Predicting Ligand Binding Sites on Protein Surfaces by 3-Dimensional Probability Density Distributions of Interacting Atoms

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Jian, Jhih-Wei; Elumalai, Pavadai; Pitti, Thejkiran; Wu, Chih Yuan; Tsai, Keng-Chang; Chang, Jeng-Yih; Peng, Hung-Pin; Yang, An-Suei

2016-01-01

Predicting ligand binding sites (LBSs) on protein structures, which are obtained either from experimental or computational methods, is a useful first step in functional annotation or structure-based drug design for the protein structures. In this work, the structure-based machine learning algorithm ISMBLab-LIG was developed to predict LBSs on protein surfaces with input attributes derived from the three-dimensional probability density maps of interacting atoms, which were reconstructed on the query protein surfaces and were relatively insensitive to local conformational variations of the tentative ligand binding sites. The prediction accuracy of the ISMBLab-LIG predictors is comparable to that of the best LBS predictors benchmarked on several well-established testing datasets. More importantly, the ISMBLab-LIG algorithm has substantial tolerance to the prediction uncertainties of computationally derived protein structure models. As such, the method is particularly useful for predicting LBSs not only on experimental protein structures without known LBS templates in the database but also on computationally predicted model protein structures with structural uncertainties in the tentative ligand binding sites. PMID:27513851

Fringe proteins modulate Notch-ligand cis and trans interactions to specify signaling states.

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LeBon, Lauren; Lee, Tom V; Sprinzak, David; Jafar-Nejad, Hamed; Elowitz, Michael B

2014-09-25

The Notch signaling pathway consists of multiple types of receptors and ligands, whose interactions can be tuned by Fringe glycosyltransferases. A major challenge is to determine how these components control the specificity and directionality of Notch signaling in developmental contexts. Here, we analyzed same-cell (cis) Notch-ligand interactions for Notch1, Dll1, and Jag1, and their dependence on Fringe protein expression in mammalian cells. We found that Dll1 and Jag1 can cis-inhibit Notch1, and Fringe proteins modulate these interactions in a way that parallels their effects on trans interactions. Fringe similarly modulated Notch-ligand cis interactions during Drosophila development. Based on these and previously identified interactions, we show how the design of the Notch signaling pathway leads to a restricted repertoire of signaling states that promote heterotypic signaling between distinct cell types, providing insight into the design principles of the Notch signaling system, and the specific developmental process of Drosophila dorsal-ventral boundary formation.

Ligand-receptor Interactions by NMR Spectroscopy

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Novak. P.

2008-04-01

Full Text Available Today NMR spectroscopy is a method of choice for elucidation of interactions between biomolecules and the potential ligands. Knowledge on these interactions is an essential prerequisite for the rational drug design. The most important contribution of NMR to drug design a few years ago was the 3D structure determination of proteins. Besides delivering the 3D structures of the free proteins as a raw material for the modeling studies on ligand binding, NMR can directly yield valuable experimental data on the biologically important protein-ligand complexes. In addition to X-ray diffraction, NMR spectroscopy can provide information on the internal protein dynamics ordynamics of intermolecular interactions. Changes in NMR parameters allow us to detect ("SAR by NMR" and quantitatively determine binding affinities (titration, diffusion NMR experiments, etc. of potential ligands. Also, it is possible to determine the binding site and conformations of ligands, receptors and receptor-ligand complexes with the help of NMR methods such as tr-NOESY. Epitopes or functional groups responsible for binding of ligands to the receptor can be identified by employing STD or WaterLOGSY experiments. In this review are described some of the most frequent NMR methods for the characterization of the interactions between biomolecules and ligands, together with their advantages and disadvantages.

Analysis of protein stability and ligand interactions by thermal shift assay.

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Huynh, Kathy; Partch, Carrie L

2015-02-02

Purification of recombinant proteins for biochemical assays and structural studies is time-consuming and presents inherent difficulties that depend on the optimization of protein stability. The use of dyes to monitor thermal denaturation of proteins with sensitive fluorescence detection enables rapid and inexpensive determination of protein stability using real-time PCR instruments. By screening a wide range of solution conditions and additives in a 96-well format, the thermal shift assay easily identifies conditions that significantly enhance the stability of recombinant proteins. The same approach can be used as an initial low-cost screen to discover new protein-ligand interactions by capitalizing on increases in protein stability that typically occur upon ligand binding. This unit presents a methodological workflow for small-scale, high-throughput thermal denaturation of recombinant proteins in the presence of SYPRO Orange dye. Copyright © 2015 John Wiley & Sons, Inc.

Simple Ligand-Receptor Interaction Descriptor (SILIRID) for alignment-free binding site comparison.

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Chupakhin, Vladimir; Marcou, Gilles; Gaspar, Helena; Varnek, Alexandre

2014-06-01

We describe SILIRID (Simple Ligand-Receptor Interaction Descriptor), a novel fixed size descriptor characterizing protein-ligand interactions. SILIRID can be obtained from the binary interaction fingerprints (IFPs) by summing up the bits corresponding to identical amino acids. This results in a vector of 168 integer numbers corresponding to the product of the number of entries (20 amino acids and one cofactor) and 8 interaction types per amino acid (hydrophobic, aromatic face to face, aromatic edge to face, H-bond donated by the protein, H-bond donated by the ligand, ionic bond with protein cation and protein anion, and interaction with metal ion). Efficiency of SILIRID to distinguish different protein binding sites has been examined in similarity search in sc-PDB database, a druggable portion of the Protein Data Bank, using various protein-ligand complexes as queries. The performance of retrieval of structurally and evolutionary related classes of proteins was comparable to that of state-of-the-art approaches (ROC AUC ≈ 0.91). SILIRID can efficiently be used to visualize chemogenomic space covered by sc-PDB using Generative Topographic Mapping (GTM): sc-PDB SILIRID data form clusters corresponding to different protein types.

