drosophila melanogaster eukaryotic translation initiation factor 5C domain-containing protein (ECP) is composed of two independently folded domains which belong to the basic leucine-zipper and W2 domain-containing protein (BZW) family. Based on the sequence similarity between the C-terminal W2 domain of ECP and some eukaryotic translation initiation factors (such as eIF2B∊, eIF4γ, eIF5 etc.), ECP has been speculated to participate in the translation initiation process. Structural information on the C-terminal W2 domain of ECP would be helpful in understanding the specific cellular function of this protein. Here, the W2 domain of ECP was expressed and crystallized. Crystals grown by the hanging-drop vapour-diffusion method diffracted to 2.70 Å resolution and belonged to space group I4, with unit-cell parameters a = b = 81.05, c = 57.44 Å. The Matthews coefficient suggested that there was one molecule per asymmetric unit in the crystal

Crystal Structure of a Complex of the Intracellular Domain of Interferon λ Receptor 1 (IFNLR1) and the FERM/SH2 Domains of Human JAK1.

Science.gov (United States)

Zhang, Di; Wlodawer, Alexander; Lubkowski, Jacek

2016-11-20

The crystal structure of a construct consisting of the FERM and SH2-like domains of the human Janus kinase 1 (JAK1) bound to a fragment of the intracellular domain of the interferon-λ receptor 1 (IFNLR1) has been determined at the nominal resolution of 2.1Å. In this structure, the receptor peptide forms an 85-Å-long extended chain, in which both the previously identified box1 and box2 regions bind simultaneously to the FERM and SH2-like domains of JAK1. Both domains of JAK1 are generally well ordered, with regions not seen in the crystal structure limited to loops located away from the receptor-binding regions. The structure provides a much more complete and accurate picture of the interactions between JAK1 and IFNLR1 than those given in earlier reports, illuminating the molecular basis of the JAK-cytokine receptor association. A glutamate residue adjacent to the box2 region in IFNLR1 mimics the mode of binding of a phosphotyrosine in classical SH2 domains. It was shown here that a deletion of residues within the box1 region of the receptor abolishes stable interactions with JAK1, although it was previously shown that box2 alone is sufficient to stabilize a similar complex of the interferon-α receptor and TYK2. Published by Elsevier Ltd.

Quantifying information transfer by protein domains: Analysis of the Fyn SH2 domain structure

DEFF Research Database (Denmark)

Lenaerts, Tom; Ferkinghoff-Borg, Jesper; Stricher, Francois

2008-01-01

instance of communication over a noisy channel. In particular, we analyze the conformational correlations between protein residues and apply the concept of mutual information to quantify information exchange. Mapping out changes of mutual information on the protein structure then allows visualizing how...... distal communication is achieved. We illustrate the approach by analyzing information transfer by the SH2 domain of Fyn tyrosine kinase, obtained from Monte Carlo dynamics simulations. Our analysis reveals that the Fyn SH2 domain forms a noisy communication channel that couples residues located......Background: Efficient communication between distant sites within a protein is essential for cooperative biological response. Although often associated with large allosteric movements, more subtle changes in protein dynamics can also induce long-range correlations. However, an appropriate formalism...

Barriers and facilitators to disease-modifying antirheumatic drug use in patients with inflammatory rheumatic diseases: a qualitative theory-based study.

Science.gov (United States)

Voshaar, Marieke; Vriezekolk, Johanna; van Dulmen, Sandra; van den Bemt, Bart; van de Laar, Mart

2016-10-21

Although disease-modifying anti-rheumatic drugs (DMARDs) are the cornerstone of treatment for inflammatory rheumatic diseases, medication adherence to DMARDs is often suboptimal. Effective interventions to improve adherence to DMARDs are lacking, and new targets are needed to improve adherence. The aim of the present study was to explore patients' barriers and facilitators of optimal DMARD use. These factors might be used as targets for adherence interventions. In a mixed method study design, patients (n = 120) with inflammatory arthritis (IA) completed a questionnaire based on an existing adapted Theoretical Domains Framework (TDF) to identify facilitators and barriers of DMARD use. A subgroup of these patients (n = 21) participated in focus groups to provide insights into their facilitators and barriers. The answers to the questionnaires and responses of the focus groups were thematically coded by three researchers independently and subsequently categorized. The barriers and facilitators that were reported by IA patients presented large inter-individual variations. The identified barriers and facilitators could be captured in the following domains based on an adapted TDF: (i) knowledge, (ii) emotions, (iii) attention, memory, and decision processes, (iv) social influences, (v) beliefs about capability, (vi) beliefs about consequences, (vii) motivation and goals, (viii) goal conflict, (ix) environmental context and resources, and (x) skills. Patients with IA have a variety of barriers and facilitators with regard to their DMARD use. All of these barriers and facilitators could be categorized into adapted domains of the TDF. Interventions that address individual facilitators and barriers, based on capability, opportunity, and motivation, are needed to develop strategies for medication adherence that are tailored to individual patient needs.

Direct Binding between Pre-S1 and TRP-like Domains in TRPP Channels Mediates Gating and Functional Regulation by PIP2

Directory of Open Access Journals (Sweden)

Wang Zheng

2018-02-01

Full Text Available Transient receptor potential (TRP channels are regulated by diverse stimuli comprising thermal, chemical, and mechanical modalities. They are also commonly regulated by phosphatidylinositol-4,5-bisphosphate (PIP2, with underlying mechanisms largely unknown. We here revealed an intramolecular interaction of the TRPP3 N and C termini (N-C that is functionally essential. The interaction was mediated by aromatic Trp81 in pre-S1 domain and cationic Lys568 in TRP-like domain. Structure-function analyses revealed similar N-C interaction in TRPP2 as well as TRPM8/-V1/-C4 via highly conserved tryptophan and lysine/arginine residues. PIP2 bound to cationic residues in TRPP3, including K568, thereby disrupting the N-C interaction and negatively regulating TRPP3. PIP2 had similar negative effects on TRPP2. Interestingly, we found that PIP2 facilitates the N-C interaction in TRPM8/-V1, resulting in channel potentiation. The intramolecular N-C interaction might represent a shared mechanism underlying the gating and PIP2 regulation of TRP channels.

Secretory Vesicle Priming by CAPS Is Independent of Its SNARE-Binding MUN Domain

Directory of Open Access Journals (Sweden)

Cuc Quynh Nguyen Truong

2014-11-01

Full Text Available Priming of secretory vesicles is a prerequisite for their Ca2+-dependent fusion with the plasma membrane. The key vesicle priming proteins, Munc13s and CAPSs, are thought to mediate vesicle priming by regulating the conformation of the t-SNARE syntaxin, thereby facilitating SNARE complex assembly. Munc13s execute their priming function through their MUN domain. Given that the MUN domain of Ca2+-dependent activator protein for secretion (CAPS also binds syntaxin, it was assumed that CAPSs prime vesicles through the same mechanism as Munc13s. We studied naturally occurring splice variants of CAPS2 in CAPS1/CAPS2-deficient cells and found that CAPS2 primes vesicles independently of its MUN domain. Instead, the pleckstrin homology domain of CAPS2 seemingly is essential for its priming function. Our findings indicate a priming mode for secretory vesicles. This process apparently requires membrane phospholipids, does not involve the binding or direct conformational regulation of syntaxin by MUN domains of CAPSs, and is therefore not redundant with Munc13 action.

Structural domains required for channel function of the mouse transient receptor potential protein homologue TRP1beta.

Science.gov (United States)

Engelke, Michael; Friedrich, Olaf; Budde, Petra; Schäfer, Christina; Niemann, Ursula; Zitt, Christof; Jüngling, Eberhard; Rocks, Oliver; Lückhoff, Andreas; Frey, Jürgen

2002-07-17

Transient receptor potential proteins (TRP) are supposed to participate in the formation of store-operated Ca(2+) influx channels by co-assembly. However, little is known which domains facilitate the interaction of subunits. Contribution of the N-terminal coiled-coil domain and ankyrin-like repeats and the putative pore region of the mouse TRP1beta (mTRP1beta) variant to the formation of functional cation channels were analyzed following overexpression in HEK293 (human embryonic kidney) cells. MTRP1beta expressing cells exhibited enhanced Ca(2+) influx and enhanced whole-cell membrane currents compared to mTRP1beta deletion mutants. Using a yeast two-hybrid assay only the coiled-coil domain facilitated homodimerization of the N-terminus. These results suggest that the N-terminus of mTRP1beta is required for structural organization thus forming functional channels.

SH2 Domains Serve as Lipid-Binding Modules for pTyr-Signaling Proteins.

Science.gov (United States)

Park, Mi-Jeong; Sheng, Ren; Silkov, Antonina; Jung, Da-Jung; Wang, Zhi-Gang; Xin, Yao; Kim, Hyunjin; Thiagarajan-Rosenkranz, Pallavi; Song, Seohyeon; Yoon, Youngdae; Nam, Wonhee; Kim, Ilshin; Kim, Eui; Lee, Dong-Gyu; Chen, Yong; Singaram, Indira; Wang, Li; Jang, Myoung Ho; Hwang, Cheol-Sang; Honig, Barry; Ryu, Sungho; Lorieau, Justin; Kim, You-Me; Cho, Wonhwa

2016-04-07

The Src-homology 2 (SH2) domain is a protein interaction domain that directs myriad phosphotyrosine (pY)-signaling pathways. Genome-wide screening of human SH2 domains reveals that ∼90% of SH2 domains bind plasma membrane lipids and many have high phosphoinositide specificity. They bind lipids using surface cationic patches separate from pY-binding pockets, thus binding lipids and the pY motif independently. The patches form grooves for specific lipid headgroup recognition or flat surfaces for non-specific membrane binding and both types of interaction are important for cellular function and regulation of SH2 domain-containing proteins. Cellular studies with ZAP70 showed that multiple lipids bind its C-terminal SH2 domain in a spatiotemporally specific manner and thereby exert exquisite spatiotemporal control over its protein binding and signaling activities in T cells. Collectively, this study reveals how lipids control SH2 domain-mediated cellular protein-protein interaction networks and suggest a new strategy for therapeutic modulation of pY-signaling pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

Conformational determinants of phosphotyrosine peptides complexed with the Src SH2 domain.

Directory of Open Access Journals (Sweden)

Joseph Nachman

2010-06-01

Full Text Available The inhibition of specific SH2 domain mediated protein-protein interactions as an effective chemotherapeutic approach in the treatment of diseases remains a challenge. That different conformations of peptide-ligands are preferred by different SH2 domains is an underappreciated observation from the structural analysis of phosphotyrosine peptide binding to SH2 domains that may aid in future drug design. To explore the nature of ligand binding, we use simulated annealing (SA to sample the conformational space of phosphotyrosine-containing peptides complexed with the Src SH2 domain. While in good agreement with the crystallographic and NMR studies of high-affinity phosphopeptide-SH2 domain complexes, the results suggest that the structural basis for phopsphopeptide- Src SH2 interactions is more complex than the "two-pronged plug two-hole socket" model. A systematic study of peptides of type pYEEX, where pY is phosphotyrosine and X is a hydrophobic residue, indicates that these peptides can assume two conformations, one extended and one helical, representing the balance between the interaction of residue X with the hydrophobic hole on the surface of the Src SH2 domain, and its contribution to the inherent tendency of the two glutamic acids to form an alpha-helix. In contrast, a beta-turn conformation, almost identical to that observed in the crystal structure of pYVNV bound to the Grb2 SH2 domain, predominates for pYXNX peptides, even in the presence of isoleucine at the third position. While peptide binding affinities, as measured by fluorescence polarization, correlate with the relative proportion of extended peptide conformation, these results suggest a model where all three residues C-terminal to the phosphotyrosine determine the conformation of the bound phosphopeptide. The information obtained in this work can be used in the design of specific SH2 domain inhibitors.

Critical role of the FERM domain in Pyk2 stimulated glioma cell migration

International Nuclear Information System (INIS)

Lipinski, Christopher A.; Tran, Nhan L.; Dooley, Andrea; Pang, Yuan-Ping; Rohl, Carole; Kloss, Jean; Yang, Zhongbo; McDonough, Wendy; Craig, David; Berens, Michael E.; Loftus, Joseph C.

2006-01-01

The strong tendency of malignant glioma cells to invade locally into surrounding normal brain precludes effective surgical resection, reduces the efficacy of radiotherapy, and is associated with increased resistance to chemotherapy regimens. We report that the N-terminal FERM domain of Pyk2 regulates its promigratory function. A 3-dimensional model of the Pyk2 FERM domain was generated and mutagenesis studies identified residues essential for Pyk2 promigratory function. Model-based targeted mutations within the FERM domain decreased Pyk2 phosphorylation and reduced the capacity of Pyk2 to stimulate glioma cell migration but did not significantly alter the intracellular distribution of Pyk2. Expression of autonomous Pyk2 FERM domain fragments containing analogous mutations exhibited reduced capacity to inhibit glioma cell migration and Pyk2 phosphorylation relative to expression of an autonomous wild type FERM domain fragment. These results indicate that the FERM domain plays an important role in regulating the functional competency of Pyk2 as a promigratory factor in glioma

The 1.7 Å X-ray crystal structure of the porcine factor VIII C2 domain and binding analysis to anti-human C2 domain antibodies and phospholipid surfaces.

Directory of Open Access Journals (Sweden)

Caileen M Brison

Full Text Available The factor VIII C2 domain is essential for binding to activated platelet surfaces as well as the cofactor activity of factor VIII in blood coagulation. Inhibitory antibodies against the C2 domain commonly develop following factor VIII replacement therapy for hemophilia A patients, or they may spontaneously arise in cases of acquired hemophilia. Porcine factor VIII is an effective therapeutic for hemophilia patients with inhibitor due to its low cross-reactivity; however, the molecular basis for this behavior is poorly understood. In this study, the X-ray crystal structure of the porcine factor VIII C2 domain was determined, and superposition of the human and porcine C2 domains demonstrates that most surface-exposed differences cluster on the face harboring the "non-classical" antibody epitopes. Furthermore, antibody-binding results illustrate that the "classical" 3E6 antibody can bind both the human and porcine C2 domains, although the inhibitory titer to human factor VIII is 41 Bethesda Units (BU/mg IgG versus 0.8 BU/mg IgG to porcine factor VIII, while the non-classical G99 antibody does not bind to the porcine C2 domain nor inhibit porcine factor VIII activity. Further structural analysis of differences between the electrostatic surface potentials suggest that the C2 domain binds to the negatively charged phospholipid surfaces of activated platelets primarily through the 3E6 epitope region. In contrast, the G99 face, which contains residue 2227, should be distal to the membrane surface. Phospholipid binding assays indicate that both porcine and human factor VIII C2 domains bind with comparable affinities, and the human K2227A and K2227E mutants bind to phospholipid surfaces with similar affinities as well. Lastly, the G99 IgG bound to PS-immobilized factor VIII C2 domain with an apparent dissociation constant of 15.5 nM, whereas 3E6 antibody binding to PS-bound C2 domain was not observed.

Autophagy Facilitates IFN-γ-induced Jak2-STAT1 Activation and Cellular Inflammation*

Science.gov (United States)

Chang, Yu-Ping; Tsai, Cheng-Chieh; Huang, Wei-Ching; Wang, Chi-Yun; Chen, Chia-Ling; Lin, Yee-Shin; Kai, Jui-In; Hsieh, Chia-Yuan; Cheng, Yi-Lin; Choi, Pui-Ching; Chen, Shun-Hua; Chang, Shih-Ping; Liu, Hsiao-Sheng; Lin, Chiou-Feng

2010-01-01

Autophagy is regulated for IFN-γ-mediated antimicrobial efficacy; however, its molecular effects for IFN-γ signaling are largely unknown. Here, we show that autophagy facilitates IFN-γ-activated Jak2-STAT1. IFN-γ induces autophagy in wild-type but not in autophagy protein 5 (Atg5−/−)-deficient mouse embryonic fibroblasts (MEFs), and, autophagy-dependently, IFN-γ induces IFN regulatory factor 1 and cellular inflammatory responses. Pharmacologically inhibiting autophagy using 3-methyladenine, a known inhibitor of class III phosphatidylinositol 3-kinase, confirms these effects. Either Atg5−/− or Atg7−/− MEFs are, independent of changes in IFN-γ receptor expression, resistant to IFN-γ-activated Jak2-STAT1, which suggests that autophagy is important for IFN-γ signal transduction. Lentivirus-based short hairpin RNA for Atg5 knockdown confirmed the importance of autophagy for IFN-γ-activated STAT1. Without autophagy, reactive oxygen species increase and cause SHP2 (Src homology-2 domain-containing phosphatase 2)-regulated STAT1 inactivation. Inhibiting SHP2 reversed both cellular inflammation and the IFN-γ-induced activation of STAT1 in Atg5−/− MEFs. Our study provides evidence that there is a link between autophagy and both IFN-γ signaling and cellular inflammation and that autophagy, because it inhibits the expression of reactive oxygen species and SHP2, is pivotal for Jak2-STAT1 activation. PMID:20592027

Structure of the interleukin-2 tyrosine kinase Src homology 2 domain; comparison between X-ray and NMR-derived structures

International Nuclear Information System (INIS)

Joseph, Raji E.; Ginder, Nathaniel D.; Hoy, Julie A.; Nix, Jay C.; Fulton, D. Bruce; Honzatko, Richard B.; Andreotti, Amy H.

2012-01-01

The interleukin-2 tyrosine kinase Src homology 2 domain was crystallized and its structure was solved to 2.35 Å resolution. The structure reveals a domain-swapped dimer that is related to other dimeric SH2 domains solved previously. The cis–trans-prolyl isomerization that is evident from solution studies of Itk SH2 cannot be observed in the crystal structure. The crystal structure of the interleukin-2 tyrosine kinase Src homology domain (Itk SH2) is described and it is found that unlike in studies of this domain using NMR spectroscopy, cis–trans-prolyl isomerization is not readily detected in the crystal structure. Based on similarities between the Itk SH2 crystal form and the cis form of the Itk SH2 NMR structure, it is concluded that it is likely that the prolyl imide bond at least in part adopts the cis conformation in the crystal form. However, the lack of high-resolution data and the dynamic nature of the proline-containing loop mean that the precise imide-bond conformation cannot be determined and prolyl cis–trans isomerization in the crystal cannot be ruled out. Given the preponderance of structures that have been solved by X-ray crystallography in the Protein Data Bank, this result supports the notion that prolyl isomerization in folded proteins has been underestimated among known structures. Interestingly, while the precise status of the proline residue is ambiguous, Itk SH2 crystallizes as a domain-swapped dimer. The domain-swapped structure of Itk SH2 is similar to the domain-swapped SH2 domains of Grb2 and Nck, with domain swapping occurring at the β-meander region of all three SH2 domains. Thus, for Itk SH2 structural analysis by NMR spectroscopy and X-ray crystallography revealed very different structural features: proline isomerization versus domain-swapped dimerization, respectively

Arid5b facilitates chondrogenesis by recruiting the histone demethylase Phf2 to Sox9-regulated genes

Science.gov (United States)

Hata, Kenji; Takashima, Rikako; Amano, Katsuhiko; Ono, Koichiro; Nakanishi, Masako; Yoshida, Michiko; Wakabayashi, Makoto; Matsuda, Akio; Maeda, Yoshinobu; Suzuki, Yutaka; Sugano, Sumio; Whitson, Robert H.; Nishimura, Riko; Yoneda, Toshiyuki

2013-11-01

Histone modification, a critical step for epigenetic regulation, is an important modulator of biological events. Sox9 is a transcription factor critical for endochondral ossification; however, proof of its epigenetic regulation remains elusive. Here we identify AT-rich interactive domain 5b (Arid5b) as a transcriptional co-regulator of Sox9. Arid5b physically associates with Sox9 and synergistically induces chondrogenesis. Growth of Arid5b-/- mice is retarded with delayed endochondral ossification. Sox9-dependent chondrogenesis is attenuated in Arid5b-deficient cells. Arid5b recruits Phf2, a histone lysine demethylase, to the promoter region of Sox9 target genes and stimulates H3K9me2 demethylation of these genes. In the promoters of chondrogenic marker genes, H3K9me2 levels are increased in Arid5b-/- chondrocytes. Finally, we show that Phf2 knockdown inhibits Sox9-induced chondrocyte differentiation. Our findings establish an epigenomic mechanism of skeletal development, whereby Arid5b promotes chondrogenesis by facilitating Phf2-mediated histone demethylation of Sox9-regulated chondrogenic gene promoters.

Exercise in patients with Type 2 diabetes: Facilitators and barriers - A qualitative study

Directory of Open Access Journals (Sweden)

T S Advika

2017-01-01

Full Text Available Introduction: Diabetes is a major noncommunicable disease affecting more than 65 million Indians. Although treatment algorithms suggest lifestyle measures (diet and exercise along with medications data regarding adherence to exercise as well as facilitators and barriers to the practice of physical activity in such patients are limited. Hence, this qualitative study was conducted. Objectives: The objective of this study is to describe the factors which (1 Facilitated and (2 hindered the practice of regular exercise in patients with Type 2 diabetes. Methodology: The study was conducted on 13 diabetic patients admitted to a tertiary care center in Bengaluru - St. John's Medical College Hospital, to explore factors that acted as facilitators and barriers to physical activity. Data saturation with the coded themes was achieved on interviewing 13 patients, after which, thematic analysis was done, and final themes reported. Results: The age of the study participants (7 males, 6 females ranged from 40 to 80 years. Among those who did exercise, factors such as awareness regarding the benefits of exercise and complications linked with diabetes, positive family support, and emphasis by nursing staff emerged as facilitators. Lack of time, obligations to others, inability to link exercise with blood sugar control, lack of perception of obesity as a health issue, inadequate emphasis by physicians, social/cultural issues, lack of infrastructure, and physical restriction were the factors that acted as barriers to physical activity. In addition to the above, a clear lack of adherence to standard guidelines, while advising patients by physicians was also noted. Conclusion: A comprehensive approach by both doctors and nurses, based on standard guidelines, could help in implementing adherence to exercise in patients with diabetes.

Jarid2 binds mono-ubiquitylated H2A lysine 119 to mediate crosstalk between Polycomb complexes PRC1 and PRC2

DEFF Research Database (Denmark)

Cooper, Sarah; Grijzenhout, Anne; Underwood, Elizabeth

2016-01-01

crosstalk between these modifications is critical for the formation of stable Polycomb domains at target gene loci. While the molecular mechanism for recognition of H3K27me3 by PRC1 is well defined, the interaction of PRC2 with H2AK119u1 is poorly understood. Here we demonstrate a critical role for the PRC2...... cofactor Jarid2 in mediating the interaction of PRC2 with H2AK119u1. We identify a ubiquitin interaction motif at the amino-terminus of Jarid2, and demonstrate that this domain facilitates PRC2 localization to H2AK119u1 both in vivo and in vitro. Our findings ascribe a critical function to Jarid2...... and define a key mechanism that links PRC1 and PRC2 in the establishment of Polycomb domains....

Itk tyrosine kinase substrate docking is mediated by a nonclassical SH2 domain surface of PLCgamma1.

Science.gov (United States)

Min, Lie; Joseph, Raji E; Fulton, D Bruce; Andreotti, Amy H

2009-12-15

Interleukin-2 tyrosine kinase (Itk) is a Tec family tyrosine kinase that mediates signaling processes after T cell receptor engagement. Activation of Itk requires recruitment to the membrane via its pleckstrin homology domain, phosphorylation of Itk by the Src kinase, Lck, and binding of Itk to the SLP-76/LAT adapter complex. After activation, Itk phosphorylates and activates phospholipase C-gamma1 (PLC-gamma1), leading to production of two second messengers, DAG and IP(3). We have previously shown that phosphorylation of PLC-gamma1 by Itk requires a direct, phosphotyrosine-independent interaction between the Src homology 2 (SH2) domain of PLC-gamma1 and the kinase domain of Itk. We now define this docking interface using a combination of mutagenesis and NMR spectroscopy and show that disruption of the Itk/PLCgamma1 docking interaction attenuates T cell signaling. The binding surface on PLCgamma1 that mediates recognition by Itk highlights a nonclassical binding activity of the well-studied SH2 domain providing further evidence that SH2 domains participate in important signaling interactions beyond recognition of phosphotyrosine.

Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain

Science.gov (United States)

Mistry, Pragnesh; Laird, Michelle H. W.; Schwarz, Ryan S.; Greene, Shannon; Dyson, Tristan; Snyder, Greg A.; Xiao, Tsan Sam; Chauhan, Jay; Fletcher, Steven; Toshchakov, Vladimir Y.; MacKerell, Alexander D.; Vogel, Stefanie N.

2015-01-01

Toll-like receptor (TLR) signaling is initiated by dimerization of intracellular Toll/IL-1 receptor resistance (TIR) domains. For all TLRs except TLR3, recruitment of the adapter, myeloid differentiation primary response gene 88 (MyD88), to TLR TIR domains results in downstream signaling culminating in proinflammatory cytokine production. Therefore, blocking TLR TIR dimerization may ameliorate TLR2-mediated hyperinflammatory states. The BB loop within the TLR TIR domain is critical for mediating certain protein–protein interactions. Examination of the human TLR2 TIR domain crystal structure revealed a pocket adjacent to the highly conserved P681 and G682 BB loop residues. Using computer-aided drug design (CADD), we sought to identify a small molecule inhibitor(s) that would fit within this pocket and potentially disrupt TLR2 signaling. In silico screening identified 149 compounds and 20 US Food and Drug Administration-approved drugs based on their predicted ability to bind in the BB loop pocket. These compounds were screened in HEK293T-TLR2 transfectants for the ability to inhibit TLR2-mediated IL-8 mRNA. C16H15NO4 (C29) was identified as a potential TLR2 inhibitor. C29, and its derivative, ortho-vanillin (o-vanillin), inhibited TLR2/1 and TLR2/6 signaling induced by synthetic and bacterial TLR2 agonists in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages. C29 failed to inhibit signaling induced by other TLR agonists and TNF-α. Mutagenesis of BB loop pocket residues revealed an indispensable role for TLR2/1, but not TLR2/6, signaling, suggesting divergent roles. Mice treated with o-vanillin exhibited reduced TLR2-induced inflammation. Our data provide proof of principle that targeting the BB loop pocket is an effective approach for identification of TLR2 signaling inhibitors. PMID:25870276

Making cognitive decision support work: Facilitating adoption, knowledge and behavior change through QI.