Forging the Basis for Developing Protein-Ligand Interaction Scoring Functions.

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Liu, Zhihai; Su, Minyi; Han, Li; Liu, Jie; Yang, Qifan; Li, Yan; Wang, Renxiao

2017-02-21

In structure-based drug design, scoring functions are widely used for fast evaluation of protein-ligand interactions. They are often applied in combination with molecular docking and de novo design methods. Since the early 1990s, a whole spectrum of protein-ligand interaction scoring functions have been developed. Regardless of their technical difference, scoring functions all need data sets combining protein-ligand complex structures and binding affinity data for parametrization and validation. However, data sets of this kind used to be rather limited in terms of size and quality. On the other hand, standard metrics for evaluating scoring function used to be ambiguous. Scoring functions are often tested in molecular docking or even virtual screening trials, which do not directly reflect the genuine quality of scoring functions. Collectively, these underlying obstacles have impeded the invention of more advanced scoring functions. In this Account, we describe our long-lasting efforts to overcome these obstacles, which involve two related projects. On the first project, we have created the PDBbind database. It is the first database that systematically annotates the protein-ligand complexes in the Protein Data Bank (PDB) with experimental binding data. This database has been updated annually since its first public release in 2004. The latest release (version 2016) provides binding data for 16 179 biomolecular complexes in PDB. Data sets provided by PDBbind have been applied to many computational and statistical studies on protein-ligand interaction and various subjects. In particular, it has become a major data resource for scoring function development. On the second project, we have established the Comparative Assessment of Scoring Functions (CASF) benchmark for scoring function evaluation. Our key idea is to decouple the "scoring" process from the "sampling" process, so scoring functions can be tested in a relatively pure context to reflect their quality. In our

Structural analysis of protein-ligand interactions: the binding of endogenous compounds and of synthetic drugs.

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Gallina, Anna M; Bork, Peer; Bordo, Domenico

2014-02-01

The large number of macromolecular structures deposited with the Protein Data Bank (PDB) describing complexes between proteins and either physiological compounds or synthetic drugs made it possible a systematic analysis of the interactions occurring between proteins and their ligands. In this work, the binding pockets of about 4000 PDB protein-ligand complexes were investigated and amino acid and interaction types were analyzed. The residues observed with lowest frequency in protein sequences, Trp, His, Met, Tyr, and Phe, turned out to be the most abundant in binding pockets. Significant differences between drug-like and physiological compounds were found. On average, physiological compounds establish with respect to drugs about twice as many hydrogen bonds with protein atoms, whereas drugs rely more on hydrophobic interactions to establish target selectivity. The large number of PDB structures describing homologous proteins in complex with the same ligand made it possible to analyze the conservation of binding pocket residues among homologous protein structures bound to the same ligand, showing that Gly, Glu, Arg, Asp, His, and Thr are more conserved than other amino acids. Also in the cases in which the same ligand is bound to unrelated proteins, the binding pockets showed significant conservation in the residue types. In this case, the probability of co-occurrence of the same amino acid type in the binding pockets could be up to thirteen times higher than that expected on a random basis. The trends identified in this study may provide an useful guideline in the process of drug design and lead optimization. Copyright © 2014 John Wiley & Sons, Ltd.

Descriptor Data Bank (DDB): A Cloud Platform for Multiperspective Modeling of Protein-Ligand Interactions.

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Ashtawy, Hossam M; Mahapatra, Nihar R

2018-01-22

Protein-ligand (PL) interactions play a key role in many life processes such as molecular recognition, molecular binding, signal transmission, and cell metabolism. Examples of interaction forces include hydrogen bonding, hydrophobic effects, steric clashes, electrostatic contacts, and van der Waals attractions. Currently, a large number of hypotheses and perspectives to model these interaction forces are scattered throughout the literature and largely forgotten. Instead, had they been assembled and utilized collectively, they would have substantially improved the accuracy of predicting binding affinity of protein-ligand complexes. In this work, we present Descriptor Data Bank (DDB), a data-driven platform on the cloud for facilitating multiperspective modeling of PL interactions. DDB is an open-access hub for depositing, hosting, executing, and sharing descriptor extraction tools and data for a large number of interaction modeling hypotheses. The platform also implements a machine-learning (ML) toolbox for automatic descriptor filtering and analysis and scoring function (SF) fitting and prediction. The descriptor filtering module is used to filter out irrelevant and/or noisy descriptors and to produce a compact subset from all available features. We seed DDB with 16 diverse descriptor extraction tools developed in-house and collected from the literature. The tools altogether generate over 2700 descriptors that characterize (i) proteins, (ii) ligands, and (iii) protein-ligand complexes. The in-house descriptors we extract are protein-specific which are based on pairwise primary and tertiary alignment of protein structures followed by clustering and trilateration. We built and used DDB's ML library to fit SFs to the in-house descriptors and those collected from the literature. We then evaluated them on several data sets that were constructed to reflect real-world drug screening scenarios. We found that multiperspective SFs that were constructed using a large number

Electrostatic similarities between protein and small molecule ligands facilitate the design of protein-protein interaction inhibitors.

Directory of Open Access Journals (Sweden)

Arnout Voet

Full Text Available One of the underlying principles in drug discovery is that a biologically active compound is complimentary in shape and molecular recognition features to its receptor. This principle infers that molecules binding to the same receptor may share some common features. Here, we have investigated whether the electrostatic similarity can be used for the discovery of small molecule protein-protein interaction inhibitors (SMPPIIs. We have developed a method that can be used to evaluate the similarity of electrostatic potentials between small molecules and known protein ligands. This method was implemented in a software called EleKit. Analyses of all available (at the time of research SMPPII structures indicate that SMPPIIs bear some similarities of electrostatic potential with the ligand proteins of the same receptor. This is especially true for the more polar SMPPIIs. Retrospective analysis of several successful SMPPIIs has shown the applicability of EleKit in the design of new SMPPIIs.

Molecular dynamics simulation of S100B protein to explore ligand blockage of the interaction with p53 protein

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Zhou, Zhigang; Li, Yumin

2009-10-01

As a tumor suppressor, p53 plays an important role in cancer suppression. The biological function of p53 as a tumor suppressor is disabled when it binds to S100B. Developing the ligands to block the S100B-p53 interaction has been proposed as one of the most important approaches to the development of anti-cancer agents. We screened a small compound library against the binding interface of S100B and p53 to identify potential compounds to interfere with the interaction. The ligand-binding effect on the S100B-p53 interaction was explored by molecular dynamics at the atomic level. The results show that the ligand bound between S100B and p53 propels the two proteins apart by about 2 Å compared to the unligated S100B-p53 complex. The binding affinity of S100B and p53 decreases by 8.5-14.6 kcal/mol after a ligand binds to the interface from the original unligated state of the S100B-p53 complex. Ligand-binding interferes with the interaction of S100B and p53. Such interference could impact the association of S100B and p53, which would free more p53 protein from the pairing with S100B and restore the biological function of p53 as a tumor suppressor. The analysis of the binding mode and ligand structural features would facilitate our effort to identify and design ligands to block S100B-p53 interaction effectively. The results from the work suggest that developing ligands targeting the interface of S100B and p53 could be a promising approach to recover the normal function of p53 as a tumor suppressor.

Crystallization of bi-functional ligand protein complexes.

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Antoni, Claudia; Vera, Laura; Devel, Laurent; Catalani, Maria Pia; Czarny, Bertrand; Cassar-Lajeunesse, Evelyn; Nuti, Elisa; Rossello, Armando; Dive, Vincent; Stura, Enrico Adriano

2013-06-01

Homodimerization is important in signal transduction and can play a crucial role in many other biological systems. To obtaining structural information for the design of molecules able to control the signalization pathways, the proteins involved will have to be crystallized in complex with ligands that induce dimerization. Bi-functional drugs have been generated by linking two ligands together chemically and the relative crystallizability of complexes with mono-functional and bi-functional ligands has been evaluated. There are problems associated with crystallization with such ligands, but overall, the advantages appear to be greater than the drawbacks. The study involves two matrix metalloproteinases, MMP-12 and MMP-9. Using flexible and rigid linkers we show that it is possible to control the crystal packing and that by changing the ligand-enzyme stoichiometric ratio, one can toggle between having one bi-functional ligand binding to two enzymes and having the same ligand bound to each enzyme. The nature of linker and its point of attachment on the ligand can be varied to aid crystallization, and such variations can also provide valuable structural information about the interactions made by the linker with the protein. We report here the crystallization and structure determination of seven ligand-dimerized complexes. These results suggest that the use of bi-functional drugs can be extended beyond the realm of protein dimerization to include all drug design projects. Copyright © 2013 Elsevier Inc. All rights reserved.

Discovering protein-ligand chalcogen bonding in the protein data bank using endocyclic sulfur-containing heterocycles as ligand search subsets.

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Mitchell, Miguel O

2017-09-24

The chalcogen bond, the noncovalent, electrostatic attraction between covalently bonded atoms in group 16 and Lewis bases, is present in protein-ligand interactions based on X-ray structures deposited in the Protein Data Bank (PDB). Discovering protein-ligand chalcogen bonding in the PDB employed a strategy that focused on searching the database for protein complexes of five-membered, heterocyclic ligands containing endocyclic sulfur with endo electron-withdrawing groups (isothiazoles; thiazoles; 1,2,3-, 1,2.4-, 1,2,5-, 1,3,4-thiadiazoles) and thiophenes with exo electron-withdrawing groups, e.g., 2-chloro, 2-bromo, 2-amino, 2-alkylthio. Out of 930 ligands investigated, 33 or 3.5% have protein-ligand S---O interactions of which 31 are chalcogen bonds and two appear to be S---HO hydrogen bonds. The bond angles for some of the chalcogen bonds found in the PDB are less than 90°, and an electrostatic model is proposed to explain this phenomenon.

Accuracy and precision of protein-ligand interaction kinetics determined from chemical shift titrations.