Science.gov (United States)

Weir, Charlene; Brunker, Cherie; Butler, Jorie; Supiano, Mark A

2017-07-01

This paper evaluates the role of facilitation in the successful implementation of Computerized Decision Support (CDS). Facilitation processes include education, specialized computerized decision support, and work process reengineering. These techniques, as well as modeling and feedback enhance self-efficacy, which we propose is one of the factors that mediate the effectiveness of any CDS. In this study, outpatient clinics implemented quality improvement (QI) projects focused on improving geriatric care. Quality Improvement is the systematic process of improving quality through continuous measurement and targeted actions. The program, entitled "Advancing Geriatric Education through Quality Improvement" (AGE QI), consisted of a 6-month, QI based, intervention: (1) 2h didactic session, (2) 1h QI planning session, (3) computerized decision support design and implementation, (4) QI facilitation activities, (5) outcome feedback, and (6) 20h of CME. Specifically, we examined the impact of the QI based program on clinician's perceived self-efficacy in caring for older adults and the relationship of implementation support and facilitation on perceived success. The intervention was implemented at 3 institutions, 27 community healthcare system clinics, and 134 providers. This study reports the results of pre/post surveys for the forty-nine clinicians who completed the full CME program. Self-efficacy ratings for specific clinical behaviors related to care of older adults were assessed using a Likert based instrument. Self-ratings of efficacy improved across the following domains (depression, falls, end-of-life, functional status and medication management) and specifically in QI targeted domains and were associated with overall clinic improvements. Published by Elsevier Inc.

SH2-catalytic domain linker heterogeneity influences allosteric coupling across the SFK family.

Science.gov (United States)

Register, A C; Leonard, Stephen E; Maly, Dustin J

2014-11-11

Src-family kinases (SFKs) make up a family of nine homologous multidomain tyrosine kinases whose misregulation is responsible for human disease (cancer, diabetes, inflammation, etc.). Despite overall sequence homology and identical domain architecture, differences in SH3 and SH2 regulatory domain accessibility and ability to allosterically autoinhibit the ATP-binding site have been observed for the prototypical SFKs Src and Hck. Biochemical and structural studies indicate that the SH2-catalytic domain (SH2-CD) linker, the intramolecular binding epitope for SFK SH3 domains, is responsible for allosterically coupling SH3 domain engagement to autoinhibition of the ATP-binding site through the conformation of the αC helix. As a relatively unconserved region between SFK family members, SH2-CD linker sequence variability across the SFK family is likely a source of nonredundant cellular functions between individual SFKs via its effect on the availability of SH3 and SH2 domains for intermolecular interactions and post-translational modification. Using a combination of SFKs engineered with enhanced or weakened regulatory domain intramolecular interactions and conformation-selective inhibitors that report αC helix conformation, this study explores how SH2-CD sequence heterogeneity affects allosteric coupling across the SFK family by examining Lyn, Fyn1, and Fyn2. Analyses of Fyn1 and Fyn2, isoforms that are identical but for a 50-residue sequence spanning the SH2-CD linker, demonstrate that SH2-CD linker sequence differences can have profound effects on allosteric coupling between otherwise identical kinases. Most notably, a dampened allosteric connection between the SH3 domain and αC helix leads to greater autoinhibitory phosphorylation by Csk, illustrating the complex effects of SH2-CD linker sequence on cellular function.

Structure of a new crystal form of human Hsp70 ATPase domain.

Science.gov (United States)

Osipiuk, J; Walsh, M A; Freeman, B C; Morimoto, R I; Joachimiak, A

1999-05-01

Hsp70 proteins are highly conserved proteins induced by heat shock and other stress conditions. An ATP-binding domain of human Hsp70 protein has been crystallized in two major morphological forms at pH 7.0 in the presence of PEG 8000 and CaCl2. Both crystal forms belong to the orthorhombic space group P212121, but show no resemblance in unit-cell parameters. Analysis of the crystal structures for both forms shows a 1-2 A shift of one of the subdomains of the protein. This conformational change could reflect a 'natural' flexibility of the protein which might be relevant to ATP binding and may facilitate the interaction of other proteins with Hsp70 protein.

In vitro guanine nucleotide exchange activity of DHR-2/DOCKER/CZH2 domains.

Science.gov (United States)

Côté, Jean-François; Vuori, Kristiina

2006-01-01

Rho family GTPases regulate a large variety of biological processes, including the reorganization of the actin cytoskeleton. Like other members of the Ras superfamily of small GTP-binding proteins, Rho GTPases cycle between a GDP-bound (inactive) and a GTP-bound (active) state, and, when active, the GTPases relay extracellular signals to a large number of downstream effectors. Guanine nucleotide exchange factors (GEFs) promote the exchange of GDP for GTP on Rho GTPases, thereby activating them. Most Rho-GEFs mediate their effects through their signature domain known as the Dbl Homology-Pleckstrin Homology (DH-PH) module. Recently, we and others identified a family of evolutionarily conserved, DOCK180-related proteins that also display GEF activity toward Rho GTPases. The DOCK180-family of proteins lacks the canonical DH-PH module. Instead, they rely on a novel domain, termed DHR-2, DOCKER, or CZH2, to exchange GDP for GTP on Rho targets. In this chapter, the experimental approach that we used to uncover the exchange activity of the DHR-2 domain of DOCK180-related proteins will be described.

MERTK interactions with SH2-domain proteins in the retinal pigment epithelium.

Science.gov (United States)

Shelby, Shameka J; Colwill, Karen; Dhe-Paganon, Sirano; Pawson, Tony; Thompson, Debra A

2013-01-01

The receptor tyrosine kinase MERTK plays an essential role in the phagocytic uptake of shed photoreceptor membranes by the retinal pigment epithelium (RPE). A fundamental aspect of signal transduction by receptor tyrosine kinases involves autophosphorylation of tyrosine residues that recruit Src-homology 2 (SH2)-domain proteins to the receptor intracellular domain. The goal of the current study was to evaluate the interactions of human MERTK with SH2-domain proteins present in the RPE. The MERTK intracellular domain was expressed as a 6xHis-fusion protein (6xHis-rMERTK(571-999)), purified and phosphorylated. Ni(2+)-NTA pull downs were performed using 6xHis-rMERTK(571-999) in incubations with recombinant phosphotyrosine-recognition sequences expressed as GST-fusion proteins. In addition, pull downs of native SH2-domain proteins were performed using 6xHis-rMERTK(571-999) and protein homogenates from rat RPE/choroid. For both recombinant and native proteins, western analysis detected MERTK interactions with GRB2, PIK3R1 (P85α), VAV3, and SRC. Immunohistochemical analysis localized each protein to mouse RPE. In cultured RPE-J cells incubated with rod outer segments (OS), siRNA knockdown of Grb2 had no effect on OS binding, but significantly reduced OS uptake. Pik3r1 localized to early phagosomes along with Rab5 and Eea1. Phosphorylation and activation of Src was detected downstream of phagocytosis and Mertk activation. These findings suggest that MERTK signaling in the RPE involves a cohort of SH2-domain proteins with the potential to regulate both cytoskeletal rearrangement and membrane movement. Identification of the SH2-domain signaling partners of MERTK is an important step toward further defining the mechanism of RPE phagocytosis that is central to the function and survival of the retina.

MERTK interactions with SH2-domain proteins in the retinal pigment epithelium.

Directory of Open Access Journals (Sweden)

Shameka J Shelby

Full Text Available The receptor tyrosine kinase MERTK plays an essential role in the phagocytic uptake of shed photoreceptor membranes by the retinal pigment epithelium (RPE. A fundamental aspect of signal transduction by receptor tyrosine kinases involves autophosphorylation of tyrosine residues that recruit Src-homology 2 (SH2-domain proteins to the receptor intracellular domain. The goal of the current study was to evaluate the interactions of human MERTK with SH2-domain proteins present in the RPE. The MERTK intracellular domain was expressed as a 6xHis-fusion protein (6xHis-rMERTK(571-999, purified and phosphorylated. Ni(2+-NTA pull downs were performed using 6xHis-rMERTK(571-999 in incubations with recombinant phosphotyrosine-recognition sequences expressed as GST-fusion proteins. In addition, pull downs of native SH2-domain proteins were performed using 6xHis-rMERTK(571-999 and protein homogenates from rat RPE/choroid. For both recombinant and native proteins, western analysis detected MERTK interactions with GRB2, PIK3R1 (P85α, VAV3, and SRC. Immunohistochemical analysis localized each protein to mouse RPE. In cultured RPE-J cells incubated with rod outer segments (OS, siRNA knockdown of Grb2 had no effect on OS binding, but significantly reduced OS uptake. Pik3r1 localized to early phagosomes along with Rab5 and Eea1. Phosphorylation and activation of Src was detected downstream of phagocytosis and Mertk activation. These findings suggest that MERTK signaling in the RPE involves a cohort of SH2-domain proteins with the potential to regulate both cytoskeletal rearrangement and membrane movement. Identification of the SH2-domain signaling partners of MERTK is an important step toward further defining the mechanism of RPE phagocytosis that is central to the function and survival of the retina.

Direct Binding between Pre-S1 and TRP-like Domains in TRPP Channels Mediates Gating and Functional Regulation by PIP2.

Science.gov (United States)

Zheng, Wang; Cai, Ruiqi; Hofmann, Laura; Nesin, Vasyl; Hu, Qiaolin; Long, Wentong; Fatehi, Mohammad; Liu, Xiong; Hussein, Shaimaa; Kong, Tim; Li, Jingru; Light, Peter E; Tang, Jingfeng; Flockerzi, Veit; Tsiokas, Leonidas; Chen, Xing-Zhen

2018-02-06

Transient receptor potential (TRP) channels are regulated by diverse stimuli comprising thermal, chemical, and mechanical modalities. They are also commonly regulated by phosphatidylinositol-4,5-bisphosphate (PIP2), with underlying mechanisms largely unknown. We here revealed an intramolecular interaction of the TRPP3 N and C termini (N-C) that is functionally essential. The interaction was mediated by aromatic Trp81 in pre-S1 domain and cationic Lys568 in TRP-like domain. Structure-function analyses revealed similar N-C interaction in TRPP2 as well as TRPM8/-V1/-C4 via highly conserved tryptophan and lysine/arginine residues. PIP2 bound to cationic residues in TRPP3, including K568, thereby disrupting the N-C interaction and negatively regulating TRPP3. PIP2 had similar negative effects on TRPP2. Interestingly, we found that PIP2 facilitates the N-C interaction in TRPM8/-V1, resulting in channel potentiation. The intramolecular N-C interaction might represent a shared mechanism underlying the gating and PIP2 regulation of TRP channels. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

A novel mutation in the tyrosine kinase domain of ERBB2 in hepatocellular carcinoma

International Nuclear Information System (INIS)

Bekaii-Saab, Tanios; Williams, Nita; Plass, Christoph; Calero, Miguel Villalona; Eng, Charis

2006-01-01

Several studies showed that gain-of-function somatic mutations affecting the catalytic domain of EGFR in non-small cell lung carcinomas were associated with response to gefitinib and erlotinib, both EGFR-tyrosine kinase inhibitors. In addition, 4% of non-small cell lung carcinomas were shown to have ERBB2 mutations in the kinase domain. In our study, we sought to determine if similar respective gain-of-function EGFR and ERBB2 mutations were present in hepatoma and/or biliary cancers. We extracted genomic DNA from 40 hepatoma (18) and biliary cancers (22) samples, and 44 adenocarcinomas of the lung, this latter as a positive control for mutation detection. We subjected those samples to PCR-based semi-automated double stranded nucleotide sequencing targeting exons 18–21 of EGFR and ERBB2. All samples were tested against matched normal DNA. We found 11% of hepatoma, but no biliary cancers, harbored a novel ERBB2 H878Y mutation in the activating domain. These newly described mutations may play a role in predicting response to EGFR-targeted therapy in hepatoma and their role should be explored in prospective studies

Cerebellar Kainate Receptor-Mediated Facilitation of Glutamate Release Requires Ca2+-Calmodulin and PKA

Directory of Open Access Journals (Sweden)

Rafael Falcón-Moya

2018-06-01

Full Text Available We elucidated the mechanisms underlying the kainate receptor (KAR-mediated facilitatory modulation of synaptic transmission in the cerebellum. In cerebellar slices, KA (3 μM increased the amplitude of evoked excitatory postsynaptic currents (eEPSCs at synapses between axon terminals of parallel fibers (PF and Purkinje neurons. KA-mediated facilitation was antagonized by NBQX under condition where AMPA receptors were previously antagonized. Inhibition of protein kinase A (PKA suppressed the effect of KA on glutamate release, which was also obviated by the prior stimulation of adenylyl cyclase (AC. KAR-mediated facilitation of synaptic transmission was prevented by blocking Ca2+ permeant KARs using philanthotoxin. Furthermore, depletion of intracellular Ca2+ stores by thapsigargin, or inhibition of Ca2+-induced Ca2+-release by ryanodine, abrogated the synaptic facilitation by KA. Thus, the KA-mediated modulation was conditional on extracellular Ca2+ entry through Ca2+-permeable KARs, as well as and mobilization of Ca2+ from intracellular stores. Finally, KAR-mediated facilitation was sensitive to calmodulin inhibitors, W-7 and calmidazolium, indicating that the increased cytosolic [Ca2+] sustaining KAR-mediated facilitation of synaptic transmission operates through a downstream Ca2+/calmodulin coupling. We conclude that, at cerebellar parallel fiber-Purkinje cell synapses, presynaptic KARs mediate glutamate release facilitation, and thereby enhance synaptic transmission through Ca2+-calmodulin dependent activation of adenylyl cyclase/cAMP/protein kinase A signaling.

Characterizing SH2 Domain Specificity and Network Interactions Using SPOT Peptide Arrays.

Science.gov (United States)

Liu, Bernard A

2017-01-01

Src Homology 2 (SH2) domains are protein interaction modules that recognize and bind tyrosine phosphorylated ligands. Their ability to distinguish binding to over thousands of potential phosphotyrosine (pTyr) ligands within the cell is critical for the fidelity of receptor tyrosine kinase (RTK) signaling. Within humans there are over a hundred SH2 domains with more than several thousand potential ligands across many cell types and cell states. Therefore, defining the specificity of individual SH2 domains is critical for predicting and identifying their physiological ligands. Here, in this chapter, I describe the broad use of SPOT peptide arrays for examining SH2 domain specificity. An orientated peptide array library (OPAL) approach can uncover both favorable and non-favorable residues, thus providing an in-depth analysis to SH2 specificity. Moreover, I discuss the application of SPOT arrays for paneling SH2 ligand binding with physiological peptides.

Functional significance of SRJ domain mutations in CITED2.

Directory of Open Access Journals (Sweden)

Chiann-mun Chen

Full Text Available CITED2 is a transcriptional co-activator with 3 conserved domains shared with other CITED family members and a unique Serine-Glycine Rich Junction (SRJ that is highly conserved in placental mammals. Loss of Cited2 in mice results in cardiac and aortic arch malformations, adrenal agenesis, neural tube and placental defects, and partially penetrant defects in left-right patterning. By screening 1126 sporadic congenital heart disease (CHD cases and 1227 controls, we identified 19 variants, including 5 unique non-synonymous sequence variations (N62S, R92G, T166N, G180-A187del and A187T in patients. Many of the CHD-specific variants identified in this and previous studies cluster in the SRJ domain. Transient transfection experiments show that T166N mutation impairs TFAP2 co-activation function and ES cell proliferation. We find that CITED2 is phosphorylated by MAPK1 in vitro at T166, and that MAPK1 activation enhances the coactivation function of CITED2 but not of CITED2-T166N. In order to investigate the functional significance in vivo, we generated a T166N mutation of mouse Cited2. We also used PhiC31 integrase-mediated cassette exchange to generate a Cited2 knock-in allele replacing the mouse Cited2 coding sequence with human CITED2 and with a mutant form deleting the entire SRJ domain. Mouse embryos expressing only CITED2-T166N or CITED2-SRJ-deleted alleles surprisingly show no morphological abnormalities, and mice are viable and fertile. These results indicate that the SRJ domain is dispensable for these functions of CITED2 in mice and that mutations clustering in the SRJ region are unlikely to be the sole cause of the malformations observed in patients with sporadic CHD. Our results also suggest that coding sequence mutations observed in case-control studies need validation using in vivo models and that predictions based on structural conservation and in vitro functional assays, or even in vivo global loss of function models, may be

A single amino acid difference between the intracellular domains of amyloid precursor protein and amyloid-like precursor protein 2 enables induction of synaptic depression and block of long-term potentiation.

Science.gov (United States)

Trillaud-Doppia, Emilie; Paradis-Isler, Nicolas; Boehm, Jannic

2016-07-01

Alzheimer disease (AD) is initially characterized as a disease of the synapse that affects synaptic transmission and synaptic plasticity. While amyloid-beta and tau have been traditionally implicated in causing AD, recent studies suggest that other factors, such as the intracellular domain of the amyloid-precursor protein (APP-ICD), can also play a role in the development of AD. Here, we show that the expression of APP-ICD induces synaptic depression, while the intracellular domain of its homolog amyloid-like precursor protein 2 (APLP2-ICD) does not. We are able to show that this effect by APP-ICD is due to a single alanine vs. proline difference between APP-ICD and APLP2-ICD. The alanine in APP-ICD and the proline in APLP2-ICD lie directly behind a conserved caspase cleavage site. Inhibition of caspase cleavage of APP-ICD prevents the induction of synaptic depression. Finally, we show that the expression of APP-ICD increases and facilitates long-term depression and blocks induction of long-term potentiation. The block in long-term potentiation can be overcome by mutating the aforementioned alanine in APP-ICD to the proline of APLP2. Based on our results, we propose the emergence of a new APP critical domain for the regulation of synaptic plasticity and in consequence for the development of AD. Copyright © 2016 Elsevier Inc. All rights reserved.

Investigation of phosphotyrosine recognition by the SH2 domain of the Src kinase.

Science.gov (United States)

Bradshaw, J M; Mitaxov, V; Waksman, G

1999-11-05

The binding of tyrosine phosphorylated targets by SH2 domains is required for propagation of many cellular signals in higher eukaryotes; however, the determinants of phosphotyrosine (pTyr) recognition by SH2 domains are not well understood. In order to identify the attributes of pTyr required for high affinity interaction with SH2 domains, the binding of the SH2 domain of the Src kinase (Src SH2 domain) to a dephosphorylated peptide, a phosphoserine-containing peptide, and the amino acid pTyr was studied using titration calorimetry and compared with the binding of a high affinity tyrosyl phosphopeptide. The dephosphorylated peptide and the phosphoserine containing peptide both bind extremely weakly to the Src SH2 domain (DeltaGo (dephosphorylated)=-3.6 kcal/mol, DeltaGo (phosphoserine) >-3.7 kcal/mol); however, the DeltaGo value of pTyr binding is more favorable (-4.7 kcal/mol, or 50 % of the entire binding free energy of a high affinity tyrosyl phosphopeptide). These results indicate that both the phosphate and the tyrosine ring of the pTyr are critical determinants of high affinity binding. Alanine mutagenesis was also used to evaluate the energetic contribution to binding of ten residues located in the pTyr-binding site. Mutation of the strictly conserved Arg betaB5 resulted in a large increase in DeltaGo (DeltaDeltaGo=3.2 kcal/mol) while elimination of the other examined residues each resulted in a significantly smaller (DeltaDeltaGoSH2 domain, surprisingly increased affinity by eightfold (DeltaDeltaGo=-1.1 kcal/mol). Using a double mutant cycle analysis, it was revealed that residues of the pTyr-binding pocket are not coupled to the peptide residues C-terminal to the pTyr. In addition, comparison of each residue's DeltaDeltaGo value upon mutation with that residue's sequence conservation among SH2 domains revealed only a modest correlation between a residue's energetic contribution to pTyr recognition and its conservation throughout evolution. The results of

2D acoustic-elastic coupled waveform inversion in the Laplace domain

KAUST Repository

Bae, Hoseuk

2010-04-01

Although waveform inversion has been intensively studied in an effort to properly delineate the Earth\\'s structures since the early 1980s, most of the time- and frequency-domain waveform inversion algorithms still have critical limitations in their applications to field data. This may be attributed to the highly non-linear objective function and the unreliable low-frequency components. To overcome the weaknesses of conventional waveform inversion algorithms, the acoustic Laplace-domain waveform inversion has been proposed. The Laplace-domain waveform inversion has been known to provide a long-wavelength velocity model even for field data, which may be because it employs the zero-frequency component of the damped wavefield and a well-behaved logarithmic objective function. However, its applications have been confined to 2D acoustic media.We extend the Laplace-domain waveform inversion algorithm to a 2D acoustic-elastic coupled medium, which is encountered in marine exploration environments. In 2D acoustic-elastic coupled media, the Laplace-domain pressures behave differently from those of 2D acoustic media, although the overall features are similar to each other. The main differences are that the pressure wavefields for acoustic-elastic coupled media show negative values even for simple geological structures unlike in acoustic media, when the Laplace damping constant is small and the water depth is shallow. The negative values may result from more complicated wave propagation in elastic media and at fluid-solid interfaces.Our Laplace-domain waveform inversion algorithm is also based on the finite-element method and logarithmic wavefields. To compute gradient direction, we apply the back-propagation technique. Under the assumption that density is fixed, P- and S-wave velocity models are inverted from the pressure data. We applied our inversion algorithm to the SEG/EAGE salt model and the numerical results showed that the Laplace-domain waveform inversion

Solution NMR structure of the HLTF HIRAN domain: a conserved module in SWI2/SNF2 DNA damage tolerance proteins

International Nuclear Information System (INIS)

Korzhnev, Dmitry M.; Neculai, Dante; Dhe-Paganon, Sirano; Arrowsmith, Cheryl H.; Bezsonova, Irina

2016-01-01

HLTF is a SWI2/SNF2-family ATP-dependent chromatin remodeling enzyme that acts in the error-free branch of DNA damage tolerance (DDT), a cellular mechanism that enables replication of damaged DNA while leaving damage repair for a later time. Human HLTF and a closely related protein SHPRH, as well as their yeast homologue Rad5, are multi-functional enzymes that share E3 ubiquitin-ligase activity required for activation of the error-free DDT. HLTF and Rad5 also function as ATP-dependent dsDNA translocases and possess replication fork reversal activities. Thus, they can convert Y-shaped replication forks into X-shaped Holliday junction structures that allow error-free replication over DNA lesions. The fork reversal activity of HLTF is dependent on 3′-ssDNA-end binding activity of its N-terminal HIRAN domain. Here we present the solution NMR structure of the human HLTF HIRAN domain, an OB-like fold module found in organisms from bacteria (as a stand-alone domain) to plants, fungi and metazoan (in combination with SWI2/SNF2 helicase-like domain). The obtained structure of free HLTF HIRAN is similar to recently reported structures of its DNA bound form, while the NMR analysis also reveals that the DNA binding site of the free domain exhibits conformational heterogeneity. Sequence comparison of N-terminal regions of HLTF, SHPRH and Rad5 aided by knowledge of the HLTF HIRAN structure suggests that the SHPRH N-terminus also includes an uncharacterized structured module, exhibiting weak sequence similarity with HIRAN regions of HLTF and Rad5, and potentially playing a similar functional role.

Solution NMR structure of the HLTF HIRAN domain: a conserved module in SWI2/SNF2 DNA damage tolerance proteins

Energy Technology Data Exchange (ETDEWEB)

Korzhnev, Dmitry M. [University of Connecticut Health, Department of Molecular Biology and Biophysics (United States); Neculai, Dante [Zhejiang University, School of Medicine (China); Dhe-Paganon, Sirano [Dana-Farber Cancer Institute, Department of Cancer Biology (United States); Arrowsmith, Cheryl H. [University of Toronto, Structural Genomics Consortium (Canada); Bezsonova, Irina, E-mail: bezsonova@uchc.edu [University of Connecticut Health, Department of Molecular Biology and Biophysics (United States)

2016-11-15

HLTF is a SWI2/SNF2-family ATP-dependent chromatin remodeling enzyme that acts in the error-free branch of DNA damage tolerance (DDT), a cellular mechanism that enables replication of damaged DNA while leaving damage repair for a later time. Human HLTF and a closely related protein SHPRH, as well as their yeast homologue Rad5, are multi-functional enzymes that share E3 ubiquitin-ligase activity required for activation of the error-free DDT. HLTF and Rad5 also function as ATP-dependent dsDNA translocases and possess replication fork reversal activities. Thus, they can convert Y-shaped replication forks into X-shaped Holliday junction structures that allow error-free replication over DNA lesions. The fork reversal activity of HLTF is dependent on 3′-ssDNA-end binding activity of its N-terminal HIRAN domain. Here we present the solution NMR structure of the human HLTF HIRAN domain, an OB-like fold module found in organisms from bacteria (as a stand-alone domain) to plants, fungi and metazoan (in combination with SWI2/SNF2 helicase-like domain). The obtained structure of free HLTF HIRAN is similar to recently reported structures of its DNA bound form, while the NMR analysis also reveals that the DNA binding site of the free domain exhibits conformational heterogeneity. Sequence comparison of N-terminal regions of HLTF, SHPRH and Rad5 aided by knowledge of the HLTF HIRAN structure suggests that the SHPRH N-terminus also includes an uncharacterized structured module, exhibiting weak sequence similarity with HIRAN regions of HLTF and Rad5, and potentially playing a similar functional role.

Structural Characterization of Monomeric/Dimeric State of p59fyn SH2 Domain.

Science.gov (United States)

Huculeci, Radu; Kieken, Fabien; Garcia-Pino, Abel; Buts, Lieven; van Nuland, Nico; Lenaerts, Tom

2017-01-01

Src homology 2 (SH2) domains are key modulators in various signaling pathways allowing the recognition of phosphotyrosine sites of different proteins. Despite the fact that SH2 domains acquire their biological functions in a monomeric state, a multitude of reports have shown their tendency to dimerize. Here, we provide a technical description on how to isolate and characterize by gel filtration, circular dichroism (CD), and nuclear magnetic resonance (NMR) each conformational state of p59 fyn SH2 domain.

Occupancy of a C2-C2 type 'zinc-finger' protein domain by copper. Direct observation by electrospray ionization mass spectrometry.