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Markin, Craig J; Spyracopoulos, Leo

2012-12-01

NMR-monitored chemical shift titrations for the study of weak protein-ligand interactions represent a rich source of information regarding thermodynamic parameters such as dissociation constants (K ( D )) in the micro- to millimolar range, populations for the free and ligand-bound states, and the kinetics of interconversion between states, which are typically within the fast exchange regime on the NMR timescale. We recently developed two chemical shift titration methods wherein co-variation of the total protein and ligand concentrations gives increased precision for the K ( D ) value of a 1:1 protein-ligand interaction (Markin and Spyracopoulos in J Biomol NMR 53: 125-138, 2012). In this study, we demonstrate that classical line shape analysis applied to a single set of (1)H-(15)N 2D HSQC NMR spectra acquired using precise protein-ligand chemical shift titration methods we developed, produces accurate and precise kinetic parameters such as the off-rate (k ( off )). For experimentally determined kinetics in the fast exchange regime on the NMR timescale, k ( off ) ~ 3,000 s(-1) in this work, the accuracy of classical line shape analysis was determined to be better than 5 % by conducting quantum mechanical NMR simulations of the chemical shift titration methods with the magnetic resonance toolkit GAMMA. Using Monte Carlo simulations, the experimental precision for k ( off ) from line shape analysis of NMR spectra was determined to be 13 %, in agreement with the theoretical precision of 12 % from line shape analysis of the GAMMA simulations in the presence of noise and protein concentration errors. In addition, GAMMA simulations were employed to demonstrate that line shape analysis has the potential to provide reasonably accurate and precise k ( off ) values over a wide range, from 100 to 15,000 s(-1). The validity of line shape analysis for k ( off ) values approaching intermediate exchange (~100 s(-1)), may be facilitated by more accurate K ( D ) measurements

NMR Studies of Protein Hydration and Protein-Ligand Interactions

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Chong, Yuan

Water on the surface of a protein is called hydration water. Hydration water is known to play a crucial role in a variety of biological processes including protein folding, enzymatic activation, and drug binding. Although the significance of hydration water has been recognized, the underlying mechanism remains far from being understood. This dissertation employs a unique in-situ nuclear magnetic resonance (NMR) technique to study the mechanism of protein hydration and the role of hydration in alcohol-protein interactions. Water isotherms in proteins are measured at different temperatures via the in-situ NMR technique. Water is found to interact differently with hydrophilic and hydrophobic groups on the protein. Water adsorption on hydrophilic groups is hardly affected by the temperature, while water adsorption on hydrophobic groups strongly depends on the temperature around 10 C, below which the adsorption is substantially reduced. This effect is induced by the dramatic decrease in the protein flexibility below 10 C. Furthermore, nanosecond to microsecond protein dynamics and the free energy, enthalpy, and entropy of protein hydration are studied as a function of hydration level and temperature. A crossover at 10 C in protein dynamics and thermodynamics is revealed. The effect of water at hydrophilic groups on protein dynamics and thermodynamics shows little temperature dependence, whereas water at hydrophobic groups has stronger effect above 10 C. In addition, I investigate the role of water in alcohol binding to the protein using the in-situ NMR detection. The isotherms of alcohols are first measured on dry proteins, then on proteins with a series of controlled hydration levels. The free energy, enthalpy, and entropy of alcohol binding are also determined. Two distinct types of alcohol binding are identified. On the one hand, alcohols can directly bind to a few specific sites on the protein. This type of binding is independent of temperature and can be

A web server for analysis, comparison and prediction of protein ligand binding sites.

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Singh, Harinder; Srivastava, Hemant Kumar; Raghava, Gajendra P S

2016-03-25

One of the major challenges in the field of system biology is to understand the interaction between a wide range of proteins and ligands. In the past, methods have been developed for predicting binding sites in a protein for a limited number of ligands. In order to address this problem, we developed a web server named 'LPIcom' to facilitate users in understanding protein-ligand interaction. Analysis, comparison and prediction modules are available in the "LPIcom' server to predict protein-ligand interacting residues for 824 ligands. Each ligand must have at least 30 protein binding sites in PDB. Analysis module of the server can identify residues preferred in interaction and binding motif for a given ligand; for example residues glycine, lysine and arginine are preferred in ATP binding sites. Comparison module of the server allows comparing protein-binding sites of multiple ligands to understand the similarity between ligands based on their binding site. This module indicates that ATP, ADP and GTP ligands are in the same cluster and thus their binding sites or interacting residues exhibit a high level of similarity. Propensity-based prediction module has been developed for predicting ligand-interacting residues in a protein for more than 800 ligands. In addition, a number of web-based tools have been integrated to facilitate users in creating web logo and two-sample between ligand interacting and non-interacting residues. In summary, this manuscript presents a web-server for analysis of ligand interacting residue. This server is available for public use from URL http://crdd.osdd.net/raghava/lpicom .

Accuracy and precision of protein-ligand interaction kinetics determined from chemical shift titrations

Energy Technology Data Exchange (ETDEWEB)

Markin, Craig J.; Spyracopoulos, Leo, E-mail: leo.spyracopoulos@ualberta.ca [University of Alberta, Department of Biochemistry (Canada)