Science.gov (United States)

Hutchens, T W; Allen, M H; Li, C M; Yip, T T

1992-09-07

The metal ion specificity of most 'zinc-finger' metal binding domains is unknown. The human estrogen receptor protein contains two different C2-C2 type 'zinc-finger' sequences within its DNA-binding domain (ERDBD). Copper inhibits the function of this protein by mechanisms which remain unclear. We have used electrospray ionization mass spectrometry to evaluate directly the 71-residue ERDBD (K180-M250) in the absence and presence of Cu(II) ions. The ERDBD showed a high affinity for Cu and was completely occupied with 4 Cu bound; each Cu ion was evidently bound to only two ligand residues (net loss of only 2 Da per bound Cu). The Cu binding stoichiometry was confirmed by atomic absorption. These results (i) provide the first direct physical evidence for the ability of the estrogen receptor DNA-binding domain to bind Cu and (ii) document a twofold difference in the Zn- and Cu-binding capacity. Differences in the ERDBD domain structure with bound Zn and Cu are predicted. Given the relative intracellular contents of Zn and Cu, our findings demonstrate the need to investigate further the Cu occupancy of this and other zinc-finger domains both in vitro and in vivo.

The ShcA SH2 domain engages a 14-3-3/PI3'K signaling complex and promotes breast cancer cell survival.

Science.gov (United States)

Ursini-Siegel, J; Hardy, W R; Zheng, Y; Ling, C; Zuo, D; Zhang, C; Podmore, L; Pawson, T; Muller, W J

2012-11-29

The ShcA adapter protein transmits activating signals downstream of receptor and cytoplasmic tyrosine kinases through the establishment of phosphotyrosine-dependent complexes. In this regard, ShcA possesses both a phosphotyrosine-binding domain (PTB) and Src homology 2 domain (SH2), which bind phosphotyrosine residues in a sequence-specific manner. Although the majority of receptor tyrosine kinases expressed in breast cancer cells bind the PTB domain, very little is known regarding the biological importance of SH2-driven ShcA signaling during mammary tumorigenesis. To address this, we employed transgenic mice expressing a mutant ShcA allele harboring a non-functional SH2 domain (ShcR397K) under the transcriptional control of the endogenous ShcA promoter. Using transplantation approaches, we demonstrate that SH2-dependent ShcA signaling within the mammary epithelial compartment is essential for breast tumor outgrowth, survival and the development of lung metastases. We further show that the ShcA SH2 domain activates the AKT pathway, potentially through a novel SH2-mediated complex between ShcA, 14-3-3ζ and the p85 regulatory subunit of phosphatidylinositol 3 (PI3') kinase. This study is the first to demonstrate that the SH2 domain of ShcA is critical for tumor survival during mammary tumorigenesis.

Solution structure of telomere binding domain of AtTRB2 derived from Arabidopsis thaliana

International Nuclear Information System (INIS)

Yun, Ji-Hye; Lee, Won Kyung; Kim, Heeyoun; Kim, Eunhee; Cheong, Chaejoon; Cho, Myeon Haeng; Lee, Weontae

2014-01-01

Highlights: • We have determined solution structure of Myb domain of AtTRB2. • The Myb domain of AtTRB2 is located in the N-terminal region. • The Myb domain of AtTRB2 binds to plant telomeric DNA without fourth helix. • Helix 2 and 3 of the Myb domain of AtTRB2 are involved in DNA recognition. • AtTRB2 is a novel protein distinguished from other known plant TBP. - Abstract: Telomere homeostasis is regulated by telomere-associated proteins, and the Myb domain is well conserved for telomere binding. AtTRB2 is a member of the SMH (Single-Myb-Histone)-like family in Arabidopsis thaliana, having an N-terminal Myb domain, which is responsible for DNA binding. The Myb domain of AtTRB2 contains three α-helices and loops for DNA binding, which is unusual given that other plant telomere-binding proteins have an additional fourth helix that is essential for DNA binding. To understand the structural role for telomeric DNA binding of AtTRB2, we determined the solution structure of the Myb domain of AtTRB2 (AtTRB2 1–64 ) using nuclear magnetic resonance (NMR) spectroscopy. In addition, the inter-molecular interaction between AtTRB2 1–64 and telomeric DNA has been characterized by the electrophoretic mobility shift assay (EMSA) and NMR titration analyses for both plant (TTTAGGG)n and human (TTAGGG)n telomere sequences. Data revealed that Trp28, Arg29, and Val47 residues located in Helix 2 and Helix 3 are crucial for DNA binding, which are well conserved among other plant telomere binding proteins. We concluded that although AtTRB2 is devoid of the additional fourth helix in the Myb-extension domain, it is able to bind to plant telomeric repeat sequences as well as human telomeric repeat sequences

Purification, crystallization and preliminary crystallographic analysis of the SH2 domain of IL-2-inducible T-cell kinase

International Nuclear Information System (INIS)

Joseph, Raji E.; Ginder, Nathaniel D.; Hoy, Julie A.; Nix, Jay C.; Honzatko, Richard B.; Andreotti, Amy H.

2011-01-01

Crystallization conditions are described for the cis- and trans-imide bond-containing SH2 domain of IL-2-inducible T-cell kinase. Proline is a unique amino acid owing to the relatively small energy difference between the cis and trans conformations of its peptide bond. The X–Pro imide bond readily undergoes cis–trans isomerization in the context of short peptides as well as some proteins. However, the direct detection of cis–trans proline isomerization in folded proteins is technically challenging. NMR spectroscopy is well suited to the direct detection of proline isomerization in folded proteins. It is less clear how well X-ray crystallography can reveal this conformational exchange event in folded proteins. Conformational heterogeneity owing to cis–trans proline isomerization in the Src homology 2 (SH2) domain of the IL-2-inducible T-cell kinase (ITK) has been extensively characterized by NMR. Using the ITK SH2 domain as a test system, an attempt was made to determine whether proline isomerization could be detected in a crystal structure of the ITK SH2 domain. As a first step towards this goal, the purification, crystallization and preliminary characterization of the ITK SH2 domain are described

Presence of an SH2 domain in the actin-binding protein tensin.

Science.gov (United States)

Davis, S; Lu, M L; Lo, S H; Lin, S; Butler, J A; Druker, B J; Roberts, T M; An, Q; Chen, L B

1991-05-03

The molecular cloning of the complementary DNA coding for a 90-kilodalton fragment of tensin, an actin-binding component of focal contacts and other submembraneous cytoskeletal structures, is reported. The derived amino acid sequence revealed the presence of a Src homology 2 (SH2) domain. This domain is shared by a number of signal transduction proteins including nonreceptor tyrosine kinases such as Abl, Fps, Src, and Src family members, the transforming protein Crk, phospholipase C-gamma 1, PI-3 (phosphatidylinositol) kinase, and guanosine triphosphatase-activating protein (GAP). Like the SH2 domain found in Src, Crk, and Abl, the SH2 domain of tensin bound specifically to a number of phosphotyrosine-containing proteins from v-src-transformed cells. Tensin was also found to be phosphorylated on tyrosine residues. These findings suggest that by possessing both actin-binding and phosphotyrosine-binding activities and being itself a target for tyrosine kinases, tensin may link signal transduction pathways with the cytoskeleton.

The PH Domain of PDK1 Exhibits a Novel, Phospho-Regulated Monomer-Dimer Equilibrium With Important Implications for Kinase Domain Activation: Single Molecule and Ensemble Studies†

Science.gov (United States)

Ziemba, Brian P.; Pilling, Carissa; Calleja, Véronique; Larijani, Banafshé; Falke, Joseph J.

2013-01-01

Phosphoinositide-Dependent Kinase-1 (PDK1) is an essential master kinase recruited to the plasma membrane by the binding of its C-terminal PH domain to the signaling lipid phosphatidylinositol-3,4-5-trisphosphate (PIP3). Membrane binding leads to PDK1 phospho-activation, but despite the central role of PDK1 in signaling and cancer biology this activation mechanism remains poorly understood. PDK1 has been shown to exist as a dimer in cells, and one crystal structure of its isolated PH domain exhibits a putative dimer interface. It has been proposed that phosphorylation of PH domain residue T513 (or the phospho-mimetic T513E mutation) may regulate a novel PH domain dimer-monomer equilibrium, thereby converting an inactive PDK1 dimer to an active monomer. However, the oligomeric state(s) of the PH domain on the membrane have not yet been determined, nor whether a negative charge at position 513 is sufficient to regulate its oligomeric state. The present study investigates the binding of purified WT and T513E PDK1 PH domains to lipid bilayers containing the PIP3 target lipid, using both single molecule and ensemble measurements. Single molecule analysis of the brightness of fluorescent PH domain shows that the PIP3-bound WT PH domain on membranes is predominantly dimeric, while the PIP3-bound T513E PH domain is monomeric, demonstrating that negative charge at the T513 position is sufficient to dissociate the PH domain dimer and is thus likely to play a central role in PDK1 monomerization and activation. Single molecule analysis of 2-D diffusion of PH domain-PIP3 complexes reveals that the dimeric WT PH domain diffuses at the same rate a single lipid molecule, indicating that only one of its two PIP3 binding sites is occupied and there is little protein penetration into the bilayer as observed for other PH domains. The 2-D diffusion of T513E PH domain is slower, suggesting the negative charge disrupts local structure in a way that enables greater protein insertion into

Quantitation of the calcium and membrane binding properties of the C2 domains of dysferlin.

Science.gov (United States)

Abdullah, Nazish; Padmanarayana, Murugesh; Marty, Naomi J; Johnson, Colin P

2014-01-21

Dysferlin is a large membrane protein involved in calcium-triggered resealing of the sarcolemma after injury. Although it is generally accepted that dysferlin is Ca(2+) sensitive, the Ca(2+) binding properties of dysferlin have not been characterized. In this study, we report an analysis of the Ca(2+) and membrane binding properties of all seven C2 domains of dysferlin as well as a multi-C2 domain construct. Isothermal titration calorimetry measurements indicate that all seven dysferlin C2 domains interact with Ca(2+) with a wide range of binding affinities. The C2A and C2C domains were determined to be the most sensitive, with Kd values in the tens of micromolar, whereas the C2D domain was least sensitive, with a near millimolar Kd value. Mutagenesis of C2A demonstrates the requirement for negatively charged residues in the loop regions for divalent ion binding. Furthermore, dysferlin displayed significantly lower binding affinity for the divalent cations magnesium and strontium. Measurement of a multidomain construct indicates that the solution binding affinity does not change when C2 domains are linked. Finally, sedimentation assays suggest all seven C2 domains bind lipid membranes, and that Ca(2+) enhances but is not required for interaction. This report reveals for the first time, to our knowledge, that all dysferlin domains bind Ca(2+) albeit with varying affinity and stoichiometry. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Online interprofessional education facilitation: A scoping review.

Science.gov (United States)

Evans, Sherryn Maree; Ward, Catherine; Reeves, Scott

2018-04-22

The use of online media to deliver interprofessional education (IPE) is becoming more prevalent across health professions education settings. Facilitation of IPE activities is known to be critical to the effective delivery of IPE, however, specifics about the nature of online IPE facilitation remains unclear. To explore the health professions education literature to understand the extent, range and nature of research on online IPE facilitation. Scoping review methodology was used to guide a search of four electronic databases for relevant papers. Of the 2095 abstracts initially identified, after screening of both abstracts and full-text papers, 10 studies were selected for inclusion in this review. Following abstraction of key information from each study, a thematic analysis was undertaken. Three key themes emerged to describe the nature of the IPE facilitation literature: (1) types of online IPE facilitation contributions, (2) the experience of online IPE facilitation and (3) personal outcomes of online IPE facilitation. These IPE facilitation themes were particularly focused on facilitation of interprofessional student teams on an asynchronous basis. While the included studies provide some insight into the nature of online IPE facilitation, future research is needed to better understand facilitator contributions, and the facilitation experience and associated outcomes, both relating to synchronous and asynchronous online environments.

MiDas: automatic extraction of a common domain of discourse in sleep medicine for multi-center data integration.

Science.gov (United States)

Sahoo, Satya S; Ogbuji, Chimezie; Luo, Lingyun; Dong, Xiao; Cui, Licong; Redline, Susan S; Zhang, Guo-Qiang

2011-01-01

Clinical studies often use data dictionaries with controlled sets of terms to facilitate data collection, limited interoperability and sharing at a local site. Multi-center retrospective clinical studies require that these data dictionaries, originating from individual participating centers, be harmonized in preparation for the integration of the corresponding clinical research data. Domain ontologies are often used to facilitate multi-center data integration by modeling terms from data dictionaries in a logic-based language, but interoperability among domain ontologies (using automated techniques) is an unresolved issue. Although many upper-level reference ontologies have been proposed to address this challenge, our experience in integrating multi-center sleep medicine data highlights the need for an upper level ontology that models a common set of terms at multiple-levels of abstraction, which is not covered by the existing upper-level ontologies. We introduce a methodology underpinned by a Minimal Domain of Discourse (MiDas) algorithm to automatically extract a minimal common domain of discourse (upper-domain ontology) from an existing domain ontology. Using the Multi-Modality, Multi-Resource Environment for Physiological and Clinical Research (Physio-MIMI) multi-center project in sleep medicine as a use case, we demonstrate the use of MiDas in extracting a minimal domain of discourse for sleep medicine, from Physio-MIMI's Sleep Domain Ontology (SDO). We then extend the resulting domain of discourse with terms from the data dictionary of the Sleep Heart and Health Study (SHHS) to validate MiDas. To illustrate the wider applicability of MiDas, we automatically extract the respective domains of discourse from 6 sample domain ontologies from the National Center for Biomedical Ontologies (NCBO) and the OBO Foundry.

Purification of SOCS (Suppressor of Cytokine Signaling) SH2 Domains for Structural and Functional Studies.

Science.gov (United States)

Liau, Nicholas P D; Laktyushin, Artem; Babon, Jeffrey J

2017-01-01

Src Homology 2 (SH2) domains are protein domains which have a high binding affinity for specific amino acid sequences containing a phosphorylated tyrosine residue. The Suppressors of Cytokine Signaling (SOCS) proteins use an SH2 domain to bind to components of certain cytokine signaling pathways to downregulate the signaling cascade. The recombinantly produced SH2 domains of various SOCS proteins have been used to undertake structural and functional studies elucidating the method of how such targeting occurs. Here, we describe the protocol for the recombinant production and purification of SOCS SH2 domains, with an emphasis on SOCS3.

Two zinc-binding domains in the transporter AdcA from Streptococcus pyogenes facilitate high-affinity binding and fast transport of zinc.

Science.gov (United States)

Cao, Kun; Li, Nan; Wang, Hongcui; Cao, Xin; He, Jiaojiao; Zhang, Bing; He, Qing-Yu; Zhang, Gong; Sun, Xuesong

2018-04-20

Zinc is an essential metal in bacteria. One important bacterial zinc transporter is AdcA, and most bacteria possess AdcA homologs that are single-domain small proteins due to better efficiency of protein biogenesis. However, a double-domain AdcA with two zinc-binding sites is significantly overrepresented in Streptococcus species, many of which are major human pathogens. Using molecular simulation and experimental validations of AdcA from Streptococcus pyogenes , we found here that the two AdcA domains sequentially stabilize the structure upon zinc binding, indicating an organization required for both increased zinc affinity and transfer speed. This structural organization appears to endow Streptococcus species with distinct advantages in zinc-depleted environments, which would not be achieved by each single AdcA domain alone. This enhanced zinc transport mechanism sheds light on the significance of the evolution of the AdcA domain fusion, provides new insights into double-domain transporter proteins with two binding sites for the same ion, and indicates a potential target of antimicrobial drugs against pathogenic Streptococcus species. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Site descriptive modelling Forsmark, stage 2.2. A fracture domain concept as a basis for the statistical modelling of fractures and minor deformation zones, and interdisciplinary coordination

Energy Technology Data Exchange (ETDEWEB)

Olofsson, Isabelle; Simeonov, Assen [Swedish Nuclear Fuel and Waste Manageme nt Co., Stockholm (Sweden); Stephens, Michael [Geological Survey of Sweden (SGU), U ppsala (Sweden); Follin, Sven [SF GeoLogic AB, Taeby (Sweden); Nilsson, Ann-Chatrin [G eosigma AB, Uppsala (Sweden); Roeshoff, Kennert; Lindberg, Ulrika; Lanaro, Flavio [Bergbygg konsult AB, Haesselby (Sweden); Fredriksson, Anders; Persson, Lars [Golder Associat es AB (Sweden)

2007-04-15

The Swedish Nuclear Fuel and Waste Management Company (SKB) is undertaking site characterization at two different locations, Forsmark and Simpevarp/Laxemar, with the objective of siting a final waste repository at depth for spent nuclear fuel. The programme is built upon the development of site descriptive models after each data freeze. This report describes the first attempt to define fracture domains for the Forsmark site modelling in stage 2.2. Already during model version 1.2 at Forsmark, significant spatial variability in the fracture pattern was observed. The variability appeared to be so significant that it provoked the need for a subdivision of the model volume for the treatment of geological and hydrogeological data into sub-volumes. Subsequent analyses of data collected up to data freeze 2.1 led to a better understanding of the site and a concept for the definition of fracture domains based on geological characteristics matured. The main objectives of this report are to identify and describe fracture domains at the site on the basis of geological data and to compile hydrogeological, hydrogeochemical and rock mechanics data within each fracture domain and address the implications of this integration activity. On the basis of borehole data, six fracture domains (FFM01-FFM06) have been recognized inside and immediately around the candidate volume. Three of these domains (FFM01, FFM02 and FFM06) lie inside the target volume for a potential repository in the northwestern part of the candidate area, and need to be addressed in the geological DFN modelling work. The hydrogeological data support the subdivision of the bedrock into fracture domains FFM01, FFM02 and FFM03. Few or no data are available for the other three domains. The hydrogeochemical data also support the subdivision into fracture domains FFM01 and FFM02. Since few data are available from the bedrock between deformation zones inside FFM03, there is little information on the hydrogeochemical

Structures of the Sgt2/SGTA Dimerization Domain with the Get5/UBL4A UBL Domain Reveal an Interaction that Forms a Conserved Dynamic Interface

Directory of Open Access Journals (Sweden)

Justin W. Chartron

2012-12-01

Full Text Available In the cytoplasm, the correct delivery of membrane proteins is an essential and highly regulated process. The posttranslational targeting of the important tail-anchor membrane (TA proteins has recently been under intense investigation. A specialized pathway, called the guided entry of TA proteins (GET pathway in yeast and the transmembrane domain recognition complex (TRC pathway in vertebrates, recognizes endoplasmic-reticulum-targeted TA proteins and delivers them through a complex series of handoffs. An early step is the formation of a complex between Sgt2/SGTA, a cochaperone with a presumed ubiquitin-like-binding domain (UBD, and Get5/UBL4A, a ubiquitin-like domain (UBL-containing protein. We structurally characterize this UBD/UBL interaction for both yeast and human proteins. This characterization is supported by biophysical studies that demonstrate that complex formation is mediated by electrostatics, generating an interface that has high-affinity with rapid kinetics. In total, this work provides a refined model of the interplay of Sgt2 homologs in TA targeting.

Crystal structure of Src-like adaptor protein 2 reveals close association of SH3 and SH2 domains through β-sheet formation.

Science.gov (United States)

Wybenga-Groot, Leanne E; McGlade, C Jane

2013-12-01

The Src-like adaptor proteins (SLAP/SLAP2) are key components of Cbl-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling in hematopoietic cells. SLAP and SLAP2 consist of adjacent SH3 and SH2 domains that are most similar in sequence to Src family kinases (SFKs). Notably, the SH3-SH2 connector sequence is significantly shorter in SLAP/SLAP2 than in SFKs. To understand the structural implication of a short SH3-SH2 connector sequence, we solved the crystal structure of a protein encompassing the SH3 domain, SH3-SH2 connector, and SH2 domain of SLAP2 (SLAP2-32). While both domains adopt typical folds, the short SH3-SH2 connector places them in close association. Strand βe of the SH3 domain interacts with strand βA of the SH2 domain, resulting in the formation of a continuous β sheet that spans the length of the protein. Disruption of the SH3/SH2 interface through mutagenesis decreases SLAP-32 stability in vitro, consistent with inter-domain binding being an important component of SLAP2 structure and function. The canonical peptide binding pockets of the SH3 and SH2 domains are fully accessible, in contrast to other protein structures that display direct interaction between SH3 and SH2 domains, in which either peptide binding surface is obstructed by the interaction. Our results reveal potential sites of novel interaction for SH3 and SH2 domains, and illustrate the adaptability of SH2 and SH3 domains in mediating interactions. As well, our results suggest that the SH3 and SH2 domains of SLAP2 function interdependently, with implications on their mode of substrate binding. © 2013.

SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins.

Science.gov (United States)

Koch, C A; Anderson, D; Moran, M F; Ellis, C; Pawson, T

1991-05-03

Src homology (SH) regions 2 and 3 are noncatalytic domains that are conserved among a series of cytoplasmic signaling proteins regulated by receptor protein-tyrosine kinases, including phospholipase C-gamma, Ras GTPase (guanosine triphosphatase)-activating protein, and Src-like tyrosine kinases. The SH2 domains of these signaling proteins bind tyrosine phosphorylated polypeptides, implicated in normal signaling and cellular transformation. Tyrosine phosphorylation acts as a switch to induce the binding of SH2 domains, thereby mediating the formation of heteromeric protein complexes at or near the plasma membrane. The formation of these complexes is likely to control the activation of signal transduction pathways by tyrosine kinases. The SH3 domain is a distinct motif that, together with SH2, may modulate interactions with the cytoskeleton and membrane. Some signaling and transforming proteins contain SH2 and SH3 domains unattached to any known catalytic element. These noncatalytic proteins may serve as adaptors to link tyrosine kinases to specific target proteins. These observations suggest that SH2 and SH3 domains participate in the control of intracellular responses to growth factor stimulation.

A qualitative theory guided analysis of stroke survivors' perceived barriers and facilitators to physical activity.

Science.gov (United States)

Nicholson, Sarah L; Donaghy, Marie; Johnston, Marie; Sniehotta, Falko F; van Wijck, Frederike; Johnston, Derek; Greig, Carolyn; McMurdo, Marion E T; Mead, Gillian

2014-01-01

After stroke, physical activity and physical fitness levels are low, impacting on health, activity and participation. It is unclear how best to support stroke survivors to increase physical activity. Little is known about the barriers and facilitators to physical activity after stroke. Thus, our aim was to explore stroke survivors' perceived barriers and facilitators to physical activity. Semi-structured interviews with 13 ambulatory stroke survivors exploring perceived barriers and facilitators to physical activity post stroke were conducted in participants' homes, audio-recorded and transcribed verbatim. The Theoretical Domains Framework (TDF) informed content analysis of the interview transcripts. Data saturation was reached after interviews with 13 participants (median age of 76 years (inter-quartile range (IQR) = 69-83 years). The median time since stroke was 345 d (IQR = 316-366 d). The most commonly reported TDF domains were "beliefs about capabilities", "environmental context and resources" and "social influence". The most commonly reported perceived motivators were: social interaction, beliefs of benefits of exercise, high self-efficacy and the necessity of routine behaviours. The most commonly reported perceived barriers were: lack of professional support on discharge from hospital and follow-up, transport issues to structured classes/interventions, lack of control and negative affect. Stroke survivors perceive several different barriers and facilitators to physical activity. Stroke services need to address barriers to physical activity and to build on facilitators to promote physical activity after stroke. Physical activity post stroke can improve physical fitness and function, yet physical activity remains low among stroke survivors. Understanding stroke survivors' perceived barriers and facilitators to physical activity is essential to develop targeted interventions to increase physical activity. Beliefs about capabilities, environmental

Interaction of moving domain boundaries with a magnetic field in GdΛ2 (MoOΛ4)Λ3

International Nuclear Information System (INIS)

Popov, S.A.; Tikhomirova, N.A.; Phlerova, S.A.

1985-01-01

Results obtained during the investigation of gadolinium molybdate Gd 2 (MoO 4 ) 3 (GMo) crystal repolarization by the electric field at the background of simultaneous action of permanent magnetic fields with a strength up to 20kOe are presented. The magnetic field is oriented in different directions in respect to crystallographic sample directions. Polarization- optical control of a domain structure was conducted in synchronism with sample repolarization. Study of the effect of magnetic field on integral rate of domain boundaries motion in GMO has shown, that a speed of domain wall motion changes as a function of magnetic field orientation with respect to moving domain wall. So, if the wall is oriented paralled to magnetic field force lines, at H=20kOe speed of its motion increases a 1.2-1.5 times, and decreases a 2-2.5 times in the case of perpendicular orientation

Management and control applications in Agriculture domain via a Future Internet Business-to-Business platform

Directory of Open Access Journals (Sweden)

Sokratis Barmpounakis

2015-05-01

Full Text Available The Agriculture business domain, as a vital part of the overall supply chain, is expected to highly evolve in the upcoming years via the developments, which are taking place on the side of the Future Internet. This paper presents a novel Business-to-Business collaboration platform from the agri-food sector perspective, which aims to facilitate the collaboration of numerous stakeholders belonging to associated business domains, in an effective and flexible manner. The contemporary B2B collaboration schemes already place the requirements for swift deployment of cloud applications, capable of both integrating diverse legacy systems, as well as developing in a rapid way new services and systems, which will be able to instantly communicate and provide complete, “farm-to-fork” solutions for farmers, agri-food and logistics service providers, ICT companies, end-product producers, etc. To this end, this conceptual paper describes how these requirements are addressed via the FIspace B2B platform, focusing on the Greenhouse Management & Control scenarios.

Inhibition of tumor metastasis by a growth factor receptor bound protein 2 Src homology 2 domain-binding antagonist.

Science.gov (United States)

Giubellino, Alessio; Gao, Yang; Lee, Sunmin; Lee, Min-Jung; Vasselli, James R; Medepalli, Sampath; Trepel, Jane B; Burke, Terrence R; Bottaro, Donald P

2007-07-01

Metastasis, the primary cause of death in most forms of cancer, is a multistep process whereby cells from the primary tumor spread systemically and colonize distant new sites. Blocking critical steps in this process could potentially inhibit tumor metastasis and dramatically improve cancer survival rates; however, our understanding of metastasis at the molecular level is still rudimentary. Growth factor receptor binding protein 2 (Grb2) is a widely expressed adapter protein with roles in epithelial cell growth and morphogenesis, as well as angiogenesis, making it a logical target for anticancer drug development. We have previously shown that a potent antagonist of Grb2 Src homology-2 domain-binding, C90, blocks growth factor-driven cell motility in vitro and angiogenesis in vivo. We now report that C90 inhibits metastasis in vivo in two aggressive tumor models, without affecting primary tumor growth rate. These results support the potential efficacy of this compound in reducing the metastatic spread of primary solid tumors and establish a critical role for Grb2 Src homology-2 domain-mediated interactions in this process.

Specific phosphopeptide binding regulates a conformational change in the PI 3-kinase SH2 domain associated with enzyme activation.

Science.gov (United States)

Shoelson, S E; Sivaraja, M; Williams, K P; Hu, P; Schlessinger, J; Weiss, M A

1993-01-01

SH2 (src-homology 2) domains define a newly recognized binding motif that mediates the physical association of target phosphotyrosyl proteins with downstream effector enzymes. An example of such phosphoprotein-effector coupling is provided by the association of phosphatidylinositol 3-kinase (PI 3-kinase) with specific phosphorylation sites within the PDGF receptor, the c-Src/polyoma virus middle T antigen complex and the insulin receptor substrate IRS-1. Notably, phosphoprotein association with the SH2 domains of p85 also stimulates an increase in catalytic activity of the PI 3-kinase p110 subunit, which can be mimicked by phosphopeptides corresponding to targeted phosphoprotein phosphorylation sites. To investigate how phosphoprotein binding to the p85 SH2 domain stimulates p110 catalytic activation, we have examined the differential effects of phosphotyrosine and PDGF receptor-, IRS-1- and c-Src-derived phosphopeptides on the conformation of an isolated SH2 domain of PI 3-kinase. Although phosphotyrosine and both activating and non-activating phosphopeptides bind to the SH2 domain, activating phosphopeptides bind with higher affinity and induce a qualitatively distinct conformational change as monitored by CD and NMR spectroscopy. Amide proton exchange and protease protection assays further show that high affinity, specific phosphopeptide binding induces non-local dynamic SH2 domain stabilization. Based on these findings we propose that specific phosphoprotein binding to the p85 subunit induces a change in SH2 domain structure which is transmitted to the p110 subunit and regulates enzymatic activity by an allosteric mechanism. Images PMID:8382612

Influenza Polymerase Can Adopt an Alternative Configuration Involving a Radical Repacking of PB2 Domains.

Science.gov (United States)

Thierry, Eric; Guilligay, Delphine; Kosinski, Jan; Bock, Thomas; Gaudon, Stephanie; Round, Adam; Pflug, Alexander; Hengrung, Narin; El Omari, Kamel; Baudin, Florence; Hart, Darren J; Beck, Martin; Cusack, Stephen

2016-01-07

Influenza virus polymerase transcribes or replicates the segmented RNA genome (vRNA) into respectively viral mRNA or full-length copies and initiates RNA synthesis by binding the conserved 3' and 5' vRNA ends (the promoter). In recent structures of promoter-bound polymerase, the cap-binding and endonuclease domains are configured for cap snatching, which generates capped transcription primers. Here, we present a FluB polymerase structure with a bound complementary cRNA 5' end that exhibits a major rearrangement of the subdomains within the C-terminal two-thirds of PB2 (PB2-C). Notably, the PB2 nuclear localization signal (NLS)-containing domain translocates ∼90 Å to bind to the endonuclease domain. FluA PB2-C alone and RNA-free FluC polymerase are similarly arranged. Biophysical and cap-dependent endonuclease assays show that in solution the polymerase explores different conformational distributions depending on which RNA is bound. The inherent flexibility of the polymerase allows it to adopt alternative conformations that are likely important during polymerase maturation into active progeny RNPs. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

A potent transrepression domain in the retinoblastoma protein induces a cell cycle arrest when bound to E2F sites.

Science.gov (United States)

Sellers, W R; Rodgers, J W; Kaelin, W G

1995-01-01

An intact T/E1A-binding domain (the pocket) is necessary, but not sufficient, for the retinoblastoma protein (RB) to bind to DNA-protein complexes containing E2F and for RB to induce a G1/S block. Indirect evidence suggests that the binding of RB to E2F may, in addition to inhibiting E2F transactivation function, generate a complex capable of functioning as a transrepressor. Here we show that a chimera in which the E2F1 transactivation domain was replaced with the RB pocket could, in a DNA-binding and pocket-dependent manner, mimic the ability of RB to repress transcription and induce a cell cycle arrest. In contrast, a transdominant negative E2F1 mutant that is capable of blocking E2F-dependent transactivation did not. Fusion of the RB pocket to a heterologous DNA-binding domain unrelated to E2F likewise generated a transrepressor protein when scored against a suitable reporter. These results suggest that growth suppression by RB is due, at least in part, to transrepression mediated by the pocket domain bound to certain promoters via E2F. Images Fig. 4 Fig. 5 PMID:8524800

Solution structure of telomere binding domain of AtTRB2 derived from Arabidopsis thaliana

Energy Technology Data Exchange (ETDEWEB)

Yun, Ji-Hye [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Lee, Won Kyung [Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Kim, Heeyoun [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Kim, Eunhee; Cheong, Chaejoon [Magnetic Resonance Team, Korea Basic Science Institute (KBSI), Ochang, Chungbuk 363-883 (Korea, Republic of); Cho, Myeon Haeng [Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Lee, Weontae, E-mail: wlee@spin.yonsei.ac.kr [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of)

2014-09-26

Highlights: • We have determined solution structure of Myb domain of AtTRB2. • The Myb domain of AtTRB2 is located in the N-terminal region. • The Myb domain of AtTRB2 binds to plant telomeric DNA without fourth helix. • Helix 2 and 3 of the Myb domain of AtTRB2 are involved in DNA recognition. • AtTRB2 is a novel protein distinguished from other known plant TBP. - Abstract: Telomere homeostasis is regulated by telomere-associated proteins, and the Myb domain is well conserved for telomere binding. AtTRB2 is a member of the SMH (Single-Myb-Histone)-like family in Arabidopsis thaliana, having an N-terminal Myb domain, which is responsible for DNA binding. The Myb domain of AtTRB2 contains three α-helices and loops for DNA binding, which is unusual given that other plant telomere-binding proteins have an additional fourth helix that is essential for DNA binding. To understand the structural role for telomeric DNA binding of AtTRB2, we determined the solution structure of the Myb domain of AtTRB2 (AtTRB2{sub 1–64}) using nuclear magnetic resonance (NMR) spectroscopy. In addition, the inter-molecular interaction between AtTRB2{sub 1–64} and telomeric DNA has been characterized by the electrophoretic mobility shift assay (EMSA) and NMR titration analyses for both plant (TTTAGGG)n and human (TTAGGG)n telomere sequences. Data revealed that Trp28, Arg29, and Val47 residues located in Helix 2 and Helix 3 are crucial for DNA binding, which are well conserved among other plant telomere binding proteins. We concluded that although AtTRB2 is devoid of the additional fourth helix in the Myb-extension domain, it is able to bind to plant telomeric repeat sequences as well as human telomeric repeat sequences.

Structures of BIR domains from human NAIP and cIAP2

International Nuclear Information System (INIS)

Herman, Maria Dolores; Moche, Martin; Flodin, Susanne; Welin, Martin; Trésaugues, Lionel; Johansson, Ida; Nilsson, Martina; Nordlund, Pär; Nyman, Tomas

2009-01-01

The crystal structures of the human NAIP BIR2 and cIAP2 BIR3 domains have been determined. Both BIR domains harbors an amino-terminal tetrapeptide in its peptide-binding groove. The inhibitor of apoptosis (IAP) family of proteins contains key modulators of apoptosis and inflammation that interact with caspases through baculovirus IAP-repeat (BIR) domains. Overexpression of IAP proteins frequently occurs in cancer cells, thus counteracting the activated apoptotic program. The IAP proteins have therefore emerged as promising targets for cancer therapy. In this work, X-ray crystallography was used to determine the first structures of BIR domains from human NAIP and cIAP2. Both structures harbour an N-terminal tetrapeptide in the conserved peptide-binding groove. The structures reveal that these two proteins bind the tetrapeptides in a similar mode as do other BIR domains. Detailed interactions are described for the P1′–P4′ side chains of the peptide, providing a structural basis for peptide-specific recognition. An arginine side chain in the P3′ position reveals favourable interactions with its hydrophobic moiety in the binding pocket, while hydrophobic residues in the P2′ and P4′ pockets make similar interactions to those seen in other BIR domain–peptide complexes. The structures also reveal how a serine in the P1′ position is accommodated in the binding pockets of NAIP and cIAP2. In addition to shedding light on the specificity determinants of these two proteins, the structures should now also provide a framework for future structure-based work targeting these proteins

Membrane Restructuring by Phospholipase A2 Is Regulated by the Presence of Lipid Domains

DEFF Research Database (Denmark)

Leidy, Chad; Ocampo, Jackson; Duelund, Lars

2011-01-01

Secretory phospholipase A2 (sPLA2) catalyzes the hydrolysis of glycerophospholipids. This enzyme is sensitive to membrane structure, and its activity has been shown to increase in the presence of liquid-crystalline/gel (Lα/Lβ) lipid domains. In this work, we explore whether lipid domains can also...... without necessarily destroying the membrane. We confirm by high-performance liquid chromatography the preferential hydrolysis of DMPC within the phase coexistence region of the DMPC/DSPC phase diagram, showing that this preferential hydrolysis is accentuated close to the solidus phase boundary...

An incoherent feedforward loop facilitates adaptive tuning of gene expression.

Science.gov (United States)

Hong, Jungeui; Brandt, Nathan; Abdul-Rahman, Farah; Yang, Ally; Hughes, Tim; Gresham, David

2018-04-05

We studied adaptive evolution of gene expression using long-term experimental evolution of Saccharomyces cerevisiae in ammonium-limited chemostats. We found repeated selection for non-synonymous variation in the DNA binding domain of the transcriptional activator, GAT1, which functions with the repressor, DAL80 in an incoherent type-1 feedforward loop (I1-FFL) to control expression of the high affinity ammonium transporter gene, MEP2. Missense mutations in the DNA binding domain of GAT1 reduce its binding to the GATAA consensus sequence. However, we show experimentally, and using mathematical modeling, that decreases in GAT1 binding result in increased expression of MEP2 as a consequence of properties of I1-FFLs. Our results show that I1-FFLs, one of the most commonly occurring network motifs in transcriptional networks, can facilitate adaptive tuning of gene expression through modulation of transcription factor binding affinities. Our findings highlight the importance of gene regulatory architectures in the evolution of gene expression. © 2018, Hong et al.

Contextualized analysis of a needs assessment using the Theoretical Domains Framework: a case example in endocrinology.

Science.gov (United States)

Lazure, Patrice; Bartel, Robert C; Biller, Beverly M K; Molitch, Mark E; Rosenthal, Stephen M; Ross, Judith L; Bernsten, Brock D; Hayes, Sean M

2014-07-24

The Theoretical Domains Framework (TDF) is a set of 14 domains of behavior change that provide a framework for the critical issues and factors influencing optimal knowledge translation. Considering that a previous study has identified optimal knowledge translation techniques for each TDF domain, it was hypothesized that the TDF could be used to contextualize and interpret findings from a behavioral and educational needs assessment. To illustrate this hypothesis, findings and recommendations drawn from a 2012 national behavioral and educational needs assessment conducted with healthcare providers who treat and manage Growth and Growth Hormone Disorders, will be discussed using the TDF. This needs assessment utilized a mixed-methods research approach that included a combination of: [a] data sources (Endocrinologists (n:120), Pediatric Endocrinologists (n:53), Pediatricians (n:52)), [b] data collection methods (focus groups, interviews, online survey), [c] analysis methodologies (qualitative - analyzed through thematic analysis, quantitative - analyzed using frequencies, cross-tabulations, and gap analysis). Triangulation was used to generate trustworthy findings on the clinical practice gaps of endocrinologists, pediatric endocrinologists, and general pediatricians in their provision of care to adult patients with adult growth hormone deficiency or acromegaly, or children/teenagers with pediatric growth disorders. The identified gaps were then broken into key underlying determinants, categorized according to the TDF domains, and linked to optimal behavioral change techniques. The needs assessment identified 13 gaps, each with one or more underlying determinant(s). Overall, these determinants were mapped to 9 of the 14 TDF domains. The Beliefs about Consequences domain was identified as a contributing determinant to 7 of the 13 challenges. Five of the gaps could be related to the Skills domain, while three were linked to the Knowledge domain. The TDF categorization of

Contextualized analysis of a needs assessment using the Theoretical Domains Framework: a case example in endocrinology

Science.gov (United States)

2014-01-01

Background The Theoretical Domains Framework (TDF) is a set of 14 domains of behavior change that provide a framework for the critical issues and factors influencing optimal knowledge translation. Considering that a previous study has identified optimal knowledge translation techniques for each TDF domain, it was hypothesized that the TDF could be used to contextualize and interpret findings from a behavioral and educational needs assessment. To illustrate this hypothesis, findings and recommendations drawn from a 2012 national behavioral and educational needs assessment conducted with healthcare providers who treat and manage Growth and Growth Hormone Disorders, will be discussed using the TDF. Methods This needs assessment utilized a mixed-methods research approach that included a combination of: [a] data sources (Endocrinologists (n:120), Pediatric Endocrinologists (n:53), Pediatricians (n:52)), [b] data collection methods (focus groups, interviews, online survey), [c] analysis methodologies (qualitative - analyzed through thematic analysis, quantitative - analyzed using frequencies, cross-tabulations, and gap analysis). Triangulation was used to generate trustworthy findings on the clinical practice gaps of endocrinologists, pediatric endocrinologists, and general pediatricians in their provision of care to adult patients with adult growth hormone deficiency or acromegaly, or children/teenagers with pediatric growth disorders. The identified gaps were then broken into key underlying determinants, categorized according to the TDF domains, and linked to optimal behavioral change techniques. Results The needs assessment identified 13 gaps, each with one or more underlying determinant(s). Overall, these determinants were mapped to 9 of the 14 TDF domains. The Beliefs about Consequences domain was identified as a contributing determinant to 7 of the 13 challenges. Five of the gaps could be related to the Skills domain, while three were linked to the Knowledge domain

Comparison of structure, function and regulation of plant cold shock domain proteins to bacterial and animal cold shock domain proteins.

Science.gov (United States)

Chaikam, Vijay; Karlson, Dale T

2010-01-01

The cold shock domain (CSD) is among the most ancient and well conserved nucleic acid binding domains from bacteria to higher animals and plants. The CSD facilitates binding to RNA, ssDNA and dsDNA and most functions attributed to cold shock domain proteins are mediated by this nucleic acid binding activity. In prokaryotes, cold shock domain proteins only contain a single CSD and are termed cold shock proteins (Csps). In animal model systems, various auxiliary domains are present in addition to the CSD and are commonly named Y-box proteins. Similar to animal CSPs, plant CSPs contain auxiliary C-terminal domains in addition to their N-terminal CSD. Cold shock domain proteins have been shown to play important roles in development and stress adaptation in wide variety of organisms. In this review, the structure, function and regulation of plant CSPs are compared and contrasted to the characteristics of bacterial and animal CSPs. [BMB reports 2010; 43(1): 1-8].

Effective elements of school health promotion across behavioral domains: a systematic review of reviews

Directory of Open Access Journals (Sweden)

Peters Louk WH

2009-06-01

Full Text Available Abstract Background Most school health education programs focus on a single behavioral domain. Integrative programs that address multiple behaviors may be more efficient, but only if the elements of change are similar for these behaviors. The objective of this study was to examine which effective elements of school health education are similar across three particular behavioral domains. Methods A systematic review of reviews of the effectiveness of school-based health promotion programs was conducted for the domains of substance abuse, sexual behavior, and nutrition. The literature search spanned the time period between 1995 and October 2006 and included three databases, websites of review centers and backward search. Fifty-five reviews and meta-analyses met predetermined relevance and publication criteria and were included. Data was extracted by one reviewer and checked by a second reviewer. A standardized data extraction form was used, with detailed attention to effective elements pertaining to program goals, development, content, methods, facilitator, components and intensity. Two assessors rated the quality of reviews as strong, moderate or weak. We included only strong and moderate reviews in two types of analysis: one based on interpretation of conflicting results, the other on a specific vote-counting rule. Results Thirty six reviews were rated strong, 6 moderate, and 13 weak. A multitude of effective elements was identified in the included reviews and many elements were similar for two or more domains. In both types of analysis, five elements with evidence from strong reviews were found to be similar for all three domains: use of theory; addressing social influences, especially social norms; addressing cognitive-behavioral skills; training of facilitators; and multiple components. Two additional elements had positive results in all domains with the rule-based method of analysis, but had inconclusive results in at least one domain with

Hydrogeological characterisation and modelling of deformation zones and fracture domains, Forsmark modelling stage 2.2

Energy Technology Data Exchange (ETDEWEB)

Follin, Sven (SF GeoLogic AB, Taeby (SE)); Leven, Jakob (Swedish Nuclear Fuel and Waste Management Co., Stockholm (SE)); Hartley, Lee; Jackson, Peter; Joyce, Steve; Roberts, David; Swift, Ben (Serco Assurance, Harwell (GB))

2007-09-15

The work reported here collates the structural-hydraulic information gathered in 21 cored boreholes and 32 percussion-drilled boreholes belonging to Forsmark site description, modelling stage 2.2. The analyses carried out provide the hydrogeological input descriptions of the bedrock in Forsmark needed by the end users Repository Engineering, Safety Assessment and Environmental Impact Assessment; that is, hydraulic properties of deformation zones and fracture domains. The same information is also needed for constructing 3D groundwater flow models of the Forsmark site and surrounding area. The analyses carried out render the following conceptual model regarding the observed heterogeneity in deformation zone transmissivity: We find the geological division of the deterministically modelled deformation zones into eight categories (sets) useful from a hydrogeological point of view. Seven of the eight categories are steeply dipping, WNW, NW, NNW, NNE, NE, ENE and EW, and on is gently dipping, G. All deformation zones, regardless of orientation (strike and dip), are subjected to a substantial decrease in transmissivity with depth. The data gathered suggest a contrast of c. 20,000 times for the uppermost one kilometre of bedrock, i.e. more than four orders of magnitude. The hydraulic properties below this depth are not investigated. The lateral heterogeneity is also substantial but more irregular in its appearance. For instance, for a given elevation and deformation zone category (orientation), the spatial variability in transmissivity within a particular deformation zone appears to be as large as the variability between all deformation zones. This suggests that the lateral correlation length is shorter than the shortest distance between two adjacent observation points and shorter than the category spacing. The observation that the mean transmissivity of the gently-dipping deformation zones is c. one to two orders of magnitude greater than the mean transmissivities of all

Enhanced Prediction of Src Homology 2 (SH2) Domain Binding Potentials Using a Fluorescence Polarization-derived c-Met, c-Kit, ErbB, and Androgen Receptor Interactome*

Science.gov (United States)

Leung, Kin K.; Hause, Ronald J.; Barkinge, John L.; Ciaccio, Mark F.; Chuu, Chih-Pin; Jones, Richard B.

2014-01-01

Many human diseases are associated with aberrant regulation of phosphoprotein signaling networks. Src homology 2 (SH2) domains represent the major class of protein domains in metazoans that interact with proteins phosphorylated on the amino acid residue tyrosine. Although current SH2 domain prediction algorithms perform well at predicting the sequences of phosphorylated peptides that are likely to result in the highest possible interaction affinity in the context of random peptide library screens, these algorithms do poorly at predicting the interaction potential of SH2 domains with physiologically derived protein sequences. We employed a high throughput interaction assay system to empirically determine the affinity between 93 human SH2 domains and phosphopeptides abstracted from several receptor tyrosine kinases and signaling proteins. The resulting interaction experiments revealed over 1000 novel peptide-protein interactions and provided a glimpse into the common and specific interaction potentials of c-Met, c-Kit, GAB1, and the human androgen receptor. We used these data to build a permutation-based logistic regression classifier that performed considerably better than existing algorithms for predicting the interaction potential of several SH2 domains. PMID:24728074

Data-Aided Frequency-Domain 2×2 MIMO Equalizer for 112 Gbit/s PDM-QPSK Coherent Transmission Systems

DEFF Research Database (Denmark)

Pittalà, Fabio; Hauske, Fabian N.; Ye, Yabin

2012-01-01

Benefits of a low-complexity adaptive 32-tap 2×2 MIMO frequency-domain filter update by data-aided channel estimation over a time-domain filter with DD-LMS are shown. Superior stability and convergence speed is demonstrated with identical impairment tolerance....

Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation

Directory of Open Access Journals (Sweden)

Pero Stephanie C

2007-09-01

Full Text Available Abstract Background Human growth factor receptor bound protein 7 (Grb7 is an adapter protein that mediates the coupling of tyrosine kinases with their downstream signaling pathways. Grb7 is frequently overexpressed in invasive and metastatic human cancers and is implicated in cancer progression via its interaction with the ErbB2 receptor and focal adhesion kinase (FAK that play critical roles in cell proliferation and migration. It is thus a prime target for the development of novel anti-cancer therapies. Recently, an inhibitory peptide (G7-18NATE has been developed which binds specifically to the Grb7 SH2 domain and is able to attenuate cancer cell proliferation and migration in various cancer cell lines. Results As a first step towards understanding how Grb7 may be inhibited by G7-18NATE, we solved the crystal structure of the Grb7 SH2 domain to 2.1 Å resolution. We describe the details of the peptide binding site underlying target specificity, as well as the dimer interface of Grb 7 SH2. Dimer formation of Grb7 was determined to be in the μM range using analytical ultracentrifugation for both full-length Grb7 and the SH2 domain alone, suggesting the SH2 domain forms the basis of a physiological dimer. ITC measurements of the interaction of the G7-18NATE peptide with the Grb7 SH2 domain revealed that it binds with a binding affinity of Kd = ~35.7 μM and NMR spectroscopy titration experiments revealed that peptide binding causes perturbations to both the ligand binding surface of the Grb7 SH2 domain as well as to the dimer interface, suggesting that dimerisation of Grb7 is impacted on by peptide binding. Conclusion Together the data allow us to propose a model of the Grb7 SH2 domain/G7-18NATE interaction and to rationalize the basis for the observed binding specificity and affinity. We propose that the current study will assist with the development of second generation Grb7 SH2 domain inhibitors, potentially leading to novel inhibitors of

Vortex Ring Dynamics in Radially Confined Domains

Science.gov (United States)

Stewart, Kelley; Niebel, Casandra; Jung, Sunghwan; Vlachos, Pavlos

2010-11-01

Vortex ring dynamics have been studied extensively in semi-infinite quiescent volumes. However, very little is known about vortex-ring formation in wall-bounded domains where vortex wall interaction will affect both the vortex ring pinch-off and propagation velocity. This study addresses this limitation and studies vortex formation in radially confined domains to analyze the affect of vortex-ring wall interaction on the formation and propagation of the vortex ring. Vortex rings were produced using a pneumatically driven piston cylinder arrangement and were ejected into a long cylindrical tube which defined the confined downstream domain. A range of confinement domains were studied with varying confinement diameters Velocity field measurements were performed using planar Time Resolved Digital Particle Image Velocimetry (TRDPIV) and were processed using an in-house developed cross-correlation PIV algorithm. The experimental analysis was used to facilitate the development of a theoretical model to predict the variations in vortex ring circulation over time within confined domains.

The CC2D1A, a member of a new gene family with C2 domains, is involved in autosomal recessive non-syndromic mental retardation.

Science.gov (United States)

Basel-Vanagaite, L; Attia, R; Yahav, M; Ferland, R J; Anteki, L; Walsh, C A; Olender, T; Straussberg, R; Magal, N; Taub, E; Drasinover, V; Alkelai, A; Bercovich, D; Rechavi, G; Simon, A J; Shohat, M

2006-03-01

The molecular basis of autosomal recessive non-syndromic mental retardation (NSMR) is poorly understood, mostly owing to heterogeneity and absence of clinical criteria for grouping families for linkage analysis. Only two autosomal genes, the PRSS12 gene on chromosome 4q26 and the CRBN on chromosome 3p26, have been shown to cause autosomal recessive NSMR, each gene in only one family. To identify the gene causing autosomal recessive NSMR on chromosome 19p13.12. The candidate region established by homozygosity mapping was narrowed down from 2.4 Mb to 0.9 Mb on chromosome 19p13.12. A protein truncating mutation was identified in the gene CC2D1A in nine consanguineous families with severe autosomal recessive NSMR. The absence of the wild type protein in the lymphoblastoid cells of the patients was confirmed. CC2D1A is a member of a previously uncharacterised gene family that carries two conserved motifs, a C2 domain and a DM14 domain. The C2 domain is found in proteins which function in calcium dependent phospholipid binding; the DM14 domain is unique to the CC2D1A protein family and its role is unknown. CC2D1A is a putative signal transducer participating in positive regulation of I-kappaB kinase/NFkappaB cascade. Expression of CC2D1A mRNA was shown in the embryonic ventricular zone and developing cortical plate in staged mouse embryos, persisting into adulthood, with highest expression in the cerebral cortex and hippocampus. A previously unknown signal transduction pathway is important in human cognitive development.

The PH domain of phosphoinositide-dependent kinase-1 exhibits a novel, phospho-regulated monomer-dimer equilibrium with important implications for kinase domain activation: single-molecule and ensemble studies.