2012-12-15

NMR-monitored chemical shift titrations for the study of weak protein-ligand interactions represent a rich source of information regarding thermodynamic parameters such as dissociation constants (K{sub D}) in the micro- to millimolar range, populations for the free and ligand-bound states, and the kinetics of interconversion between states, which are typically within the fast exchange regime on the NMR timescale. We recently developed two chemical shift titration methods wherein co-variation of the total protein and ligand concentrations gives increased precision for the K{sub D} value of a 1:1 protein-ligand interaction (Markin and Spyracopoulos in J Biomol NMR 53: 125-138, 2012). In this study, we demonstrate that classical line shape analysis applied to a single set of {sup 1}H-{sup 15}N 2D HSQC NMR spectra acquired using precise protein-ligand chemical shift titration methods we developed, produces accurate and precise kinetic parameters such as the off-rate (k{sub off}). For experimentally determined kinetics in the fast exchange regime on the NMR timescale, k{sub off} {approx} 3,000 s{sup -1} in this work, the accuracy of classical line shape analysis was determined to be better than 5 % by conducting quantum mechanical NMR simulations of the chemical shift titration methods with the magnetic resonance toolkit GAMMA. Using Monte Carlo simulations, the experimental precision for k{sub off} from line shape analysis of NMR spectra was determined to be 13 %, in agreement with the theoretical precision of 12 % from line shape analysis of the GAMMA simulations in the presence of noise and protein concentration errors. In addition, GAMMA simulations were employed to demonstrate that line shape analysis has the potential to provide reasonably accurate and precise k{sub off} values over a wide range, from 100 to 15,000 s{sup -1}. The validity of line shape analysis for k{sub off} values approaching intermediate exchange ({approx}100 s{sup -1}), may be facilitated by

Residue preference mapping of ligand fragments in the Protein Data Bank.

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Wang, Lirong; Xie, Zhaojun; Wipf, Peter; Xie, Xiang-Qun

2011-04-25

The interaction between small molecules and proteins is one of the major concerns for structure-based drug design because the principles of protein-ligand interactions and molecular recognition are not thoroughly understood. Fortunately, the analysis of protein-ligand complexes in the Protein Data Bank (PDB) enables unprecedented possibilities for new insights. Herein, we applied molecule-fragmentation algorithms to split the ligands extracted from PDB crystal structures into small fragments. Subsequently, we have developed a ligand fragment and residue preference mapping (LigFrag-RPM) algorithm to map the profiles of the interactions between these fragments and the 20 proteinogenic amino acid residues. A total of 4032 fragments were generated from 71 798 PDB ligands by a ring cleavage (RC) algorithm. Among these ligand fragments, 315 unique fragments were characterized with the corresponding fragment-residue interaction profiles by counting residues close to these fragments. The interaction profiles revealed that these fragments have specific preferences for certain types of residues. The applications of these interaction profiles were also explored and evaluated in case studies, showing great potential for the study of protein-ligand interactions and drug design. Our studies demonstrated that the fragment-residue interaction profiles generated from the PDB ligand fragments can be used to detect whether these fragments are in their favorable or unfavorable environments. The algorithm for a ligand fragment and residue preference mapping (LigFrag-RPM) developed here also has the potential to guide lead chemistry modifications as well as binding residues predictions.

Protein-Ligand Informatics Force Field (PLIff): Toward a Fully Knowledge Driven "Force Field" for Biomolecular Interactions.

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Verdonk, Marcel L; Ludlow, R Frederick; Giangreco, Ilenia; Rathi, Prakash Chandra

2016-07-28

The Protein Data Bank (PDB) contains a wealth of data on nonbonded biomolecular interactions. If this information could be distilled down to nonbonded interaction potentials, these would have some key advantages over standard force fields. However, there are some important outstanding issues to address in order to do this successfully. This paper introduces the protein-ligand informatics "force field", PLIff, which begins to address these key challenges ( https://bitbucket.org/AstexUK/pli ). As a result of their knowledge-based nature, the next-generation nonbonded potentials that make up PLIff automatically capture a wide range of interaction types, including special interactions that are often poorly described by standard force fields. We illustrate how PLIff may be used in structure-based design applications, including interaction fields, fragment mapping, and protein-ligand docking. PLIff performs at least as well as state-of-the art scoring functions in terms of pose predictions and ranking compounds in a virtual screening context.

Architecture effects on multivalent interactions by polypeptide-based multivalent ligands

Science.gov (United States)

Liu, Shuang

Multivalent interactions are characterized by the simultaneous binding between multiple ligands and multiple binding sites, either in solutions or at interfaces. In biological systems, most multivalent interactions occur between protein receptors and carbohydrate ligands through hydrogen-bonding and hydrophobic interactions. Compared with weak affinity binding between one ligand and one binding site, i.e. monovalent interaction, multivalent interactioins provide greater avidity and specificity, and therefore play unique roles in a broad range of biological activities. Moreover, the studies of multivalent interactions are also essential for producing effective inhibitors and effectors of biological processes that could have important therapeutic applications. Synthetic multivalent ligands have been designed to mimic the biological functions of natural multivalent interactions, and various types of scaffolds have been used to display multiple ligands, including small molecules, linear polymers, dendrimers, nanoparticle surfaces, monolayer surfaces and liposomes. Studies have shown that multivalent interactions can be highly affected by various architectural parameters of these multivalent ligands, including ligand identities, valencies, spacing, ligand densities, nature of linker arms, scaffold length and scaffold conformation. Most of these multivalent ligands are chemically synthesized and have limitations of controlling over sequence and conformation, which is a barrier for mimicking ordered and controlled natural biological systems. Therefore, multivalent ligands with precisely controlled architecture are required for improved structure-function relationship studies. Protein engineering methods with subsequent chemical coupling of ligands provide significant advantages of controlling over backbone conformation and functional group placement, and therefore have been used to synthesize recombinant protein-based materials with desired properties similar to natural

Composition of Overlapping Protein-Protein and Protein-Ligand Interfaces.

Directory of Open Access Journals (Sweden)

Ruzianisra Mohamed

Full Text Available Protein-protein interactions (PPIs play a major role in many biological processes and they represent an important class of targets for therapeutic intervention. However, targeting PPIs is challenging because often no convenient natural substrates are available as starting point for small-molecule design. Here, we explored the characteristics of protein interfaces in five non-redundant datasets of 174 protein-protein (PP complexes, and 161 protein-ligand (PL complexes from the ABC database, 436 PP complexes, and 196 PL complexes from the PIBASE database and a dataset of 89 PL complexes from the Timbal database. In all cases, the small molecule ligands must bind at the respective PP interface. We observed similar amino acid frequencies in all three datasets. Remarkably, also the characteristics of PP contacts and overlapping PL contacts are highly similar.

Automated identification of protein-ligand interaction features using Inductive Logic Programming: a hexose binding case study.

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A Santos, Jose C; Nassif, Houssam; Page, David; Muggleton, Stephen H; E Sternberg, Michael J

2012-07-11

There is a need for automated methods to learn general features of the interactions of a ligand class with its diverse set of protein receptors. An appropriate machine learning approach is Inductive Logic Programming (ILP), which automatically generates comprehensible rules in addition to prediction. The development of ILP systems which can learn rules of the complexity required for studies on protein structure remains a challenge. In this work we use a new ILP system, ProGolem, and demonstrate its performance on learning features of hexose-protein interactions. The rules induced by ProGolem detect interactions mediated by aromatics and by planar-polar residues, in addition to less common features such as the aromatic sandwich. The rules also reveal a previously unreported dependency for residues cys and leu. They also specify interactions involving aromatic and hydrogen bonding residues. This paper shows that Inductive Logic Programming implemented in ProGolem can derive rules giving structural features of protein/ligand interactions. Several of these rules are consistent with descriptions in the literature. In addition to confirming literature results, ProGolem's model has a 10-fold cross-validated predictive accuracy that is superior, at the 95% confidence level, to another ILP system previously used to study protein/hexose interactions and is comparable with state-of-the-art statistical learners.

Recent Progress in Treating Protein-Ligand Interactions with Quantum-Mechanical Methods.

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Yilmazer, Nusret Duygu; Korth, Martin

2016-05-16

We review the first successes and failures of a "new wave" of quantum chemistry-based approaches to the treatment of protein/ligand interactions. These approaches share the use of "enhanced", dispersion (D), and/or hydrogen-bond (H) corrected density functional theory (DFT) or semi-empirical quantum mechanical (SQM) methods, in combination with ensemble weighting techniques of some form to capture entropic effects. Benchmark and model system calculations in comparison to high-level theoretical as well as experimental references have shown that both DFT-D (dispersion-corrected density functional theory) and SQM-DH (dispersion and hydrogen bond-corrected semi-empirical quantum mechanical) perform much more accurately than older DFT and SQM approaches and also standard docking methods. In addition, DFT-D might soon become and SQM-DH already is fast enough to compute a large number of binding modes of comparably large protein/ligand complexes, thus allowing for a more accurate assessment of entropic effects.

Analysis of ligand-protein exchange by Clustering of Ligand Diffusion Coefficient Pairs (CoLD-CoP)

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Snyder, David A.; Chantova, Mihaela; Chaudhry, Saadia

2015-06-01

NMR spectroscopy is a powerful tool in describing protein structures and protein activity for pharmaceutical and biochemical development. This study describes a method to determine weak binding ligands in biological systems by using hierarchic diffusion coefficient clustering of multidimensional data obtained with a 400 MHz Bruker NMR. Comparison of DOSY spectrums of ligands of the chemical library in the presence and absence of target proteins show translational diffusion rates for small molecules upon interaction with macromolecules. For weak binders such as compounds found in fragment libraries, changes in diffusion rates upon macromolecular binding are on the order of the precision of DOSY diffusion measurements, and identifying such subtle shifts in diffusion requires careful statistical analysis. The "CoLD-CoP" (Clustering of Ligand Diffusion Coefficient Pairs) method presented here uses SAHN clustering to identify protein-binders in a chemical library or even a not fully characterized metabolite mixture. We will show how DOSY NMR and the "CoLD-CoP" method complement each other in identifying the most suitable candidates for lysozyme and wheat germ acid phosphatase.

Automated identification of protein-ligand interaction features using Inductive Logic Programming: a hexose binding case study

Directory of Open Access Journals (Sweden)

A Santos Jose C

2012-07-01

Full Text Available Abstract Background There is a need for automated methods to learn general features of the interactions of a ligand class with its diverse set of protein receptors. An appropriate machine learning approach is Inductive Logic Programming (ILP, which automatically generates comprehensible rules in addition to prediction. The development of ILP systems which can learn rules of the complexity required for studies on protein structure remains a challenge. In this work we use a new ILP system, ProGolem, and demonstrate its performance on learning features of hexose-protein interactions. Results The rules induced by ProGolem detect interactions mediated by aromatics and by planar-polar residues, in addition to less common features such as the aromatic sandwich. The rules also reveal a previously unreported dependency for residues cys and leu. They also specify interactions involving aromatic and hydrogen bonding residues. This paper shows that Inductive Logic Programming implemented in ProGolem can derive rules giving structural features of protein/ligand interactions. Several of these rules are consistent with descriptions in the literature. Conclusions In addition to confirming literature results, ProGolem’s model has a 10-fold cross-validated predictive accuracy that is superior, at the 95% confidence level, to another ILP system previously used to study protein/hexose interactions and is comparable with state-of-the-art statistical learners.