Science.gov (United States)

Ziemba, Brian P; Pilling, Carissa; Calleja, Véronique; Larijani, Banafshé; Falke, Joseph J

2013-07-16

Phosphoinositide-dependent kinase-1 (PDK1) is an essential master kinase recruited to the plasma membrane by the binding of its C-terminal PH domain to the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3). Membrane binding leads to PDK1 phospho-activation, but despite the central role of PDK1 in signaling and cancer biology, this activation mechanism remains poorly understood. PDK1 has been shown to exist as a dimer in cells, and one crystal structure of its isolated PH domain exhibits a putative dimer interface. It has been proposed that phosphorylation of PH domain residue T513 (or the phospho-mimetic T513E mutation) may regulate a novel PH domain dimer-monomer equilibrium, thereby converting an inactive PDK1 dimer to an active monomer. However, the oligomeric states of the PH domain on the membrane have not yet been determined, nor whether a negative charge at position 513 is sufficient to regulate its oligomeric state. This study investigates the binding of purified wild-type (WT) and T513E PDK1 PH domains to lipid bilayers containing the PIP3 target lipid, using both single-molecule and ensemble measurements. Single-molecule analysis of the brightness of the fluorescent PH domain shows that the PIP3-bound WT PH domain on membranes is predominantly dimeric while the PIP3-bound T513E PH domain is monomeric, demonstrating that negative charge at the T513 position is sufficient to dissociate the PH domain dimer and is thus likely to play a central role in PDK1 monomerization and activation. Single-molecule analysis of two-dimensional (2D) diffusion of PH domain-PIP3 complexes reveals that the dimeric WT PH domain diffuses at the same rate as a single lipid molecule, indicating that only one of its two PIP3 binding sites is occupied and there is little penetration of the protein into the bilayer as observed for other PH domains. The 2D diffusion of T513E PH domain is slower, suggesting the negative charge disrupts local structure in a way that allows

The SH2 Domain Regulates c-Abl Kinase Activation by a Cyclin-Like Mechanism and Remodulation of the Hinge Motion

Science.gov (United States)

Dölker, Nicole; Górna, Maria W.; Sutto, Ludovico; Torralba, Antonio S.; Superti-Furga, Giulio; Gervasio, Francesco L.

2014-01-01

Regulation of the c-Abl (ABL1) tyrosine kinase is important because of its role in cellular signaling, and its relevance in the leukemiogenic counterpart (BCR-ABL). Both auto-inhibition and full activation of c-Abl are regulated by the interaction of the catalytic domain with the Src Homology 2 (SH2) domain. The mechanism by which this interaction enhances catalysis is not known. We combined computational simulations with mutagenesis and functional analysis to find that the SH2 domain conveys both local and global effects on the dynamics of the catalytic domain. Locally, it regulates the flexibility of the αC helix in a fashion reminiscent of cyclins in cyclin-dependent kinases, reorienting catalytically important motifs. At a more global level, SH2 binding redirects the hinge motion of the N and C lobes and changes the conformational equilibrium of the activation loop. The complex network of subtle structural shifts that link the SH2 domain with the activation loop and the active site may be partially conserved with other SH2-domain containing kinases and therefore offer additional parameters for the design of conformation-specific inhibitors. PMID:25299346

The SH2 domain regulates c-Abl kinase activation by a cyclin-like mechanism and remodulation of the hinge motion.

Science.gov (United States)

Dölker, Nicole; Górna, Maria W; Sutto, Ludovico; Torralba, Antonio S; Superti-Furga, Giulio; Gervasio, Francesco L

2014-10-01

Regulation of the c-Abl (ABL1) tyrosine kinase is important because of its role in cellular signaling, and its relevance in the leukemiogenic counterpart (BCR-ABL). Both auto-inhibition and full activation of c-Abl are regulated by the interaction of the catalytic domain with the Src Homology 2 (SH2) domain. The mechanism by which this interaction enhances catalysis is not known. We combined computational simulations with mutagenesis and functional analysis to find that the SH2 domain conveys both local and global effects on the dynamics of the catalytic domain. Locally, it regulates the flexibility of the αC helix in a fashion reminiscent of cyclins in cyclin-dependent kinases, reorienting catalytically important motifs. At a more global level, SH2 binding redirects the hinge motion of the N and C lobes and changes the conformational equilibrium of the activation loop. The complex network of subtle structural shifts that link the SH2 domain with the activation loop and the active site may be partially conserved with other SH2-domain containing kinases and therefore offer additional parameters for the design of conformation-specific inhibitors.

The SH2 domain regulates c-Abl kinase activation by a cyclin-like mechanism and remodulation of the hinge motion.

Directory of Open Access Journals (Sweden)

Nicole Dölker

2014-10-01

Full Text Available Regulation of the c-Abl (ABL1 tyrosine kinase is important because of its role in cellular signaling, and its relevance in the leukemiogenic counterpart (BCR-ABL. Both auto-inhibition and full activation of c-Abl are regulated by the interaction of the catalytic domain with the Src Homology 2 (SH2 domain. The mechanism by which this interaction enhances catalysis is not known. We combined computational simulations with mutagenesis and functional analysis to find that the SH2 domain conveys both local and global effects on the dynamics of the catalytic domain. Locally, it regulates the flexibility of the αC helix in a fashion reminiscent of cyclins in cyclin-dependent kinases, reorienting catalytically important motifs. At a more global level, SH2 binding redirects the hinge motion of the N and C lobes and changes the conformational equilibrium of the activation loop. The complex network of subtle structural shifts that link the SH2 domain with the activation loop and the active site may be partially conserved with other SH2-domain containing kinases and therefore offer additional parameters for the design of conformation-specific inhibitors.

Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases.

Science.gov (United States)

Jadwin, Joshua A; Oh, Dongmyung; Curran, Timothy G; Ogiue-Ikeda, Mari; Jia, Lin; White, Forest M; Machida, Kazuya; Yu, Ji; Mayer, Bruce J

2016-04-12

While the affinities and specificities of SH2 domain-phosphotyrosine interactions have been well characterized, spatio-temporal changes in phosphosite availability in response to signals, and their impact on recruitment of SH2-containing proteins in vivo, are not well understood. To address this issue, we used three complementary experimental approaches to monitor phosphorylation and SH2 binding in human A431 cells stimulated with epidermal growth factor (EGF): 1) phospho-specific mass spectrometry; 2) far-Western blotting; and 3) live cell single-molecule imaging of SH2 membrane recruitment. Far-Western and MS analyses identified both well-established and previously undocumented EGF-dependent tyrosine phosphorylation and binding events, as well as dynamic changes in binding patterns over time. In comparing SH2 binding site phosphorylation with SH2 domain membrane recruitment in living cells, we found in vivo binding to be much slower. Delayed SH2 domain recruitment correlated with clustering of SH2 domain binding sites on the membrane, consistent with membrane retention via SH2 rebinding.

Amino-acid composition after loop deletion drives domain swapping.

Science.gov (United States)

Nandwani, Neha; Surana, Parag; Udgaonkar, Jayant B; Das, Ranabir; Gosavi, Shachi

2017-10-01

Rational engineering of a protein to enable domain swapping requires an understanding of the sequence, structural and energetic factors that favor the domain-swapped oligomer over the monomer. While it is known that the deletion of loops between β-strands can promote domain swapping, the spliced sequence at the position of the loop deletion is thought to have a minimal role to play in such domain swapping. Here, two loop-deletion mutants of the non-domain-swapping protein monellin, frame-shifted by a single residue, were designed. Although the spliced sequence in the two mutants differed by only one residue at the site of the deletion, only one of them (YEIKG) promoted domain swapping. The mutant containing the spliced sequence YENKG was entirely monomeric. This new understanding that the domain swapping propensity after loop deletion may depend critically on the chemical composition of the shortened loop will facilitate the rational design of domain swapping. © 2017 The Protein Society.

Direct EIT reconstructions of complex admittivities on a chest-shaped domain in 2-D.

Science.gov (United States)

Hamilton, Sarah J; Mueller, Jennifer L

2013-04-01

Electrical impedance tomography (EIT) is a medical imaging technique in which current is applied on electrodes on the surface of the body, the resulting voltage is measured, and an inverse problem is solved to recover the conductivity and/or permittivity in the interior. Images are then formed from the reconstructed conductivity and permittivity distributions. In the 2-D geometry, EIT is clinically useful for chest imaging. In this work, an implementation of a D-bar method for complex admittivities on a general 2-D domain is presented. In particular, reconstructions are computed on a chest-shaped domain for several realistic phantoms including a simulated pneumothorax, hyperinflation, and pleural effusion. The method demonstrates robustness in the presence of noise. Reconstructions from trigonometric and pairwise current injection patterns are included.

Combining biophysical methods to analyze the disulfide bond in SH2 domain of C-terminal Src kinase.

Science.gov (United States)

Liu, Dongsheng; Cowburn, David

2016-01-01

The Src Homology 2 (SH2) domain is a structurally conserved protein domain that typically binds to a phosphorylated tyrosine in a peptide motif from the target protein. The SH2 domain of C-terminal Src kinase (Csk) contains a single disulfide bond, which is unusual for most SH2 domains. Although the global motion of SH2 domain regulates Csk function, little is known about the relationship between the disulfide bond and binding of the ligand. In this study, we combined X-ray crystallography, solution NMR, and other biophysical methods to reveal the interaction network in Csk. Denaturation studies have shown that disulfide bond contributes significantly to the stability of SH2 domain, and crystal structures of the oxidized and C122S mutant showed minor conformational changes. We further investigated the binding of SH2 domain to a phosphorylated peptide from Csk-binding protein upon reduction and oxidation using both NMR and fluorescence approaches. This work employed NMR, X-ray cryptography, and other biophysical methods to study a disulfide bond in Csk SH2 domain. In addition, this work provides in-depth understanding of the structural dynamics of Csk SH2 domain.

Importance of Hypervariable Region 2 for Stability and Affinity of a Shark Single-Domain Antibody Specific for Ebola Virus Nucleoprotein.

Directory of Open Access Journals (Sweden)

George P Anderson

Full Text Available Single-domain antibodies derived from the unique New Antigen Receptor found in sharks have numerous potential applications, ranging from diagnostic reagents to therapeutics. Shark-derived single-domain antibodies possess the same characteristic ability to refold after heat denaturation found in single-domain antibodies derived from camelid heavy-chain-only antibodies. Recently, two shark derived single-domain antibodies specific for the nucleoprotein of Ebola virus were described. Our evaluation confirmed their high affinity for the nucleoprotein, but found their melting temperatures to be low relative to most single-domain antibodies. Our first approach towards improving their stability was grafting antigen-binding regions (complementarity determining regions of one of these single-domain antibodies onto a high melting temperature shark single-domain antibody. This resulted in two variants: one that displayed excellent affinity with a low melting temperature, while the other had poor affinity but a higher melting temperature. These new proteins, however, differed in only 3 amino acids within the complementarity determining region 2 sequence. In shark single-domain antibodies, the complementarity determining region 2 is often referred to as hypervariable region 2, as this segment of the antibody domain is truncated compared to the sequence in camelid single-domain antibodies and conventional heavy chain variable domains. To elucidate which of the three amino acids or combinations thereof were responsible for the affinity and stability we made the 6 double and single point mutants that covered the intermediates between these two clones. We found a single amino acid change that achieved a 10°C higher melting temperature while maintaining sub nM affinity. This research gives insights into the impact of the shark sdAb hypervariable 2 region on both stability and affinity.

Solution structure of the human Grb7-SH2 domain/erbB2 peptide complex and structural basis for Grb7 binding to ErbB2

International Nuclear Information System (INIS)

Ivancic, Monika; Daly, Roger J.; Lyons, Barbara A.

2003-01-01

The solution structure of the hGrb7-SH2 domain in complex with a ten amino acid phosphorylated peptide ligand representative of the erbB2 receptor tyrosine kinase (pY1139) is presented as determined by nuclear magnetic resonance methods. The hGrb7-SH2 domain structure reveals the Src homology 2 domain topology consisting of a central β-sheet capped at each end by an α-helix. The presence of a four residue insertion in the region between β-strand E and the EF loop and resulting influences on the SH2 domain/peptide complex structure are discussed. The binding conformation of the erbB2 peptide is in a β-turn similar to that found in phosphorylated tyrosine peptides bound to the Grb2-SH2 domain. To our knowledge this is only the second example of an SH2 domain binding its naturally occurring ligands in a turn, instead of extended, conformation. Close contacts between residues responsible for binding specificity in hGrb7-SH2 and the erbB2 peptide are characterized and the potential effect of mutation of these residues on the hGrb7-SH2 domain structure is discussed

Purification, crystallization and preliminary crystallographic analysis of the SH2 domain of IL-2-inducible T-cell kinase.

Science.gov (United States)

Joseph, Raji E; Ginder, Nathaniel D; Hoy, Julie A; Nix, Jay C; Honzatko, Richard B; Andreotti, Amy H

2011-02-01

Proline is a unique amino acid owing to the relatively small energy difference between the cis and trans conformations of its peptide bond. The X-Pro imide bond readily undergoes cis-trans isomerization in the context of short peptides as well as some proteins. However, the direct detection of cis-trans proline isomerization in folded proteins is technically challenging. NMR spectroscopy is well suited to the direct detection of proline isomerization in folded proteins. It is less clear how well X-ray crystallography can reveal this conformational exchange event in folded proteins. Conformational heterogeneity owing to cis-trans proline isomerization in the Src homology 2 (SH2) domain of the IL-2-inducible T-cell kinase (ITK) has been extensively characterized by NMR. Using the ITK SH2 domain as a test system, an attempt was made to determine whether proline isomerization could be detected in a crystal structure of the ITK SH2 domain. As a first step towards this goal, the purification, crystallization and preliminary characterization of the ITK SH2 domain are described.

Practice Facilitators' and Leaders' Perspectives on a Facilitated Quality Improvement Program.

Science.gov (United States)

McHugh, Megan; Brown, Tiffany; Liss, David T; Walunas, Theresa L; Persell, Stephen D

2018-04-01

Practice facilitation is a promising approach to helping practices implement quality improvements. Our purpose was to describe practice facilitators' and practice leaders' perspectives on implementation of a practice facilitator-supported quality improvement program and describe where their perspectives aligned and diverged. We conducted interviews with practice leaders and practice facilitators who participated in a program that included 35 improvement strategies aimed at the ABCS of heart health (aspirin use in high-risk individuals, blood pressure control, cholesterol management, and smoking cessation). Rapid qualitative analysis was used to collect, organize, and analyze the data. We interviewed 17 of the 33 eligible practice leaders, and the 10 practice facilitators assigned to those practices. Practice leaders and practice facilitators both reported value in the program's ability to bring needed, high-quality resources to practices. Practice leaders appreciated being able to set the schedule for facilitation and select among the 35 interventions. According to practice facilitators, however, relying on practice leaders to set the pace of the intervention resulted in a lower level of program intensity than intended. Practice leaders preferred targeted assistance, particularly electronic health record documentation guidance and linkages to state smoking cessation programs. Practice facilitators reported that the easiest interventions were those that did not alter care practices. The dual perspectives of practice leaders and practice facilitators provide a more holistic picture of enablers and barriers to program implementation. There may be greater opportunities to assist small practices through simple, targeted practice facilitator-supported efforts rather than larger, comprehensive quality improvement projects. © 2018 Annals of Family Medicine, Inc.

Exceptionally tight membrane-binding may explain the key role of the synaptotagmin-7 C ₂ A domain in asynchronous neurotransmitter release

Energy Technology Data Exchange (ETDEWEB)

Voleti, Rashmi; Tomchick, Diana R.; Südhof, Thomas C.; Rizo, Josep

2017-09-18

Synaptotagmins (Syts) act as Ca2+ sensors in neurotransmitter release by virtue of Ca2+-binding to their two C2 domains, but their mechanisms of action remain unclear. Puzzlingly, Ca2+-binding to the C2B domain appears to dominate Syt1 function in synchronous release, whereas Ca2+-binding to the C2A domain mediates Syt7 function in asynchronous release. Here we show that crystal structures of the Syt7 C2A domain and C2AB region, and analyses of intrinsic Ca2+-binding to the Syt7 C2 domains using isothermal titration calorimetry, did not reveal major differences that could explain functional differentiation between Syt7 and Syt1. However, using liposome titrations under Ca2+ saturating conditions, we show that the Syt7 C2A domain has a very high membrane affinity and dominates phospholipid binding to Syt7 in the presence or absence of L-α-phosphatidylinositol 4,5-diphosphate (PIP2). For Syt1, the two Ca2+-saturated C2 domains have similar affinities for membranes lacking PIP2, but the C2B domain dominates binding to PIP2-containing membranes. Mutagenesis revealed that the dramatic differences in membrane affinity between the Syt1 and Syt7 C2A domains arise in part from apparently conservative residue substitutions, showing how striking biochemical and functional differences can result from the cumulative effects of subtle residue substitutions. Viewed together, our results suggest that membrane affinity may be a key determinant of the functions of Syt C2 domains in neurotransmitter release.

Diversity in peptide recognition by the SH2 domain of SH2B1.

Science.gov (United States)

McKercher, Marissa A; Guan, Xiaoyang; Tan, Zhongping; Wuttke, Deborah S

2018-02-01

SH2B1 is a multidomain protein that serves as a key adaptor to regulate numerous cellular events, such as insulin, leptin, and growth hormone signaling pathways. Many of these protein-protein interactions are mediated by the SH2 domain of SH2B1, which recognizes ligands containing a phosphorylated tyrosine (pY), including peptides derived from janus kinase 2, insulin receptor, and insulin receptor substrate-1 and -2. Specificity for the SH2 domain of SH2B1 is conferred in these ligands either by a hydrophobic or an acidic side chain at the +3 position C-terminal to the pY. This specificity for chemically disparate species suggests that SH2B1 relies on distinct thermodynamic or structural mechanisms to bind to peptides. Using binding and structural strategies, we have identified unique thermodynamic signatures for each peptide binding mode, and several SH2B1 residues, including K575 and R578, that play distinct roles in peptide binding. The high-resolution structure of the SH2 domain of SH2B1 further reveals conformationally plastic protein loops that may contribute to the ability of the protein to recognize dissimilar ligands. Together, numerous hydrophobic and electrostatic interactions, in addition to backbone conformational flexibility, permit the recognition of diverse peptides by SH2B1. An understanding of this expanded peptide recognition will allow for the identification of novel physiologically relevant SH2B1/peptide interactions, which can contribute to the design of obesity and diabetes pharmaceuticals to target the ligand-binding interface of SH2B1 with high specificity. © 2017 Wiley Periodicals, Inc.

A novel PKD2L1 C-terminal domain critical for trimerization and channel function.

Science.gov (United States)

Zheng, Wang; Hussein, Shaimaa; Yang, JungWoo; Huang, Jun; Zhang, Fan; Hernandez-Anzaldo, Samuel; Fernandez-Patron, Carlos; Cao, Ying; Zeng, Hongbo; Tang, Jingfeng; Chen, Xing-Zhen

2015-03-30

As a transient receptor potential (TRP) superfamily member, polycystic kidney disease 2-like-1 (PKD2L1) is also called TRPP3 and has similar membrane topology as voltage-gated cation channels. PKD2L1 is involved in hedgehog signaling, intestinal development, and sour tasting. PKD2L1 and PKD1L3 form heterotetramers with 3:1 stoichiometry. C-terminal coiled-coil-2 (CC2) domain (G699-W743) of PKD2L1 was reported to be important for its trimerization but independent studies showed that CC2 does not affect PKD2L1 channel function. It thus remains unclear how PKD2L1 proteins oligomerize into a functional channel. By SDS-PAGE, blue native PAGE and mutagenesis we here identified a novel C-terminal domain called C1 (K575-T622) involved in stronger homotrimerization than the non-overlapping CC2, and found that the PKD2L1 N-terminus is critical for dimerization. By electrophysiology and Xenopus oocyte expression, we found that C1, but not CC2, is critical for PKD2L1 channel function. Our co-immunoprecipitation and dynamic light scattering experiments further supported involvement of C1 in trimerization. Further, C1 acted as a blocking peptide that inhibits PKD2L1 trimerization as well as PKD2L1 and PKD2L1/PKD1L3 channel function. Thus, our study identified C1 as the first PKD2L1 domain essential for both PKD2L1 trimerization and channel function, and suggest that PKD2L1 and PKD2L1/PKD1L3 channels share the PKD2L1 trimerization process.

Molecular basis of the specific subcellular localization of the C2-like domain of 5-lipoxygenase.

Science.gov (United States)

Kulkarni, Shilpa; Das, Sudipto; Funk, Colin D; Murray, Diana; Cho, Wonhwa

2002-04-12

The activation of 5-lipoxygenase (5-LO) involves its calcium-dependent translocation to the nuclear envelope, where it catalyzes the two-step transformation of arachidonic acid into leukotriene A(4), leading to the synthesis of various leukotrienes. To understand the mechanism by which 5-LO is specifically targeted to the nuclear envelope, we studied the membrane binding properties of the amino-terminal domain of 5-LO, which has been proposed to have a C2 domain-like structure. The model building, electrostatic potential calculation, and in vitro membrane binding studies of the isolated C2-like domain of 5-LO and selected mutants show that this Ca(2+)-dependent domain selectively binds zwitterionic phosphatidylcholine, which is conferred by tryptophan residues (Trp(13), Trp(75), and Trp(102)) located in the putative Ca(2+)-binding loops. The spatiotemporal dynamics of the enhanced green fluorescence protein-tagged C2-like domain of 5-LO and mutants in living cells also show that the phosphatidylcholine selectivity of the C2-like domain accounts for the specific targeting of 5-LO to the nuclear envelope. Together, these results show that the C2-like domain of 5-LO is a genuine Ca(2+)-dependent membrane-targeting domain and that the subcellular localization of the domain is governed in large part by its membrane binding properties.

Crystallization and preliminary X-ray analysis of the V domain of human nectin-2

International Nuclear Information System (INIS)

Qian, Xiaomin; Qi, Jianxun; Chu, Fuliang; Liu, Jun; Li, Qing; Yan, Jinghua

2009-01-01

Crystals of the V domain of human nectin-2 diffracted to 1.85 Å resolution and were monoclinic, belonging to space group P2 1 , with unit-cell parameters a = 52.3, b = 43.9, c = 56.1 Å, β = 118.2°. Nectin-2 belongs to a family of immunoglobulin-like cell adhesion molecules that are characterized by the presence of three immunoglobulin-like domains (V, C2 and C2) in the extracellular region. The V domain plays important roles in cell adhesion, NK cell activation and the entry of some herpesvirus. In this study, the V domain of human nectin-2 was expressed in Escherichia coli in the form of inclusion bodies, which were subsequently denatured and refolded. The soluble protein was crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 1.85 Å resolution and belonged to space group P2 1 , with unit-cell parameters a = 52.3, b = 43.9, c = 56.1 Å, β = 118.2°

The identification of FANCD2 DNA binding domains reveals nuclear localization sequences.

Science.gov (United States)

Niraj, Joshi; Caron, Marie-Christine; Drapeau, Karine; Bérubé, Stéphanie; Guitton-Sert, Laure; Coulombe, Yan; Couturier, Anthony M; Masson, Jean-Yves

2017-08-21

Fanconi anemia (FA) is a recessive genetic disorder characterized by congenital abnormalities, progressive bone-marrow failure, and cancer susceptibility. The FA pathway consists of at least 21 FANC genes (FANCA-FANCV), and the encoded protein products interact in a common cellular pathway to gain resistance against DNA interstrand crosslinks. After DNA damage, FANCD2 is monoubiquitinated and accumulates on chromatin. FANCD2 plays a central role in the FA pathway, using yet unidentified DNA binding regions. By using synthetic peptide mapping and DNA binding screen by electromobility shift assays, we found that FANCD2 bears two major DNA binding domains predominantly consisting of evolutionary conserved lysine residues. Furthermore, one domain at the N-terminus of FANCD2 bears also nuclear localization sequences for the protein. Mutations in the bifunctional DNA binding/NLS domain lead to a reduction in FANCD2 monoubiquitination and increase in mitomycin C sensitivity. Such phenotypes are not fully rescued by fusion with an heterologous NLS, which enable separation of DNA binding and nuclear import functions within this domain that are necessary for FANCD2 functions. Collectively, our results enlighten the importance of DNA binding and NLS residues in FANCD2 to activate an efficient FA pathway. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Ultra-thin clay layers facilitate seismic slip in carbonate faults.

Science.gov (United States)

Smeraglia, Luca; Billi, Andrea; Carminati, Eugenio; Cavallo, Andrea; Di Toro, Giulio; Spagnuolo, Elena; Zorzi, Federico

2017-04-06

Many earthquakes propagate up to the Earth's surface producing surface ruptures. Seismic slip propagation is facilitated by along-fault low dynamic frictional resistance, which is controlled by a number of physico-chemical lubrication mechanisms. In particular, rotary shear experiments conducted at seismic slip rates (1 ms -1 ) show that phyllosilicates can facilitate co-seismic slip along faults during earthquakes. This evidence is crucial for hazard assessment along oceanic subduction zones, where pelagic clays participate in seismic slip propagation. Conversely, the reason why, in continental domains, co-seismic slip along faults can propagate up to the Earth's surface is still poorly understood. We document the occurrence of micrometer-thick phyllosilicate-bearing layers along a carbonate-hosted seismogenic extensional fault in the central Apennines, Italy. Using friction experiments, we demonstrate that, at seismic slip rates (1 ms -1 ), similar calcite gouges with pre-existing phyllosilicate-bearing (clay content ≤3 wt.%) micro-layers weaken faster than calcite gouges or mixed calcite-phyllosilicate gouges. We thus propose that, within calcite gouge, ultra-low clay content (≤3 wt.%) localized along micrometer-thick layers can facilitate seismic slip propagation during earthquakes in continental domains, possibly enhancing surface displacement.

Assembly and misassembly of cystic fibrosis transmembrane conductance regulator: folding defects caused by deletion of F508 occur before and after the calnexin-dependent association of membrane spanning domain (MSD) 1 and MSD2.

Science.gov (United States)

Rosser, Meredith F N; Grove, Diane E; Chen, Liling; Cyr, Douglas M

2008-11-01

Cystic fibrosis transmembrane conductance regulator (CFTR) is a polytopic membrane protein that functions as a Cl(-) channel and consists of two membrane spanning domains (MSDs), two cytosolic nucleotide binding domains (NBDs), and a cytosolic regulatory domain. Cytosolic 70-kDa heat shock protein (Hsp70), and endoplasmic reticulum-localized calnexin are chaperones that facilitate CFTR biogenesis. Hsp70 functions in both the cotranslational folding and posttranslational degradation of CFTR. Yet, the mechanism for calnexin action in folding and quality control of CFTR is not clear. Investigation of this question revealed that calnexin is not essential for CFTR or CFTRDeltaF508 degradation. We identified a dependence on calnexin for proper assembly of CFTR's membrane spanning domains. Interestingly, efficient folding of NBD2 was also found to be dependent upon calnexin binding to CFTR. Furthermore, we identified folding defects caused by deletion of F508 that occurred before and after the calnexin-dependent association of MSD1 and MSD2. Early folding defects are evident upon translation of the NBD1 and R-domain and are sensed by the RMA-1 ubiquitin ligase complex.