Interaction of calreticulin with CD40 ligand, TRAIL and Fas ligand

DEFF Research Database (Denmark)

Duus, K; Pagh, R T; Holmskov, U

2007-01-01

is utilized by many other functionally diverse molecules and in this work the interaction of calreticulin with C1q and structurally similar molecules was investigated. In addition to C1q and MBL, CD40 ligand (CD40L), tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL) were...... found to bind calreticulin strongly. A low level or no binding was observed for adiponectin, tumour necrosis factor-alpha (TNF-alpha), CD30L, surfactant protein-A and -D and collagen VIII. The interaction with calreticulin required a conformational change in CD40L, TRAIL and FasL and showed the same...

Identification of individual protein-ligand NOEs in the limit of intermediate exchange

International Nuclear Information System (INIS)

Reibarkh, Mikhail; Malia, Thomas J.; Hopkins, Brian T.; Wagner, Gerhard

2006-01-01

Interactions of proteins with small molecules or other macromolecules play key roles in many biological processes and in drug action, and NMR is an excellent tool for their structural characterization. Frequently, however, line broadening due to intermediate exchange completely eliminates the signals needed for measuring specific intermolecular NOEs. This limits the use of NMR for detailed structural studies in such kinetic situations. Here we show that an optimally chosen excess of ligand over protein can reduce the extent of line broadening for both the ligand and the protein. This makes observation of ligand resonances possible but reduces the size of the measurable NOEs due to the residual line broadening and the non-stoichiometric concentrations. Because the solubility of small molecule drug leads are often limited to high micromolar concentrations, protein concentrations are restricted to even lower values in the low micromolar range. At these non-stoichiometric concentrations and in the presence of significant residual line broadening, conventional NOESY experiments very often are not sensitive enough to observe intermolecular NOEs since the signals inverted by the NOESY preparation pulse sequence relax prior to significant NOE build up. Thus, we employ methods related to driven NOE spectroscopy to investigate protein-ligand interactions in the intermediate exchange regime. In this approach, individual protein resonances are selectively irradiated for up to five seconds to build up measurable NOEs at the ligand resonances. To enable saturation of individual protein resonances we prepare deuterated protein samples selectively protonated at a few sites so that the 1D 1 H spectrum of the protein is resolved well enough to permit irradiation of individual protein signals, which do not overlap with the ligand spectrum. This approach is suitable for measuring a sufficiently large number of protein-ligand NOEs that allow calculation of initial complex structures

PLASS: Protein-ligand affinity statistical score a knowledge-based force-field model of interaction derived from the PDB

Science.gov (United States)

Ozrin, V. D.; Subbotin, M. V.; Nikitin, S. M.

2004-04-01

We have developed PLASS (Protein-Ligand Affinity Statistical Score), a pair-wise potential of mean-force for rapid estimation of the binding affinity of a ligand molecule to a protein active site. This scoring function is derived from the frequency of occurrence of atom-type pairs in crystallographic complexes taken from the Protein Data Bank (PDB). Statistical distributions are converted into distance-dependent contributions to the Gibbs free interaction energy for 10 atomic types using the Boltzmann hypothesis, with only one adjustable parameter. For a representative set of 72 protein-ligand structures, PLASS scores correlate well with the experimentally measured dissociation constants: a correlation coefficient R of 0.82 and RMS error of 2.0 kcal/mol. Such high accuracy results from our novel treatment of the volume correction term, which takes into account the inhomogeneous properties of the protein-ligand complexes. PLASS is able to rank reliably the affinity of complexes which have as much diversity as in the PDB.

Functional structural motifs for protein-ligand, protein-protein, and protein-nucleic acid interactions and their connection to supersecondary structures.

Science.gov (United States)

Kinjo, Akira R; Nakamura, Haruki

2013-01-01

Protein functions are mediated by interactions between proteins and other molecules. One useful approach to analyze protein functions is to compare and classify the structures of interaction interfaces of proteins. Here, we describe the procedures for compiling a database of interface structures and efficiently comparing the interface structures. To do so requires a good understanding of the data structures of the Protein Data Bank (PDB). Therefore, we also provide a detailed account of the PDB exchange dictionary necessary for extracting data that are relevant for analyzing interaction interfaces and secondary structures. We identify recurring structural motifs by classifying similar interface structures, and we define a coarse-grained representation of supersecondary structures (SSS) which represents a sequence of two or three secondary structure elements including their relative orientations as a string of four to seven letters. By examining the correspondence between structural motifs and SSS strings, we show that no SSS string has particularly high propensity to be found interaction interfaces in general, indicating any SSS can be used as a binding interface. When individual structural motifs are examined, there are some SSS strings that have high propensity for particular groups of structural motifs. In addition, it is shown that while the SSS strings found in particular structural motifs for nonpolymer and protein interfaces are as abundant as in other structural motifs that belong to the same subunit, structural motifs for nucleic acid interfaces exhibit somewhat stronger preference for SSS strings. In regard to protein folds, many motif-specific SSS strings were found across many folds, suggesting that SSS may be a useful description to investigate the universality of ligand binding modes.

Evaluation of several two-step scoring functions based on linear interaction energy, effective ligand size, and empirical pair potentials for prediction of protein-ligand binding geometry and free energy.