Dissection of the BCR-ABL signaling network using highly specific monobody inhibitors to the SHP2 SH2 domains.

Science.gov (United States)

Sha, Fern; Gencer, Emel Basak; Georgeon, Sandrine; Koide, Akiko; Yasui, Norihisa; Koide, Shohei; Hantschel, Oliver

2013-09-10

The dysregulated tyrosine kinase BCR-ABL causes chronic myelogenous leukemia in humans and forms a large multiprotein complex that includes the Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2). The expression of SHP2 is necessary for BCR-ABL-dependent oncogenic transformation, but the precise signaling mechanisms of SHP2 are not well understood. We have developed binding proteins, termed monobodies, for the N- and C-terminal SH2 domains of SHP2. Intracellular expression followed by interactome analysis showed that the monobodies are essentially monospecific to SHP2. Two crystal structures revealed that the monobodies occupy the phosphopeptide-binding sites of the SH2 domains and thus can serve as competitors of SH2-phosphotyrosine interactions. Surprisingly, the segments of both monobodies that bind to the peptide-binding grooves run in the opposite direction to that of canonical phosphotyrosine peptides, which may contribute to their exquisite specificity. When expressed in cells, monobodies targeting the N-SH2 domain disrupted the interaction of SHP2 with its upstream activator, the Grb2-associated binder 2 adaptor protein, suggesting decoupling of SHP2 from the BCR-ABL protein complex. Inhibition of either N-SH2 or C-SH2 was sufficient to inhibit two tyrosine phosphorylation events that are critical for SHP2 catalytic activity and to block ERK activation. In contrast, targeting the N-SH2 or C-SH2 revealed distinct roles of the two SH2 domains in downstream signaling, such as the phosphorylation of paxillin and signal transducer and activator of transcription 5. Our results delineate a hierarchy of function for the SH2 domains of SHP2 and validate monobodies as potent and specific antagonists of protein-protein interactions in cancer cells.

High-Throughput Quantification of SH2 Domain-Phosphopeptide Interactions with Cellulose-Peptide Conjugate Microarrays.

Science.gov (United States)

Engelmann, Brett W

2017-01-01

The Src Homology 2 (SH2) domain family primarily recognizes phosphorylated tyrosine (pY) containing peptide motifs. The relative affinity preferences among competing SH2 domains for phosphopeptide ligands define "specificity space," and underpins many functional pY mediated interactions within signaling networks. The degree of promiscuity exhibited and the dynamic range of affinities supported by individual domains or phosphopeptides is best resolved by a carefully executed and controlled quantitative high-throughput experiment. Here, I describe the fabrication and application of a cellulose-peptide conjugate microarray (CPCMA) platform to the quantitative analysis of SH2 domain specificity space. Included herein are instructions for optimal experimental design with special attention paid to common sources of systematic error, phosphopeptide SPOT synthesis, microarray fabrication, analyte titrations, data capture, and analysis.

The CC2D1A, a member of a new gene family with C2 domains, is involved in autosomal recessive non‐syndromic mental retardation

Science.gov (United States)

Basel‐Vanagaite, L; Attia, R; Yahav, M; Ferland, R J; Anteki, L; Walsh, C A; Olender, T; Straussberg, R; Magal, N; Taub, E; Drasinover, V; Alkelai, A; Bercovich, D; Rechavi, G; Simon, A J; Shohat, M

2006-01-01

Background The molecular basis of autosomal recessive non‐syndromic mental retardation (NSMR) is poorly understood, mostly owing to heterogeneity and absence of clinical criteria for grouping families for linkage analysis. Only two autosomal genes, the PRSS12 gene on chromosome 4q26 and the CRBN on chromosome 3p26, have been shown to cause autosomal recessive NSMR, each gene in only one family. Objective To identify the gene causing autosomal recessive NSMR on chromosome 19p13.12. Results The candidate region established by homozygosity mapping was narrowed down from 2.4 Mb to 0.9 Mb on chromosome 19p13.12. A protein truncating mutation was identified in the gene CC2D1A in nine consanguineous families with severe autosomal recessive NSMR. The absence of the wild type protein in the lymphoblastoid cells of the patients was confirmed. CC2D1A is a member of a previously uncharacterised gene family that carries two conserved motifs, a C2 domain and a DM14 domain. The C2 domain is found in proteins which function in calcium dependent phospholipid binding; the DM14 domain is unique to the CC2D1A protein family and its role is unknown. CC2D1A is a putative signal transducer participating in positive regulation of I‐κB kinase/NFκB cascade. Expression of CC2D1A mRNA was shown in the embryonic ventricular zone and developing cortical plate in staged mouse embryos, persisting into adulthood, with highest expression in the cerebral cortex and hippocampus. Conclusions A previously unknown signal transduction pathway is important in human cognitive development. PMID:16033914

Domain matching epitaxy of cubic In{sub 2}O{sub 3} on r-plane sapphire

Energy Technology Data Exchange (ETDEWEB)

Vogt, Patrick; Trampert, Achim; Ramsteiner, Manfred; Bierwagen, Oliver [Paul-Drude-Institut fuer Festkoerperelektronik, Hausvogteiplatz 5-7, 10117, Berlin (Germany)

2015-07-15

Undoped, Sn-doped, and Mg-doped In{sub 2}O{sub 3} layers were grown on rhombohedral r-plane sapphire (α-Al{sub 2}O{sub 3} (10.2)) by plasma-assisted molecular beam epitaxy. X-ray diffraction and Raman scattering experiments demonstrated the formation of phase-pure, cubic (110)-oriented In{sub 2}O{sub 3} for Sn- and Mg-concentrations up to 2 x 10{sup 20} and 6 x 10{sup 20} cm{sup -3}, respectively. Scanning electron microscopy images showed facetted domains without any surface-parallel (110) facets. High Mg- or Sn-doping influenced surface morphology and the facet formation. X-ray diffraction Φ-scans indicated the formation of two rotational domains separated by an angle Φ = 86.6 due to the substrate mirror-symmetry around the in-plane-projected Al{sub 2}O{sub 3} c-axis. The in-plane epitaxial relationships to the substrate were determined for both domains. For the first domain it is Al{sub 2}O{sub 3}[01.0] parallel In{sub 2}O{sub 3}[3 anti 3 anti 4]. For the second domain the inplane epitaxial relation is Al{sub 2}O{sub 3}[01.0] parallel In{sub 2}O{sub 3}[3 anti 34]. A low-mismatch coincidence lattice of indium atoms from the film and oxygen atoms from the substrate rationalizes this epitaxial relation by domain-matched epitaxy. Cross-sectional transmission-electron microscopy showed a columnar domain-structure, indicating the vertical growth of the rotational domains after their nucleation. Coincidence structure of In{sub 2}O{sub 3} (110) (In atoms in red) grown on Al{sub 2}O{sub 3} (10.2) (O atoms in blue) showing two rotational domians. (copyright 2015 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

An SH2 domain-based tyrosine kinase assay using biotin ligase modified with a terbium(III) complex.

Science.gov (United States)

Sueda, Shinji; Shinboku, Yuki; Kusaba, Takeshi

2013-01-01

Src homology 2 (SH2) domains are modules of approximately 100 amino acids and are known to bind phosphotyrosine-containing sequences with high affinity and specificity. In the present work, we developed an SH2 domain-based assay for Src tyrosine kinase using a unique biotinylation reaction from archaeon Sulfolobus tokodaii. S. tokodaii biotinylation has a unique property that biotin protein ligase (BPL) forms a stable complex with its biotinylated substrate protein (BCCP). Here, an SH2 domain from lymphocyte-specific tyrosine kinase was genetically fused to a truncated BCCP, and the resulting fusion protein was labeled through biotinylation with BPL carrying multiple copies of a luminescent Tb(3+) complex. The labeled SH2 fusion proteins were employed to detect a phosphorylated peptide immobilized on the surface of the microtiter plate, where the phosphorylated peptide was produced by phosphorylation to the substrate peptide by Src tyrosine kinase. Our assay allows for a reliable determination of the activity of Src kinase lower than 10 pg/μL by a simple procedure.

Microstructure within domains of melt-processed YBa2Cu3O7-x superconductors

International Nuclear Information System (INIS)

Alexander, K.B.; Goyal, A.; Kroeger, D.M.; Selvamanickam, V.; Salama, K.

1992-01-01

The microstructure within single domains of melt-processed YBa 2 Cu 3 O 7-x (1:2:3) material has been examined. Rather than composing a ''brick-wall'' structure, the stacked, parallel platelets within the domains are actually portions of a single crystal. A growth mechanism is proposed that is consistent with the observed microstructural features. The anisotropic nature of the growth of 1:2:3 results in gaps separating the platelets. The gaps, however, terminate within domains, resulting in interconnected single-crystalline material. The absence of weak-link behavior for current flow along the c axis and the high critical-current densities observed within domains of melt-processed 1:2:3 material are readily explained by the fact that current flow is solely through single-crystalline material

Human alpha2-macroglobulin is composed of multiple domains, as predicted by homology with complement component C3.

Science.gov (United States)

Doan, Ninh; Gettins, Peter G W

2007-10-01

Human alpha2M (alpha2-macroglobulin) and the complement components C3 and C4 are thiol ester-containing proteins that evolved from the same ancestral gene. The recent structure determination of human C3 has allowed a detailed prediction of the location of domains within human alpha2M to be made. We describe here the expression and characterization of three alpha(2)M domains predicted to be involved in the stabilization of the thiol ester in native alpha2M and in its activation upon bait region proteolysis. The three newly expressed domains are MG2 (macroglobulin domain 2), TED (thiol ester-containing domain) and CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domain. Together with the previously characterized RBD (receptor-binding domain), they represent approx. 42% of the alpha2M polypeptide. Their expression as folded domains strongly supports the predicted domain organization of alpha2M. An X-ray crystal structure of MG2 shows it to have a fibronectin type-3 fold analogous to MG1-MG8 of C3. TED is, as predicted, an alpha-helical domain. CUB is a spliced domain composed of two stretches of polypeptide that flank TED in the primary structure. In intact C3 TED interacts with RBD, where it is in direct contact with the thiol ester, and with MG2 and CUB on opposite, flanking sides. In contrast, these alpha2M domains, as isolated species, show negligible interaction with one another, suggesting that the native conformation of alpha2M, and the consequent thiol ester-stabilizing domain-domain interactions, result from additional restraints imposed by the physical linkage of these domains or by additional domains in the protein.

Mutations in domain II of 23 S rRNA facilitate translation of a 23 S rRNA-encoded pentapeptide conferring erythromycin resistance

DEFF Research Database (Denmark)

Dam, M; Douthwaite, S; Tenson, T

1996-01-01

Mutations in domain II of Escherichia coli 23 S rRNA that cause resistance to erythromycin do so in a manner fundamentally different from mutations at the drug binding site in domain V of the 23 S rRNA. The domain II mutations are located in a hairpin structure between nucleotides 1198 and 1247...... this hypothesis, a range of point mutations was generated in domain II of 23 S rRNA in the vicinity of the E-peptide open reading frame. We find a correlation between erythromycin resistance of the mutant clones and increased accessibility of the ribosome binding site of the E-peptide gene. Furthermore......, the erythromycin resistance determinant in the mutants was shown to be confined to a small 23 S rRNA segment containing the coding region and the ribosome binding site of the E-peptide open reading frame. It thus appears that the domain II mutations mediate erythromycin resistance by increasing expression...

Activation of PI3K/Akt signaling by n-terminal SH2 domain mutants of the p85α regulatory subunit of PI3K is enhanced by deletion of its c-terminal SH2 domain.

Science.gov (United States)

Hofmann, Bianca T; Jücker, Manfred

2012-10-01

The phosphoinositide 3-kinase (PI3K) is frequently activated in human cancer cells due to gain of function mutations in the catalytic (p110) and the regulatory (p85) subunits. The regulatory subunit consists of an SH3 domain and two SH2 domains. An oncogenic form of p85α named p65 lacking the c-terminal SH2 domain (cSH2) has been cloned from an irradiation-induced murine thymic lymphoma and transgenic mice expressing p65 in T lymphocytes develop a lymphoproliferative disorder. We have recently detected a c-terminal truncated form of p85α named p76α in a human lymphoma cell line lacking most of the cSH2 domain due to a frame shift mutation. Here, we report that the deletion of the cSH2 domain enhances the activating effects of the n-terminal SH2 domain (nSH2) mutants K379E and R340E on the PI3K/Akt pathway and micro tumor formation in a focus assay. Further analysis revealed that this transforming effect is mediated by activation of the catalytic PI3K isoform p110α and downstream signaling through mTOR. Our data further support a mechanistic model in which mutations of the cSH2 domain of p85α can abrogate its negative regulatory function on PI3K activity via the nSH2 domain of p85α. Copyright © 2012 Elsevier Inc. All rights reserved.

Identification of the kinase that activates a nonmetazoan STAT gives insights into the evolution of phosphotyrosine-SH2 domain signaling.

Science.gov (United States)

Araki, Tsuyoshi; Kawata, Takefumi; Williams, Jeffrey G

2012-07-10

SH2 domains are integral to many animal signaling pathways. By interacting with specific phosphotyrosine residues, they provide regulatable protein-protein interaction domains. Dictyostelium is the only nonmetazoan with functionally characterized SH2 domains, but the cognate tyrosine kinases are unknown. There are no orthologs of the animal tyrosine kinases, but there are very many tyrosine kinase-like kinases (TKLs), a group of kinases which, despite their family name, are classified mainly as serine-threonine kinases. STATs are transcription factors that dimerize via phosphotyrosine-SH2 domain interactions. STATc is activated by phosphorylation on Tyr922 when cells are exposed to the prestalk inducer differentiation inducing factor (DIF-1), a chlorinated hexaphenone. We show that in a null mutant for Pyk2, a tyrosine-specific TKL, exposure to DIF-1 does not activate STATc. Conversely, overexpression of Pyk2 causes constitutive STATc activation. Pyk2 phosphorylates STATc on Tyr922 in vitro and complexes with STATc both in vitro and in vivo. This demonstration that a TKL directly activates a STAT has significant implications for understanding the evolutionary origins of SH2 domain-phosphotyrosine signaling. It also has mechanistic implications. Our previous work suggested that a predicted constitutive STATc tyrosine kinase activity is counterbalanced in vivo by the DIF-1-regulated activity of PTP3, a Tyr922 phosphatase. Here we show that the STATc-Pyk2 complex is formed constitutively by an interaction between the STATc SH2 domain and phosphotyrosine residues on Pyk2 that are generated by autophosphorylation. Also, as predicted, Pyk2 is constitutively active as a STATc kinase. This observation provides further evidence for this highly atypical, possibly ancestral, STAT regulation mechanism.

Structural insights into FRS2α PTB domain recognition by neurotrophin receptor TrkB.

Science.gov (United States)

Zeng, Lei; Kuti, Miklos; Mujtaba, Shiraz; Zhou, Ming-Ming

2014-07-01

The fibroblast growth factor receptor (FGFR) substrate 2 (FRS2) family proteins function as scaffolding adapters for receptor tyrosine kinases (RTKs). The FRS2α proteins interact with RTKs through the phosphotyrosine-binding (PTB) domain and transfer signals from the activated receptors to downstream effector proteins. Here, we report the nuclear magnetic resonance structure of the FRS2α PTB domain bound to phosphorylated TrkB. The structure reveals that the FRS2α-PTB domain is comprised of two distinct but adjacent pockets for its mutually exclusive interaction with either nonphosphorylated juxtamembrane region of the FGFR, or tyrosine phosphorylated peptides TrkA and TrkB. The new structural insights suggest rational design of selective small molecules through targeting of the two conjunct pockets in the FRS2α PTB domain. © 2014 Wiley Periodicals, Inc.

Structures of BIR domains from human NAIP and cIAP2.

Science.gov (United States)

Herman, Maria Dolores; Moche, Martin; Flodin, Susanne; Welin, Martin; Trésaugues, Lionel; Johansson, Ida; Nilsson, Martina; Nordlund, Pär; Nyman, Tomas

2009-11-01

The inhibitor of apoptosis (IAP) family of proteins contains key modulators of apoptosis and inflammation that interact with caspases through baculovirus IAP-repeat (BIR) domains. Overexpression of IAP proteins frequently occurs in cancer cells, thus counteracting the activated apoptotic program. The IAP proteins have therefore emerged as promising targets for cancer therapy. In this work, X-ray crystallography was used to determine the first structures of BIR domains from human NAIP and cIAP2. Both structures harbour an N-terminal tetrapeptide in the conserved peptide-binding groove. The structures reveal that these two proteins bind the tetrapeptides in a similar mode as do other BIR domains. Detailed interactions are described for the P1'-P4' side chains of the peptide, providing a structural basis for peptide-specific recognition. An arginine side chain in the P3' position reveals favourable interactions with its hydrophobic moiety in the binding pocket, while hydrophobic residues in the P2' and P4' pockets make similar interactions to those seen in other BIR domain-peptide complexes. The structures also reveal how a serine in the P1' position is accommodated in the binding pockets of NAIP and cIAP2. In addition to shedding light on the specificity determinants of these two proteins, the structures should now also provide a framework for future structure-based work targeting these proteins.

Human α2-macroglobulin is composed of multiple domains, as predicted by homology with complement component C3

Science.gov (United States)

Doan, Ninh; Gettins, Peter G. W.

2007-01-01

Human α2M (α2-macroglobulin) and the complement components C3 and C4 are thiol ester-containing proteins that evolved from the same ancestral gene. The recent structure determination of human C3 has allowed a detailed prediction of the location of domains within human α2M to be made. We describe here the expression and characterization of three α2M domains predicted to be involved in the stabilization of the thiol ester in native α2M and in its activation upon bait region proteolysis. The three newly expressed domains are MG2 (macroglobulin domain 2), TED (thiol ester-containing domain) and CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domain. Together with the previously characterized RBD (receptor-binding domain), they represent approx. 42% of the α2M polypeptide. Their expression as folded domains strongly supports the predicted domain organization of α2M. An X-ray crystal structure of MG2 shows it to have a fibronectin type-3 fold analogous to MG1–MG8 of C3. TED is, as predicted, an α-helical domain. CUB is a spliced domain composed of two stretches of polypeptide that flank TED in the primary structure. In intact C3 TED interacts with RBD, where it is in direct contact with the thiol ester, and with MG2 and CUB on opposite, flanking sides. In contrast, these α2M domains, as isolated species, show negligible interaction with one another, suggesting that the native conformation of α2M, and the consequent thiol ester-stabilizing domain–domain interactions, result from additional restraints imposed by the physical linkage of these domains or by additional domains in the protein. PMID:17608619

Coupled motions in the SH2 and kinase domains of Csk control Src phosphorylation.

Science.gov (United States)

Wong, Lilly; Lieser, Scot A; Miyashita, Osamu; Miller, Meghan; Tasken, Kjetil; Onuchic, Josè N; Adams, Joseph A; Woods, Virgil L; Jennings, Patricia A

2005-08-05

The C-terminal Src kinase (Csk) phosphorylates and down-regulates Src family tyrosine kinases. The Csk-binding protein (Cbp) localizes Csk close to its substrates at the plasma membrane, and increases the specific activity of the kinase. To investigate this long-range catalytic effect, the phosphorylation of Src and the conformation of Csk were investigated in the presence of a high-affinity phosphopeptide derived from Cbp. This peptide binds tightly to the SH2 domain and enhances Src recognition (lowers K(m)) by increasing the apparent phosphoryl transfer rate in the Csk active site, a phenomenon detected in rapid quench flow experiments. Previous studies demonstrated that the regulation of Csk activity is linked to conformational changes in the enzyme that can be probed with hydrogen-deuterium exchange methods. We show that the Cbp peptide impacts deuterium incorporation into its binding partner (the SH2 domain), and into the SH2-kinase linker and several sequences in the kinase domain, including the glycine-rich loop in the active site. These findings, along with computational data from normal mode analyses, suggest that the SH2 domain moves in a cantilever fashion with respect to the small lobe of the kinase domain, ordering the active site for catalysis. The binding of a small Cbp-derived peptide to the SH2 domain of Csk modifies these motions, enhancing Src recognition.

Expression, refolding and crystallizations of the Grb2-like (GADS) C-terminal SH3 domain complexed with a SLP-76 motif peptide

International Nuclear Information System (INIS)

Faravelli, Alessandro; Dimasi, Nazzareno

2005-01-01

Several crystals of the Grb2-like C-terminal SH3 domain in complex with a motif peptide from the SLP-76 protein were obtained and characterized. The Grb2-like adaptor protein GADS is composed of an N-terminal SH3 domain, an SH2 domain, a proline-rich region and a C-terminal SH3 domain. GADS interacts through its C-terminal SH3 domain with the adaptor protein SLP-76, thus recruiting this protein and other associated molecules to the linker for activation of T-cell (LAT) protein. The DNA encoding the C-terminal SH3 domain of GADS (GADS-cSH3) was assembled synthetically using a recursive PCR technique and the protein was overexpressed in Escherichia coli, refolded and purified. Several crystals of this domain in complex with the SLP-76 peptide were obtained and characterized

Her4 and Her2/neu tyrosine kinase domains dimerize and activate in a reconstituted in vitro system.

Science.gov (United States)

Monsey, John; Shen, Wei; Schlesinger, Paul; Bose, Ron

2010-03-05

Her4 (ErbB-4) and Her2/neu (ErbB-2) are receptor-tyrosine kinases belonging to the epidermal growth factor receptor (EGFR) family. Crystal structures of EGFR and Her4 kinase domains demonstrate kinase dimerization and activation through an allosteric mechanism. The kinase domains form an asymmetric dimer, where the C-lobe surface of one monomer contacts the N-lobe of the other monomer. EGFR kinase dimerization and activation in vitro was previously reported using a nickel-chelating lipid-liposome system, and we now apply this system to all other members of the EGFR family. Polyhistidine-tagged Her4, Her2/neu, and Her3 kinase domains are bound to these nickel-liposomes and are brought to high local concentration, mimicking what happens to full-length receptors in vivo following ligand binding. Addition of nickel-liposomes to Her4 kinase domain results in 40-fold activation in kinase activity and marked enhancement of C-terminal tail autophosphorylation. Activation of Her4 shows a sigmoidal dependence on kinase concentration, consistent with a cooperative process requiring kinase dimerization. Her2/neu kinase activity is also activated by nickel-liposomes, and is increased further by heterodimerization with Her3 or Her4. The ability of Her3 and Her4 to heterodimerize and activate other family members is studied in vitro. Her3 kinase domain readily activates Her2/neu but is a poor activator of Her4, which differs from the prediction made by the asymmetric dimer model. Mutation of Her3 residues (952)ENI(954) to the corresponding sequence in Her4 enhanced the ability of Her3 to activate Her4, demonstrating that sequence differences on the C-lobe surface influence the heterodimerization and activation of ErbB kinase domains.

The host-binding domain of the P2 phage tail spike reveals a trimeric iron-binding structure

International Nuclear Information System (INIS)

Yamashita, Eiki; Nakagawa, Atsushi; Takahashi, Junichi; Tsunoda, Kin-ichi; Yamada, Seiko; Takeda, Shigeki

2011-01-01

The C-terminal domain of a bacteriophage P2 tail-spike protein, gpV, was crystallized and its structure was solved at 1.27 Å resolution. The refined model showed a triple β-helix structure and the presence of iron, calcium and chloride ions. The adsorption and infection of bacteriophage P2 is mediated by tail fibres and tail spikes. The tail spikes on the tail baseplate are used to irreversibly adsorb to the host cells. Recently, a P2 phage tail-spike protein, gpV, was purified and it was shown that a C-terminal domain, Ser87–Leu211, is sufficient for the binding of gpV to host Escherichia coli membranes [Kageyama et al. (2009 ▶), Biochemistry, 48, 10129–10135]. In this paper, the crystal structure of the C-terminal domain of P2 gpV is reported. The structure is a triangular pyramid and looks like a spearhead composed of an intertwined β-sheet, a triple β-helix and a metal-binding region containing iron, calcium and chloride ions

An intact Pms2 ATPase domain is not essential for male fertility.

Science.gov (United States)

Fischer, Jared M; Dudley, Sandra; Miller, Ashleigh J; Liskay, R Michael

2016-03-01

The DNA mismatch repair (MMR) machinery in mammals plays critical roles in both mutation avoidance and spermatogenesis. Meiotic analysis of knockout mice of two different MMR genes, Mlh1 and Mlh3, revealed both male and female infertility associated with a defect in meiotic crossing over. In contrast, another MMR gene knockout, Pms2 (Pms2(ko/ko)), which contained a deletion of a portion of the ATPase domain, produced animals that were male sterile but female fertile. However, the meiotic phenotype of Pms2(ko/ko) males was less clear-cut than for Mlh1- or Mlh3-deficient meiosis. More recently, we generated a different Pms2 mutant allele (Pms2(cre)), which results in deletion of the same portion of the ATPase domain. Surprisingly, Pms2(cre/cre) male mice were completely fertile, suggesting that the ATPase domain of Pms2 is not required for male fertility. To explore the difference in male fertility, we examined the Pms2 RNA and found that alternative splicing of the Pms2(cre) allele results in a predicted Pms2 containing the C-terminus, which contains the Mlh1-interaction domain, a possible candidate for stabilizing Mlh1 levels. To study further the basis of male fertility, we examined Mlh1 levels in testes and found that whereas Pms2 loss in Pms2(ko/ko) mice results in severely reduced levels of Mlh1 expression in the testes, Mlh1 levels in Pms2(cre/cre) testes were reduced to a lesser extent. Thus, we propose that a primary function of Pms2 during spermatogenesis is to stabilize Mlh1 levels prior to its critical crossing over function with Mlh3. Copyright © 2015 Elsevier B.V. All rights reserved.

NovelFam3000 – Uncharacterized human protein domains conserved across model organisms

Science.gov (United States)

Kemmer, Danielle; Podowski, Raf M; Arenillas, David; Lim, Jonathan; Hodges, Emily; Roth, Peggy; Sonnhammer, Erik LL; Höög, Christer; Wasserman, Wyeth W

2006-01-01

Background Despite significant efforts from the research community, an extensive portion of the proteins encoded by human genes lack an assigned cellular function. Most metazoan proteins are composed of structural and/or functional domains, of which many appear in multiple proteins. Once a domain is characterized in one protein, the presence of a similar sequence in an uncharacterized protein serves as a basis for inference of function. Thus knowledge of a domain's function, or the protein within which it arises, can facilitate the analysis of an entire set of proteins. Description From the Pfam domain database, we extracted uncharacterized protein domains represented in proteins from humans, worms, and flies. A data centre was created to facilitate the analysis of the uncharacterized domain-containing proteins. The centre both provides researchers with links to dispersed internet resources containing gene-specific experimental data and enables them to post relevant experimental results or comments. For each human gene in the system, a characterization score is posted, allowing users to track the progress of characterization over time or to identify for study uncharacterized domains in well-characterized genes. As a test of the system, a subset of 39 domains was selected for analysis and the experimental results posted to the NovelFam3000 system. For 25 human protein members of these 39 domain families, detailed sub-cellular localizations were determined. Specific observations are presented based on the analysis of the integrated information provided through the online NovelFam3000 system. Conclusion Consistent experimental results between multiple members of a domain family allow for inferences of the domain's functional role. We unite bioinformatics resources and experimental data in order to accelerate the functional characterization of scarcely annotated domain families. PMID:16533400

NovelFam3000 – Uncharacterized human protein domains conserved across model organisms

Directory of Open Access Journals (Sweden)

Sonnhammer Erik LL

2006-03-01

Full Text Available Abstract Background Despite significant efforts from the research community, an extensive portion of the proteins encoded by human genes lack an assigned cellular function. Most metazoan proteins are composed of structural and/or functional domains, of which many appear in multiple proteins. Once a domain is characterized in one protein, the presence of a similar sequence in an uncharacterized protein serves as a basis for inference of function. Thus knowledge of a domain's function, or the protein within which it arises, can facilitate the analysis of an entire set of proteins. Description From the Pfam domain database, we extracted uncharacterized protein domains represented in proteins from humans, worms, and flies. A data centre was created to facilitate the analysis of the uncharacterized domain-containing proteins. The centre both provides researchers with links to dispersed internet resources containing gene-specific experimental data and enables them to post relevant experimental results or comments. For each human gene in the system, a characterization score is posted, allowing users to track the progress of characterization over time or to identify for study uncharacterized domains in well-characterized genes. As a test of the system, a subset of 39 domains was selected for analysis and the experimental results posted to the NovelFam3000 system. For 25 human protein members of these 39 domain families, detailed sub-cellular localizations were determined. Specific observations are presented based on the analysis of the integrated information provided through the online NovelFam3000 system. Conclusion Consistent experimental results between multiple members of a domain family allow for inferences of the domain's functional role. We unite bioinformatics resources and experimental data in order to accelerate the functional characterization of scarcely annotated domain families.

Identifying the Role of E2 Domains on Alphavirus Neutralization and Protective Immune Responses.

Directory of Open Access Journals (Sweden)

James Weger-Lucarelli

Full Text Available Chikungunya virus (CHIKV and other alphaviruses are the etiologic agents of numerous diseases in both humans and animals. Despite this, the viral mediators of protective immunity against alphaviruses are poorly understood, highlighted by the lack of a licensed human vaccine for any member of this virus genus. The alphavirus E2, the receptor-binding envelope protein, is considered to be the predominant target of the protective host immune response. Although envelope protein domains have been studied for vaccine and neutralization in flaviviruses, their role in alphaviruses is less characterized. Here, we describe the role of the alphavirus E2 domains in neutralization and protection through the use of chimeric viruses.Four chimeric viruses were constructed in which individual E2 domains of CHIKV were replaced with the corresponding domain from Semliki Forest virus (SFV (ΔDomA/ΔDomB/ΔDomC/ ΔDomA+B. Vaccination studies in mice (both live and inactivated virus revealed that domain B was the primary determinant of neutralization. Neutralization studies with CHIKV immune serum from humans were consistent with mouse studies, as ΔDomB was poorly neutralized.Using chimeric viruses, it was determined that the alphavirus E2 domain B was the critical target of neutralizing antibodies in both mice and humans. Therefore, chimeric viruses may have more relevance for vaccine discovery than peptide-based approaches, which only detect linear epitopes. This study provides new insight into the role of alphavirus E2 domains on neutralization determinants and may be useful for the design of novel therapeutic technologies.

Solution structure, dynamics and thermodynamics of the three SH3 domains of CD2AP

Energy Technology Data Exchange (ETDEWEB)

Roldan, Jose L. Ortega [Universidad de Granada, Departamento de Quimica Fisica e Instituto de Biotecnologia, Facultad de Ciencias (Spain); Blackledge, Martin [Institut de Biologie Structurale Jean-Pierre Ebel, CEA, CNRS, UJF UMR 5075, Protein Dynamics and Flexibility by NMR (France); Nuland, Nico A. J. van, E-mail: nvnuland@vub.ac.be [Vrije Universiteit Brussel, Structural Biology Brussels (Belgium); Azuaga, Ana I. [Universidad de Granada, Departamento de Quimica Fisica e Instituto de Biotecnologia, Facultad de Ciencias (Spain)

2011-06-15

CD2 associated protein (CD2AP) is an adaptor protein that plays an important role in cell to cell union needed for the kidney function. It contains three N-terminal SH3 domains that are able to interact among others with CD2, ALIX, c-Cbl and Ubiquitin. To understand the role of the individual SH3 domains of this adaptor protein we have performed a complete structural, thermodynamic and dynamic characterization of the separate domains using NMR and DSC. The energetic contributions to the stability and the backbone dynamics have been related to the structural features of each domain using the structure-based FoldX algorithm. We have found that the N-terminal SH3 domain of both adaptor proteins CD2AP and CIN85 are the most stable SH3 domains that have been studied until now. This high stability is driven by a more extensive network of intra-molecular interactions. We believe that this increased stabilization of N-terminal SH3 domains in adaptor proteins is crucial to maintain the necessary conformation to establish the proper interactions critical for the recruitment of their natural targets.

Host determinant residue lysine 627 lies on the surface of a discrete, folded domain of influenza virus polymerase PB2 subunit.

Directory of Open Access Journals (Sweden)

Franck Tarendeau

Full Text Available Understanding how avian influenza viruses adapt to human hosts is critical for the monitoring and prevention of future pandemics. Host specificity is determined by multiple sites in different viral proteins, and mutation of only a limited number of these sites can lead to inter-species transmission. Several of these sites have been identified in the viral polymerase, the best characterised being position 627 in the PB2 subunit. Efficient viral replication at the relatively low temperature of the human respiratory tract requires lysine 627 rather than the glutamic acid variant found systematically in avian viruses. However, the molecular mechanism by which any of these host specific sites determine host range are unknown, although adaptation to host factors is frequently evoked. We used ESPRIT, a library screening method, to identify a new PB2 domain that contains a high density of putative host specific sites, including residue 627. The X-ray structure of this domain (denoted the 627-domain exhibits a novel fold with the side-chain of Lys627 solvent exposed. The structure of the K627E mutated domain shows no structural differences but the charge reversal disrupts a striking basic patch on the domain surface. Five other recently proposed host determining sites of PB2 are also located on the 627-domain surface. The structure of the complete C-terminal region of PB2 comprising the 627-domain and the previously identified NLS-domain, which binds the host nuclear import factor importin alpha, was also determined. The two domains are found to pack together with a largely hydrophilic interface. These data enable a three-dimensional mapping of approximately half of PB2 sites implicated in cross-species transfer onto a single structural unit. Their surface location is consistent with roles in interactions with other viral proteins or host factors. The identification and structural characterization of these well-defined PB2 domains will help design

GUM2DFT—a software tool for uncertainty evaluation of transient signals in the frequency domain

International Nuclear Information System (INIS)

Eichstädt, S; Wilkens, V

2016-01-01

The Fourier transform and its counterpart for discrete time signals, the discrete Fourier transform (DFT), are common tools in measurement science and application. Although almost every scientific software package offers ready-to-use implementations of the DFT, the propagation of uncertainties in line with the guide to the expression of uncertainty in measurement (GUM) is typically neglected. This is of particular importance in dynamic metrology, when input estimation is carried out by deconvolution in the frequency domain. To this end, we present the new open-source software tool GUM2DFT, which utilizes closed formulas for the efficient propagation of uncertainties for the application of the DFT, inverse DFT and input estimation in the frequency domain. It handles different frequency domain representations, accounts for autocorrelation and takes advantage of the symmetry inherent in the DFT result for real-valued time domain signals. All tools are presented in terms of examples which form part of the software package. GUM2DFT will foster GUM-compliant evaluation of uncertainty in a DFT-based analysis and enable metrologists to include uncertainty evaluations in their routine work. (paper)

Optical network control plane for multi-domain networking

DEFF Research Database (Denmark)

Manolova, Anna Vasileva

This thesis focuses on multi-domain routing for traffice engineering and survivability support in optical transport networks under the Generalized Multi-Protocol Label Switching (GMPLS) control framework. First, different extensions to the Border Gateway Protocol for multi-domain Traffic...... process are not enough for efficient TE in mesh multi-domain networks. Enhancing the protocol with multi-path dissemination capability, combined with the employment of an end-to-end TE metric proves to be a highly efficient solution. Simulation results show good performance characteristics of the proposed...... is not as essential for improved network performance as the length of the provided paths. Second, the issue of multi-domain survivability support is analyzed. An AS-disjoint paths is beneficial not only for resilience support, but also for facilitating adequate network reactions to changes in the network, which...

Selective Targeting of SH2 Domain-Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies.

Science.gov (United States)

Kükenshöner, Tim; Schmit, Nadine Eliane; Bouda, Emilie; Sha, Fern; Pojer, Florence; Koide, Akiko; Seeliger, Markus; Koide, Shohei; Hantschel, Oliver

2017-05-05

The binding of Src-homology 2 (SH2) domains to phosphotyrosine (pY) sites is critical for the autoinhibition and substrate recognition of the eight Src family kinases (SFKs). The high sequence conservation of the 120 human SH2 domains poses a significant challenge to selectively perturb the interactions of even the SFK SH2 family against the rest of the SH2 domains. We have developed synthetic binding proteins, termed monobodies, for six of the SFK SH2 domains with nanomolar affinity. Most of these monobodies competed with pY ligand binding and showed strong selectivity for either the SrcA (Yes, Src, Fyn, Fgr) or SrcB subgroup (Lck, Lyn, Blk, Hck). Interactome analysis of intracellularly expressed monobodies revealed that they bind SFKs but no other SH2-containing proteins. Three crystal structures of monobody-SH2 complexes unveiled different and only partly overlapping binding modes, which rationalized the observed selectivity and enabled structure-based mutagenesis to modulate inhibition mode and selectivity. In line with the critical roles of SFK SH2 domains in kinase autoinhibition and T-cell receptor signaling, monobodies binding the Src and Hck SH2 domains selectively activated respective recombinant kinases, whereas an Lck SH2-binding monobody inhibited proximal signaling events downstream of the T-cell receptor complex. Our results show that SFK SH2 domains can be targeted with unprecedented potency and selectivity using monobodies. They are excellent tools for dissecting SFK functions in normal development and signaling and to interfere with aberrant SFK signaling networks in cancer cells. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Shapes of isolated domains and field induced evolution of regular and random 2D domain structures in LiNbO3 and LiTaO3

International Nuclear Information System (INIS)

Chernykh, A.; Shur, V.; Nikolaeva, E.; Shishkin, E.; Shur, A.; Terabe, K.; Kurimura, S.; Kitamura, K.; Gallo, K.

2005-01-01

The variety of the shapes of isolated domains, revealed in congruent and stoichiometric LiTaO 3 and LiNbO 3 by chemical etching and visualized by optical and scanning probe microscopy, was obtained by computer simulation. The kinetic nature of the domain shape was clearly demonstrated. The kinetics of domain structure with the dominance of the growth of the steps formed at the domain walls as a result of domain merging was investigated experimentally in slightly distorted artificial regular two-dimensional (2D) hexagonal domain structure and random natural one. The artificial structure has been realized in congruent LiNbO 3 by 2D electrode pattern produced by photolithography. The polarization reversal in congruent LiTaO 3 was investigated as an example of natural domain growth limited by merging. The switching process defined by domain merging was studied by computer simulation. The crucial dependence of the switching kinetics on the nuclei concentration has been revealed

Protein phosphatase 2a (PP2A binds within the oligomerization domain of striatin and regulates the phosphorylation and activation of the mammalian Ste20-Like kinase Mst3

Directory of Open Access Journals (Sweden)

Jones Candace A

2011-10-01

Full Text Available Abstract Background Striatin, a putative protein phosphatase 2A (PP2A B-type regulatory subunit, is a multi-domain scaffolding protein that has recently been linked to several diseases including cerebral cavernous malformation (CCM, which causes symptoms ranging from headaches to stroke. Striatin association with the PP2A A/C (structural subunit/catalytic subunit heterodimer alters PP2A substrate specificity, but targets and roles of striatin-associated PP2A are not known. In addition to binding the PP2A A/C heterodimer to form a PP2A holoenzyme, striatin associates with cerebral cavernous malformation 3 (CCM3 protein, the mammalian Mps one binder (MOB homolog, Mob3/phocein, the mammalian sterile 20-like (Mst kinases, Mst3, Mst4 and STK25, and several other proteins to form a large signaling complex. Little is known about the molecular architecture of the striatin complex and the regulation of these sterile 20-like kinases. Results To help define the molecular organization of striatin complexes and to determine whether Mst3 might be negatively regulated by striatin-associated PP2A, a structure-function analysis of striatin was performed. Two distinct regions of striatin are capable of stably binding directly or indirectly to Mob3--one N-terminal, including the coiled-coil domain, and another more C-terminal, including the WD-repeat domain. In addition, striatin residues 191-344 contain determinants necessary for efficient association of Mst3, Mst4, and CCM3. PP2A associates with the coiled-coil domain of striatin, but unlike Mob3 and Mst3, its binding appears to require striatin oligomerization. Deletion of the caveolin-binding domain on striatin abolishes striatin family oligomerization and PP2A binding. Point mutations in striatin that disrupt PP2A association cause hyperphosphorylation and activation of striatin-associated Mst3. Conclusions Striatin orchestrates the regulation of Mst3 by PP2A. It binds Mst3 likely as a dimer with CCM3 via

Domain-induced activation of human phospholipase A₂ type IIA: Local versus global lipid composition

DEFF Research Database (Denmark)

Leidy, C.; Linderoth, L.; Andresen, T.L.

2006-01-01

, we show that local enrichment of anionic lipids into fluid domains triggers PLA(2)-IIA activity. In addition, the compositional range of enzyme activity is shown to be related to the underlying lipid phase diagram. A comparison is done between PLA(2)-IIA and snake venom PLA(2), which in contrast...... to PLA(2)-IIA hydrolyzes both anionic and zwitterionic membranes. In general, this work shows that PLA(2)-IIA activation can be accomplished through local enrichment of anionic lipids into domains, indicating a mechanism for PLA(2)-IIA to target perturbed native membranes with low global anionic lipid...

Chemical shift assignments of the partially deuterated Fyn SH2-SH3 domain.

Science.gov (United States)

Kieken, Fabien; Loth, Karine; van Nuland, Nico; Tompa, Peter; Lenaerts, Tom

2018-04-01

Src Homology 2 and 3 (SH2 and SH3) are two key protein interaction modules involved in regulating the activity of many proteins such as tyrosine kinases and phosphatases by respective recognition of phosphotyrosine and proline-rich regions. In the Src family kinases, the inactive state of the protein is the direct result of the interaction of the SH2 and the SH3 domain with intra-molecular regions, leading to a closed structure incompetent with substrate modification. Here, we report the 1 H, 15 N and 13 C backbone- and side-chain chemical shift assignments of the partially deuterated Fyn SH3-SH2 domain and structural differences between tandem and single domains. The BMRB accession number is 27165.

Shear at Twin Domain Boundaries in YBa2Cu3O7-x

Science.gov (United States)

Caldwell, W. A.; Tamura, N.; Celestre, R. S.; MacDowell, A. A.; Padmore, H. A.; Geballe, T. H.; Koster, G.; Batterman, B. W.; Patel, J. R.

2004-05-01

The microstructure and strain state of twin domains in YBa2Cu3O7-x are discussed based upon synchrotron white-beam x-ray microdiffraction measurements. Intensity variations of the fourfold twin splitting of Laue diffraction peaks are used to determine the twin domain structure. Strain analysis shows that interfaces between neighboring twin domains are strained in shear, whereas the interior of these domains are regions of low strain. These measurements are consistent with the orientation relationships of twin boundaries within and across domains and show that basal plane shear stresses can exceed 100MPa where twin domains meet. Our results support stress field pinning of magnetic flux vortices by twin domain boundaries.

Facilitating an L2 Book Club: A Conversation-Analytic Study of Task Management

Science.gov (United States)

Ro, Eunseok

2018-01-01

This study employs conversation analysis to examine a facilitator's interactional practices in the post-expansion phase of students' presentations in the context of a book club for second language learning. The analysis shows how the facilitator establishes intersubjectivity with regard to the ongoing task and manages students' task performance.…

Barriers and Facilitators to Exercise Participation in People with Hip and/or Knee Osteoarthritis: Synthesis of the Literature Using Behavior Change Theory.

Science.gov (United States)

Dobson, Fiona; Bennell, Kim L; French, Simon D; Nicolson, Philippa J A; Klaasman, Remco N; Holden, Melanie A; Atkins, Lou; Hinman, Rana S

2016-05-01

Exercise is recommended for hip and knee osteoarthritis (OA). Patient initiation of, and adherence to, exercise is key to the success of managing symptoms. This study aimed to (1) identify modifiable barriers and facilitators to participation in intentional exercise in hip and/or knee OA, and (2) synthesize findings using behavior change theory. A scoping review with systematic searches was conducted through March 2015. Two reviewers screened studies for eligibility. Barriers and facilitators were extracted and synthesized according to the Theoretical Domains Framework (TDF) by two independent reviewers. Twenty-three studies (total of 4633 participants) were included. The greatest number of unique barriers and facilitators mapped to the Environmental Context and Resources domain. Many barriers were related to Beliefs about Consequences and Beliefs about Capabilities, whereas many facilitators were related to Reinforcement. Clinicians should take a proactive role in facilitating exercise uptake and adherence, rather than trusting patients to independently overcome barriers to exercise. Strategies that may be useful include a personalized approach to exercise prescription, considering environmental context and available resources, personalized education about beneficial consequences of exercise and reassurance about exercise capability, and use of reinforcement strategies. Future research should investigate the effectiveness of behavior change interventions that specifically target these factors.

Critical role of the Src homology 2 (SH2) domain of neuronal SH2B1 in the regulation of body weight and glucose homeostasis in mice.

Science.gov (United States)

Morris, David L; Cho, Kae Won; Rui, Liangyou

2010-08-01

SH2B1 is an SH2 domain-containing adaptor protein that plays a key role in the regulation of energy and glucose metabolism in both rodents and humans. Genetic deletion of SH2B1 in mice results in obesity and type 2 diabetes. Single-nucleotide polymorphisms in the SH2B1 loci and chromosomal deletions of the SH2B1 loci associate with obesity and insulin resistance in humans. In cultured cells, SH2B1 promotes leptin and insulin signaling by binding via its SH2 domain to phosphorylated tyrosines in Janus kinase 2 and the insulin receptor, respectively. Here we generated three lines of mice to analyze the role of the SH2 domain of SH2B1 in the central nervous system. Transgenic mice expressing wild-type, SH2 domain-defective (R555E), or SH2 domain-alone (DeltaN503) forms of SH2B1 specifically in neurons were crossed with SH2B1 knockout mice to generate KO/SH2B1, KO/R555E, or KO/DeltaN503 compound mutant mice. R555E had a replacement of Arg(555) with Glu within the SH2 domain. DeltaN503 contained an intact SH2 domain but lacked amino acids 1-503. Neuron-specific expression of recombinant SH2B1, but not R555E or DeltaN503, corrected hyperphagia, obesity, glucose intolerance, and insulin resistance in SH2B1 null mice. Neuron-specific expression of R555E in wild-type mice promoted obesity and insulin resistance. These results indicate that in addition to the SH2 domain, N-terminal regions of neuronal SH2B1 are also required for the maintenance of normal body weight and glucose metabolism. Additionally, mutations in the SH2 domain of SH2B1 may increase the susceptibility to obesity and type 2 diabetes in a dominant-negative manner.

Comparative structural analysis of lipid binding START domains.

Directory of Open Access Journals (Sweden)

Ann-Gerd Thorsell

Full Text Available Steroidogenic acute regulatory (StAR protein related lipid transfer (START domains are small globular modules that form a cavity where lipids and lipid hormones bind. These domains can transport ligands to facilitate lipid exchange between biological membranes, and they have been postulated to modulate the activity of other domains of the protein in response to ligand binding. More than a dozen human genes encode START domains, and several of them are implicated in a disease.We report crystal structures of the human STARD1, STARD5, STARD13 and STARD14 lipid transfer domains. These represent four of the six functional classes of START domains.Sequence alignments based on these and previously reported crystal structures define the structural determinants of human START domains, both those related to structural framework and those involved in ligand specificity.This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

Preparation of crystals for characterizing the Grb7 SH2 domain before and after complex formation with a bicyclic peptide antagonist.

Science.gov (United States)

Ambaye, Nigus D; Gunzburg, Menachem J; Traore, Daouda A K; Del Borgo, Mark P; Perlmutter, Patrick; Wilce, Matthew C J; Wilce, Jacqueline A

2014-02-01

Human growth factor receptor-bound protein 7 (Grb7) is an adapter protein involved in cell growth, migration and proliferation. It is now recognized that Grb7 is an emerging therapeutic target in specific cancer subtypes. Recently, the discovery of a bicyclic peptide inhibitor that targets the Grb7 SH2 domain, named G7-B1, was reported. In an attempt to probe the foundation of its interaction with Grb7, the crystallization and preliminary data collection of both the apo and G7-B1-bound forms of the Grb7 SH2 domain are reported here. Diffraction-quality crystals were obtained using the hanging-drop vapour-diffusion method. After several rounds of microseeding, crystals of the apo Grb7 SH2 domain were obtained that diffracted to 1.8 Å resolution, while those of the G7-B1-Grb7 SH2 domain complex diffracted to 2.2 Å resolution. The apo Grb7 SH2 domain crystallized in the trigonal space group P63, whereas the G7-B1-Grb7 SH2 domain complex crystallized in the monoclinic space group P21. The experimental aspects of crystallization, crystal optimization and data collection and the preliminary data are reported.

Barriers and Facilitators for Type-2 Diabetes Management in South Asians: A Systematic Review.

Directory of Open Access Journals (Sweden)

Tanveer Sohal

Full Text Available Although South Asian populations have among the highest burden of type 2 diabetes in the world, their diabetes management remains poor. We systematically reviewed studies on South Asian patient's perspectives on the barriers and facilitators to diabetes management.We conducted a literature search using OVID, CINHAL and EMBASE (January, 1990 -February, 2014 evaluating the core components of diabetes management: interactions with health care providers, diet, exercise, and medication adherence. South Asian patients were self-reported as Indian, Pakistani, Malaysian-Indian or Bangladeshi origin. From 208 abstracts reviewed, 20 studies were included (19 qualitative including mixed methods studies, 1 questionnaire. Barriers and facilitators were extracted and combined using qualitative synthesis.All studies included barriers and few facilitators were identified. Language and communication discordance with the healthcare provider was a significant barrier to receiving and understanding diabetes education. There was inconsistent willingness to partake in self-management with preference for following their physician's guidance. Barriers to adopting a diabetic diet were lack of specific details on South Asian tailored diabetic diet; social responsibilities to continue with a traditional diet, and misconceptions on the components of the diabetic diet. For exercise, South Asian patients were concerned with lack of gender specific exercise facilities and fear of injury or worsening health with exercise. Patients reported a lack of understanding about diabetes medication management, preference for folk and phytotherapy, and concerns about the long-term safety of diabetes medications. Facilitators included trust in care providers, use of culturally appropriate exercise and dietary advice and increasing family involvement. Overall themes for the barriers included lack of knowledge and misperceptions as well as lack of cultural adaptation to diabetes management

Barriers and Facilitators for Type-2 Diabetes Management in South Asians: A Systematic Review.

Science.gov (United States)

Sohal, Tanveer; Sohal, Parmjit; King-Shier, Kathryn M; Khan, Nadia A

2015-01-01

Although South Asian populations have among the highest burden of type 2 diabetes in the world, their diabetes management remains poor. We systematically reviewed studies on South Asian patient's perspectives on the barriers and facilitators to diabetes management. We conducted a literature search using OVID, CINHAL and EMBASE (January, 1990 -February, 2014) evaluating the core components of diabetes management: interactions with health care providers, diet, exercise, and medication adherence. South Asian patients were self-reported as Indian, Pakistani, Malaysian-Indian or Bangladeshi origin. From 208 abstracts reviewed, 20 studies were included (19 qualitative including mixed methods studies, 1 questionnaire). Barriers and facilitators were extracted and combined using qualitative synthesis. All studies included barriers and few facilitators were identified. Language and communication discordance with the healthcare provider was a significant barrier to receiving and understanding diabetes education. There was inconsistent willingness to partake in self-management with preference for following their physician's guidance. Barriers to adopting a diabetic diet were lack of specific details on South Asian tailored diabetic diet; social responsibilities to continue with a traditional diet, and misconceptions on the components of the diabetic diet. For exercise, South Asian patients were concerned with lack of gender specific exercise facilities and fear of injury or worsening health with exercise. Patients reported a lack of understanding about diabetes medication management, preference for folk and phytotherapy, and concerns about the long-term safety of diabetes medications. Facilitators included trust in care providers, use of culturally appropriate exercise and dietary advice and increasing family involvement. Overall themes for the barriers included lack of knowledge and misperceptions as well as lack of cultural adaptation to diabetes management. Diabetes

The study of co-citation analysis and knowledge structure on healthcare domain

Science.gov (United States)

Chu, Kuo-Chung; Liu, Wen-I.; Tsai, Ming-Yu

2012-11-01

With the prevalence of Internet and digital archives, the online e-journal database facilitates scholars to search literature in a research domain, or to cross-search an inter-disciplined field; the key literature can be efficiently traced out. This study intends to build a Web-based citation analysis system, which consists of four modules, they are: 1) literature search module; (2) statistics module; (3) articles analysis module; and (4) co-citation analysis module. The system focuses on PubMed Central dataset that has 170,000 records. In a research domain, a specific keyword searches in terms of authors, journals, and core issues. In addition, we use data mining techniques for co-citation analysis. The results assist researchers with in-depth understanding of the domain knowledge. Having an automated system for co-citation analysis, it helps to understand changes, trends, and knowledge structure of research domain. For the best of our knowledge, the proposed system differentiates from existing online electronic retrieval database analysis function. Perhaps, the proposed system is going to be a value-added database of healthcare domain, and hope to contribute the researchers.

MPEG-2 Compressed-Domain Algorithms for Video Analysis

Directory of Open Access Journals (Sweden)

Hesseler Wolfgang

2006-01-01

Full Text Available This paper presents new algorithms for extracting metadata from video sequences in the MPEG-2 compressed domain. Three algorithms for efficient low-level metadata extraction in preprocessing stages are described. The first algorithm detects camera motion using the motion vector field of an MPEG-2 video. The second method extends the idea of motion detection to a limited region of interest, yielding an efficient algorithm to track objects inside video sequences. The third algorithm performs a cut detection using macroblock types and motion vectors.

In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase.

Science.gov (United States)

Cooper, J A; Kashishian, A

1993-01-01

We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:8382774

Improving the performance of DomainDiscovery of protein domain boundary assignment using inter-domain linker index

Directory of Open Access Journals (Sweden)

Zomaya Albert Y

2006-12-01

Full Text Available Abstract Background Knowledge of protein domain boundaries is critical for the characterisation and understanding of protein function. The ability to identify domains without the knowledge of the structure – by using sequence information only – is an essential step in many types of protein analyses. In this present study, we demonstrate that the performance of DomainDiscovery is improved significantly by including the inter-domain linker index value for domain identification from sequence-based information. Improved DomainDiscovery uses a Support Vector Machine (SVM approach and a unique training dataset built on the principle of consensus among experts in defining domains in protein structure. The SVM was trained using a PSSM (Position Specific Scoring Matrix, secondary structure, solvent accessibility information and inter-domain linker index to detect possible domain boundaries for a target sequence. Results Improved DomainDiscovery is compared with other methods by benchmarking against a structurally non-redundant dataset and also CASP5 targets. Improved DomainDiscovery achieves 70% accuracy for domain boundary identification in multi-domains proteins. Conclusion Improved DomainDiscovery compares favourably to the performance of other methods and excels in the identification of domain boundaries for multi-domain proteins as a result of introducing support vector machine with benchmark_2 dataset.

The EBNA-2 N-Terminal Transactivation Domain Folds into a Dimeric Structure Required for Target Gene Activation.

Directory of Open Access Journals (Sweden)

Anders Friberg

2015-05-01

Full Text Available Epstein-Barr virus (EBV is a γ-herpesvirus that may cause infectious mononucleosis in young adults. In addition, epidemiological and molecular evidence links EBV to the pathogenesis of lymphoid and epithelial malignancies. EBV has the unique ability to transform resting B cells into permanently proliferating, latently infected lymphoblastoid cell lines. Epstein-Barr virus nuclear antigen 2 (EBNA-2 is a key regulator of viral and cellular gene expression for this transformation process. The N-terminal region of EBNA-2 comprising residues 1-58 appears to mediate multiple molecular functions including self-association and transactivation. However, it remains to be determined if the N-terminus of EBNA-2 directly provides these functions or if these activities merely depend on the dimerization involving the N-terminal domain. To address this issue, we determined the three-dimensional structure of the EBNA-2 N-terminal dimerization (END domain by heteronuclear NMR-spectroscopy. The END domain monomer comprises a small fold of four β-strands and an α-helix which form a parallel dimer by interaction of two β-strands from each protomer. A structure-guided mutational analysis showed that hydrophobic residues in the dimer interface are required for self-association in vitro. Importantly, these interface mutants also displayed severely impaired self-association and transactivation in vivo. Moreover, mutations of solvent-exposed residues or deletion of the α-helix do not impair dimerization but strongly affect the functional activity, suggesting that the EBNA-2 dimer presents a surface that mediates functionally important intra- and/or intermolecular interactions. Our study shows that the END domain is a novel dimerization fold that is essential for functional activity. Since this specific fold is a unique feature of EBNA-2 it might provide a novel target for anti-viral therapeutics.

Domain decomposition method for solving elliptic problems in unbounded domains

International Nuclear Information System (INIS)

Khoromskij, B.N.; Mazurkevich, G.E.; Zhidkov, E.P.

1991-01-01

Computational aspects of the box domain decomposition (DD) method for solving boundary value problems in an unbounded domain are discussed. A new variant of the DD-method for elliptic problems in unbounded domains is suggested. It is based on the partitioning of an unbounded domain adapted to the given asymptotic decay of an unknown function at infinity. The comparison of computational expenditures is given for boundary integral method and the suggested DD-algorithm. 29 refs.; 2 figs.; 2 tabs

Direct visualization of phase separation between superconducting and nematic domains in Co-doped CaFe2As2 close to a first-order phase transition

Science.gov (United States)

Fente, Antón; Correa-Orellana, Alexandre; Böhmer, Anna E.; Kreyssig, Andreas; Ran, S.; Bud'ko, Sergey L.; Canfield, Paul C.; Mompean, Federico J.; García-Hernández, Mar; Munuera, Carmen; Guillamón, Isabel; Suderow, Hermann

2018-01-01

We show that biaxial strain induces alternating tetragonal superconducting and orthorhombic nematic domains in Co-substituted CaFe2As2 . We use atomic force, magnetic force, and scanning tunneling microscopy to identify the domains and characterize their properties, finding in particular that tetragonal superconducting domains are very elongated, more than several tens of micrometers long and about 30 nm wide; have the same Tc as unstrained samples; and hold vortices in a magnetic field. Thus, biaxial strain produces a phase-separated state, where each phase is equivalent to what is found on either side of the first-order phase transition between antiferromagnetic orthorhombic and superconducting tetragonal phases found in unstrained samples when changing Co concentration. Having such alternating superconducting domains separated by normal conducting domains with sizes of the order of the coherence length opens opportunities to build Josephson junction networks or vortex pinning arrays and suggests that first-order quantum phase transitions lead to nanometric-size phase separation under the influence of strain.

Roles of the SH2 and SH3 domains in the regulation of neuronal Src kinase functions.

Science.gov (United States)

Groveman, Bradley R; Xue, Sheng; Marin, Vedrana; Xu, Jindong; Ali, Mohammad K; Bienkiewicz, Ewa A; Yu, Xian-Min

2011-02-01

Previous studies demonstrated that intra-domain interactions between Src family kinases (SFKs), stabilized by binding of the phosphorylated C-terminus to the SH2 domain and/or binding of the SH2 kinase linker to the SH3 domain, lock the molecules in a closed conformation, disrupt the kinase active site, and inactivate SFKs. Here we report that the up-regulation of N-methyl-D-aspartate receptors (NMDARs) induced by expression of constitutively active neuronal Src (n-Src), in which the C-terminus tyrosine is mutated to phenylalanine (n-Src/Y535F), is significantly reduced by dysfunctions of the SH2 and/or SH3 domains of the protein. Furthermore, we found that dysfunctions of SH2 and/or SH3 domains reduce auto-phosphorylation of the kinase activation loop, depress kinase activity, and decrease NMDAR phosphorylation. The SH2 domain plays a greater regulatory role than the SH3 domain. Our data also show that n-Src binds directly to the C-terminus of the NMDAR NR2A subunit in vitro, with a K(D) of 108.2 ± 13.3 nM. This binding is not Src kinase activity-dependent, and dysfunctions of the SH2 and/or SH3 domains do not significantly affect the binding. These data indicate that the SH2 and SH3 domains may function to promote the catalytic activity of active n-Src, which is important in the regulation of NMDAR functions. © 2010 The Authors Journal compilation © 2010 FEBS.

Interaction of the transactivation domain of B-Myb with the TAZ2 domain of the coactivator p300: molecular features and properties of the complex.

Directory of Open Access Journals (Sweden)

Ojore Oka

Full Text Available The transcription factor B-Myb is a key regulator of the cell cycle in vertebrates, with activation of transcription involving the recognition of specific DNA target sites and the recruitment of functional partner proteins, including the coactivators p300 and CBP. Here we report the results of detailed studies of the interaction between the transactivation domain of B-Myb (B-Myb TAD and the TAZ2 domain of p300. The B-Myb TAD was characterized using circular dichroism, fluorescence and NMR spectroscopy, which revealed that the isolated domain exists as a random coil polypeptide. Pull-down and spectroscopic experiments clearly showed that the B-Myb TAD binds to p300 TAZ2 to form a moderately tight (K(d ~1.0-10 µM complex, which results in at least partial folding of the B-Myb TAD. Significant changes in NMR spectra of p300 TAZ2 suggest that the B-Myb TAD binds to a relatively large patch on the surface of the domain (~1200 Å(2. The apparent B-Myb TAD binding site on p300 TAZ2 shows striking similarity to the surface of CBP TAZ2 involved in binding to the transactivation domain of the transcription factor signal transducer and activator of transcription 1 (STAT1, which suggests that the structure of the B-Myb TAD-p300 TAZ2 complex may share many features with that reported for STAT1 TAD-p300 TAZ2.

NMR structure of the N-terminal domain of the replication initiator protein DnaA

Energy Technology Data Exchange (ETDEWEB)

Wemmer, David E.; Lowery, Thomas J.; Pelton, Jeffrey G.; Chandonia, John-Marc; Kim, Rosalind; Yokota, Hisao; Wemmer, David E.

2007-08-07

DnaA is an essential component in the initiation of bacterial chromosomal replication. DnaA binds to a series of 9 base pair repeats leading to oligomerization, recruitment of the DnaBC helicase, and the assembly of the replication fork machinery. The structure of the N-terminal domain (residues 1-100) of DnaA from Mycoplasma genitalium was determined by NMR spectroscopy. The backbone r.m.s.d. for the first 86 residues was 0.6 +/- 0.2 Angstrom based on 742 NOE, 50 hydrogen bond, 46 backbone angle, and 88 residual dipolar coupling restraints. Ultracentrifugation studies revealed that the domain is monomeric in solution. Features on the protein surface include a hydrophobic cleft flanked by several negative residues on one side, and positive residues on the other. A negatively charged ridge is present on the opposite face of the protein. These surfaces may be important sites of interaction with other proteins involved in the replication process. Together, the structure and NMR assignments should facilitate the design of new experiments to probe the protein-protein interactions essential for the initiation of DNA replication.

15N and 31P solid-state NMR study of transmembrane domain alignment of M2 protein of influenza A virus in hydrated cylindrical lipid bilayers confined to anodic aluminum oxide nanopores.

Science.gov (United States)

Chekmenev, Eduard Y; Hu, Jun; Gor'kov, Peter L; Brey, William W; Cross, Timothy A; Ruuge, Andres; Smirnov, Alex I

2005-04-01

This communication reports the first example of a high resolution solid-state 15N 2D PISEMA NMR spectrum of a transmembrane peptide aligned using hydrated cylindrical lipid bilayers formed inside nanoporous anodic aluminum oxide (AAO) substrates. The transmembrane domain SSDPLVVA(A-15N)SIIGILHLILWILDRL of M2 protein from influenza A virus was reconstituted in hydrated 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine bilayers that were macroscopically aligned by a conventional micro slide glass support or by the AAO nanoporous substrate. 15N and 31P NMR spectra demonstrate that both the phospholipids and the protein transmembrane domain are uniformly aligned in the nanopores. Importantly, nanoporous AAO substrates may offer several advantages for membrane protein alignment in solid-state NMR studies compared to conventional methods. Specifically, higher thermal conductivity of aluminum oxide is expected to suppress thermal gradients associated with inhomogeneous radio frequency heating. Another important advantage of the nanoporous AAO substrate is its excellent accessibility to the bilayer surface for exposure to solute molecules. Such high accessibility achieved through the substrate nanochannel network could facilitate a wide range of structure-function studies of membrane proteins by solid-state NMR.

2.5-D frequency-domain viscoelastic wave modelling using finite-element method

Science.gov (United States)

Zhao, Jian-guo; Huang, Xing-xing; Liu, Wei-fang; Zhao, Wei-jun; Song, Jian-yong; Xiong, Bin; Wang, Shang-xu

2017-10-01

2-D seismic modelling has notable dynamic information discrepancies with field data because of the implicit line-source assumption, whereas 3-D modelling suffers from a huge computational burden. The 2.5-D approach is able to overcome both of the aforementioned limitations. In general, the earth model is treated as an elastic material, but the real media is viscous. In this study, we develop an accurate and efficient frequency-domain finite-element method (FEM) for modelling 2.5-D viscoelastic wave propagation. To perform the 2.5-D approach, we assume that the 2-D viscoelastic media are based on the Kelvin-Voigt rheological model and a 3-D point source. The viscoelastic wave equation is temporally and spatially Fourier transformed into the frequency-wavenumber domain. Then, we systematically derive the weak form and its spatial discretization of 2.5-D viscoelastic wave equations in the frequency-wavenumber domain through the Galerkin weighted residual method for FEM. Fixing a frequency, the 2-D problem for each wavenumber is solved by FEM. Subsequently, a composite Simpson formula is adopted to estimate the inverse Fourier integration to obtain the 3-D wavefield. We implement the stiffness reduction method (SRM) to suppress artificial boundary reflections. The results show that this absorbing boundary condition is valid and efficient in the frequency-wavenumber domain. Finally, three numerical models, an unbounded homogeneous medium, a half-space layered medium and an undulating topography medium, are established. Numerical results validate the accuracy and stability of 2.5-D solutions and present the adaptability of finite-element method to complicated geographic conditions. The proposed 2.5-D modelling strategy has the potential to address modelling studies on wave propagation in real earth media in an accurate and efficient way.

Characterization of Runella slithyformis HD-Pnk, a bifunctional DNA/RNA end-healing enzyme composed of an N-terminal 2',3' -phosphoesterase HD domain and a C-terminal 5' -OH polynucleotide kinase domain.

Science.gov (United States)

Munir, Annum; Shuman, Stewart

2016-11-28

5' and 3' end healing are key steps in nucleic acid break repair in which 5' -OH ends are phosphorylated by a polynucleotide kinase and 3' -PO 4 or 2',3' -cyclic-PO 4 ends are hydrolyzed by a phosphoesterase to generate the 5' -PO 4 and 3' -OH termini required for sealing by classic polynucleotide ligases. End healing and sealing enzymes are present in diverse bacterial taxa, often organized as modular units within a single multifunctional polypeptide or as subunits of a repair complex. Here we identify and characterize Runella slithyformis HD-Pnk as a novel bifunctional end-healing enzyme composed of an N-terminal 2',3' -phosphoesterase HD domain and a C-terminal 5' -OH polynucleotide kinase P-loop domain. HD-Pnk phosphorylates 5' -OH polynucleotides (9-mers or longer) in the presence of magnesium and any NTP donor. HD-Pnk dephosphorylates RNA 2',3' -cyclic phosphate, RNA 3' -phosphate, RNA 2' -phosphate, and DNA 3' -phosphate ends in the presence of a transition metal cofactor, which can be nickel, copper or cobalt. HD-Pnkp homologs are present in genera from eleven bacterial phyla and are often encoded in an operon with a putative ATP-dependent polynucleotide ligase. The present study provides insights to the diversity of nucleic acid repair strategies via the characterization of Runella slithyformis HD-Pnkp as the exemplar of a novel clade of dual 5' and 3' end-healing enzymes that phosphorylate 5' -OH termini and dephosphorylate 2',3' -cyclic-PO 4 , 3' -PO 4 , and 2' -PO 4 ends. The distinctive feature of HD-Pnk is its domain composition: a fusion of an N-terminal HD phosphohydrolase module to a C-terminal P-loop polynucleotide kinase module. Homologs of Runella HD-Pnk with the same domain composition, domain order, and similar polypeptide size are distributed widely among genera from eleven bacterial phyla. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Organising Sustainable Transition: Understanding the Product, Project and Service Domain of the Built Environment

DEFF Research Database (Denmark)

Thuesen, Christian; Koch-Ørvad, Nina; Maslesa, Esmir

2016-01-01

of three generic domains - the Project, Product and Service domain - with widely different markets, companies, business models and regulation. Besides identifying the characteristics of the different domains, the findings show that these domains are interdependent, but largely live their own lives......Sustainable transition of the built environment con struction industry is challenging the existing construction practices and business models. This article presents a framework for understanding and facilitating sustainable transition in the built environment. The framework was developed through...

Tyr66 acts as a conformational switch in the closed-to-open transition of the SHP-2 N-SH2-domain phosphotyrosine-peptide binding cleft

Directory of Open Access Journals (Sweden)

MacKerell Alexander D

2007-03-01

Full Text Available Abstract Background The N-terminal SH2 domain (N-SH2 of the non-receptor tyrosine phosphatase SHP-2 is involved both in localization of SHP-2 by recognition of phosphotyrosine (pY peptides and self-inhibition of SHP-2 phosphatase activity through the formation of a protein – protein interface with the phosphatase domain. Mutations that disrupt this interface break the coupling between pY-peptide binding cleft conformation and self-inhibition, thereby increasing both SHP-2 phosphatase activity and pY-peptide binding affinity, and are associated with the congenital condition Noonan syndrome and various pediatric leukemias. To better characterize the molecular process involved in N-SH2 pY-dependent binding, we have applied explicit-solvent molecular dynamics simulations to study the closed-to-open transition of the N-SH2 pY-peptide binding cleft. Results The existence of stable conformations in the left-handed helical and the extended regions of Tyr66 φ/ψ space prevent rapid interconversion of the backbone and create a conformational switch such that Tyr66 in a left-handed helical backbone conformation results in an open cleft and in an extended backbone conformation results in a closed cleft. The stable conformations arise from deep, well-localized free-energy minima in the left-handed helical and extended regions of the Tyr66 φ/ψ map. Changing the Tyr66 backbone conformation from extended to left-handed helical induces a closed-to-open transition in the cleft, and the reverse change in backbone conformation induces the reverse, open-to-closed transition. In the open-cleft state, weak solvent-exposed interactions involving the sidechains of Tyr66, Asp40, Lys55, and Gln57 serve to anchor the Tyr66 sidechain to the surface of the protein and away from the binding cleft entrance, thereby facilitating pY-peptide access to the binding cleft. Conclusion The simulations point to a regulatory role for Tyr66 and surrounding residues in SHP-2 function

Domain-Specific and Domain-General Training to Improve Kindergarten Children’s Mathematics

Directory of Open Access Journals (Sweden)

Geetha B. Ramani

2017-12-01

Full Text Available Ensuring that kindergarten children have a solid foundation in early numerical knowledge is of critical importance for later mathematical achievement. In this study, we targeted improving the numerical knowledge of kindergarteners (n = 81 from primarily low-income backgrounds using two approaches: one targeting their conceptual knowledge, specifically, their understanding of numerical magnitudes; and the other targeting their underlying cognitive system, specifically, their working memory. Both interventions involved playing game-like activities on tablet computers over the course of several sessions. As predicted, both interventions improved children’s numerical magnitude knowledge as compared to a no-contact control group, suggesting that both domain-specific and domain-general interventions facilitate mathematical learning. Individual differences in effort during the working memory game, but not the number knowledge training game predicted children’s improvements in number line estimation. The results demonstrate the potential of using a rapidly growing technology in early childhood classrooms to promote young children’s numerical knowledge.

Structural coupling of SH2-kinase domains links Fes and Abl substrate recognition and kinase activation.

Science.gov (United States)

Filippakopoulos, Panagis; Kofler, Michael; Hantschel, Oliver; Gish, Gerald D; Grebien, Florian; Salah, Eidarus; Neudecker, Philipp; Kay, Lewis E; Turk, Benjamin E; Superti-Furga, Giulio; Pawson, Tony; Knapp, Stefan

2008-09-05

The SH2 domain of cytoplasmic tyrosine kinases can enhance catalytic activity and substrate recognition, but the molecular mechanisms by which this is achieved are poorly understood. We have solved the structure of the prototypic SH2-kinase unit of the human Fes tyrosine kinase, which appears specialized for positive signaling. In its active conformation, the SH2 domain tightly interacts with the kinase N-terminal lobe and positions the kinase alphaC helix in an active configuration through essential packing and electrostatic interactions. This interaction is stabilized by ligand binding to the SH2 domain. Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions. Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface. Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.

Mutations in the LRRK2 Roc-COR tandem domain link Parkinson's disease to Wnt signalling pathways.

Science.gov (United States)

Sancho, Rosa M; Law, Bernard M H; Harvey, Kirsten

2009-10-15

Mutations in PARK8, encoding LRRK2, are the most common known cause of Parkinson's disease. The LRRK2 Roc-COR tandem domain exhibits GTPase activity controlling LRRK2 kinase activity via an intramolecular process. We report the interaction of LRRK2 with the dishevelled family of phosphoproteins (DVL1-3), key regulators of Wnt (Wingless/Int) signalling pathways important for axon guidance, synapse formation and neuronal maintenance. Interestingly, DVLs can interact with and mediate the activation of small GTPases with structural similarity to the LRRK2 Roc domain. The LRRK2 Roc-COR domain and the DVL1 DEP domain were necessary and sufficient for LRRK2-DVL1 interaction. Co-expression of DVL1 increased LRRK2 steady-state protein levels, an effect that was dependent on the DEP domain. Strikingly, LRRK2-DVL1-3 interactions were disrupted by the familial PARK8 mutation Y1699C, whereas pathogenic mutations at residues R1441 and R1728 strengthened LRRK2-DVL1 interactions. Co-expression of DVL1 with LRRK2 in mammalian cells resulted in the redistribution of LRRK2 to typical cytoplasmic DVL1 aggregates in HEK293 and SH-SY5Y cells and co-localization in neurites and growth cones of differentiated dopaminergic SH-SY5Y cells. This is the first report of the modulation of a key LRRK2-accessory protein interaction by PARK8 Roc-COR domain mutations segregating with Parkinson's disease. Since the DVL1 DEP domain is known to be involved in the regulation of small GTPases, we propose that: (i) DVLs may influence LRRK2 GTPase activity, and (ii) Roc-COR domain mutations modulating LRRK2-DVL interactions indirectly influence kinase activity. Our findings also link LRRK2 to Wnt signalling pathways, suggesting novel pathogenic mechanisms and new targets for genetic analysis in Parkinson's disease.

Changing domains in human capital measurement

Directory of Open Access Journals (Sweden)

Pharny D. Chrysler-Fox

2014-09-01

Research purpose: The aim of the study was to explore and describe changing domains within human capital management to be managed and measured. Motivation for the study: The motivation was to advance the understanding of changing measurement domains to aid practitioners to manage and measure the contribution of the human resource function and employees, in order to unlock and add value and ultimately contribute to the success of an organisation. Research design, approach and method: Unstructured, in-depth interview data of purposively selected cases from a selected panel of human resource practitioners specialising in human capital measurement was thematically analysed in this exploratory-descriptive investigation. Main findings: Findings suggested that seven domains should be managed and measured. These domains highlight new areas of impact and levels of management. In addition, crossdomain relationships in measurement allow for an understanding of the impact and potential value on which to capitalise. Practical/managerial implications: New domains to manage and measure focus the attention of practitioners beyond the transactional performance management paradigm to a transformational approach to influence the business strategy. Higher education institutions need to develop students’ cognitive skills to facilitate systems thinking. Contribution: This study suggests a new approach to managing and measuring the human capital function and the workforce.

Hydrogen diffusion in a one domain. beta. -V sub 2 H single crystal

Energy Technology Data Exchange (ETDEWEB)

Richter, D.; Mahling-Ennaoui, S. (Institut Max von Laue - Paul Langevin, 38 - Grenoble (France)); Hempelmann, R. (Forschungszentrum Juelich GmbH (Germany, F.R.). Inst. fuer Festkoerperforschung)

1989-01-01

The authors present first quasielastic neutron scattering experiments on hydrogen diffusion in a one-domain crystal of the ordered metal hydride {beta}-V{sub 2}H. The experiments led to a detailed evaluation of the microscopic jump geometries. At temperatures at which the structure is still intact the main diffusion channel leads across antistructural sites situated in empty layers in between occupied H-sheets. (orig.).

Amplification of TLO Mediator Subunit Genes Facilitate Filamentous Growth in Candida Spp.

Science.gov (United States)

Liu, Zhongle; Moran, Gary P.; Myers, Lawrence C.

2016-01-01

Filamentous growth is a hallmark of C. albicans pathogenicity compared to less-virulent ascomycetes. A multitude of transcription factors regulate filamentous growth in response to specific environmental cues. Our work, however, suggests the evolutionary history of C. albicans that resulted in its filamentous growth plasticity may be tied to a change in the general transcription machinery rather than transcription factors and their specific targets. A key genomic difference between C. albicans and its less-virulent relatives, including its closest relative C. dubliniensis, is the unique expansion of the TLO (TeLOmere-associated) gene family in C. albicans. Individual Tlo proteins are fungal-specific subunits of Mediator, a large multi-subunit eukaryotic transcriptional co-activator complex. This amplification results in a large pool of ‘free,’ non-Mediator associated, Tlo protein present in C. albicans, but not in C. dubliniensis or other ascomycetes with attenuated virulence. We show that engineering a large ‘free’ pool of the C. dubliniensis Tlo2 (CdTlo2) protein in C. dubliniensis, through overexpression, results in a number of filamentation phenotypes typically associated only with C. albicans. The amplitude of these phenotypes is proportional to the amount of overexpressed CdTlo2 protein. Overexpression of other C. dubliniensis and C. albicans Tlo proteins do result in these phenotypes. Tlo proteins and their orthologs contain a Mediator interaction domain, and a potent transcriptional activation domain. Nuclear localization of the CdTlo2 activation domain, facilitated naturally by the Tlo Mediator binding domain or artificially through an appended nuclear localization signal, is sufficient for the CdTlo2 overexpression phenotypes. A C. albicans med3 null mutant causes multiple defects including the inability to localize Tlo proteins to the nucleus and reduced virulence in a murine systemic infection model. Our data supports a model in which the