Science.gov (United States)

Rahaman, Obaidur; Estrada, Trilce P; Doren, Douglas J; Taufer, Michela; Brooks, Charles L; Armen, Roger S

2011-09-26

The performances of several two-step scoring approaches for molecular docking were assessed for their ability to predict binding geometries and free energies. Two new scoring functions designed for "step 2 discrimination" were proposed and compared to our CHARMM implementation of the linear interaction energy (LIE) approach using the Generalized-Born with Molecular Volume (GBMV) implicit solvation model. A scoring function S1 was proposed by considering only "interacting" ligand atoms as the "effective size" of the ligand and extended to an empirical regression-based pair potential S2. The S1 and S2 scoring schemes were trained and 5-fold cross-validated on a diverse set of 259 protein-ligand complexes from the Ligand Protein Database (LPDB). The regression-based parameters for S1 and S2 also demonstrated reasonable transferability in the CSARdock 2010 benchmark using a new data set (NRC HiQ) of diverse protein-ligand complexes. The ability of the scoring functions to accurately predict ligand geometry was evaluated by calculating the discriminative power (DP) of the scoring functions to identify native poses. The parameters for the LIE scoring function with the optimal discriminative power (DP) for geometry (step 1 discrimination) were found to be very similar to the best-fit parameters for binding free energy over a large number of protein-ligand complexes (step 2 discrimination). Reasonable performance of the scoring functions in enrichment of active compounds in four different protein target classes established that the parameters for S1 and S2 provided reasonable accuracy and transferability. Additional analysis was performed to definitively separate scoring function performance from molecular weight effects. This analysis included the prediction of ligand binding efficiencies for a subset of the CSARdock NRC HiQ data set where the number of ligand heavy atoms ranged from 17 to 35. This range of ligand heavy atoms is where improved accuracy of predicted ligand

Classification of Beta-lactamases and penicillin binding proteins using ligand-centric network models.

Directory of Open Access Journals (Sweden)

Hakime Öztürk

Full Text Available β-lactamase mediated antibiotic resistance is an important health issue and the discovery of new β-lactam type antibiotics or β-lactamase inhibitors is an area of intense research. Today, there are about a thousand β-lactamases due to the evolutionary pressure exerted by these ligands. While β-lactamases hydrolyse the β-lactam ring of antibiotics, rendering them ineffective, Penicillin-Binding Proteins (PBPs, which share high structural similarity with β-lactamases, also confer antibiotic resistance to their host organism by acquiring mutations that allow them to continue their participation in cell wall biosynthesis. In this paper, we propose a novel approach to include ligand sharing information for classifying and clustering β-lactamases and PBPs in an effort to elucidate the ligand induced evolution of these β-lactam binding proteins. We first present a detailed summary of the β-lactamase and PBP families in the Protein Data Bank, as well as the compounds they bind to. Then, we build two different types of networks in which the proteins are represented as nodes, and two proteins are connected by an edge with a weight that depends on the number of shared identical or similar ligands. These models are analyzed under three different edge weight settings, namely unweighted, weighted, and normalized weighted. A detailed comparison of these six networks showed that the use of ligand sharing information to cluster proteins resulted in modules comprising proteins with not only sequence similarity but also functional similarity. Consideration of ligand similarity highlighted some interactions that were not detected in the identical ligand network. Analysing the β-lactamases and PBPs using ligand-centric network models enabled the identification of novel relationships, suggesting that these models can be used to examine other protein families to obtain information on their ligand induced evolutionary paths.

Exploring the composition of protein-ligand binding sites on a large scale.

Directory of Open Access Journals (Sweden)

Nickolay A Khazanov

Full Text Available The residue composition of a ligand binding site determines the interactions available for diffusion-mediated ligand binding, and understanding general composition of these sites is of great importance if we are to gain insight into the functional diversity of the proteome. Many structure-based drug design methods utilize such heuristic information for improving prediction or characterization of ligand-binding sites in proteins of unknown function. The Binding MOAD database if one of the largest curated sets of protein-ligand complexes, and provides a source of diverse, high-quality data for establishing general trends of residue composition from currently available protein structures. We present an analysis of 3,295 non-redundant proteins with 9,114 non-redundant binding sites to identify residues over-represented in binding regions versus the rest of the protein surface. The Binding MOAD database delineates biologically-relevant "valid" ligands from "invalid" small-molecule ligands bound to the protein. Invalids are present in the crystallization medium and serve no known biological function. Contacts are found to differ between these classes of ligands, indicating that residue composition of biologically relevant binding sites is distinct not only from the rest of the protein surface, but also from surface regions capable of opportunistic binding of non-functional small molecules. To confirm these trends, we perform a rigorous analysis of the variation of residue propensity with respect to the size of the dataset and the content bias inherent in structure sets obtained from a large protein structure database. The optimal size of the dataset for establishing general trends of residue propensities, as well as strategies for assessing the significance of such trends, are suggested for future studies of binding-site composition.

Ranking docking poses by graph matching of protein-ligand interactions: lessons learned from the D3R Grand Challenge 2

Science.gov (United States)

da Silva Figueiredo Celestino Gomes, Priscila; Da Silva, Franck; Bret, Guillaume; Rognan, Didier

2018-01-01

A novel docking challenge has been set by the Drug Design Data Resource (D3R) in order to predict the pose and affinity ranking of a set of Farnesoid X receptor (FXR) agonists, prior to the public release of their bound X-ray structures and potencies. In a first phase, 36 agonists were docked to 26 Protein Data Bank (PDB) structures of the FXR receptor, and next rescored using the in-house developed GRIM method. GRIM aligns protein-ligand interaction patterns of docked poses to those of available PDB templates for the target protein, and rescore poses by a graph matching method. In agreement with results obtained during the previous 2015 docking challenge, we clearly show that GRIM rescoring improves the overall quality of top-ranked poses by prioritizing interaction patterns already visited in the PDB. Importantly, this challenge enables us to refine the applicability domain of the method by better defining the conditions of its success. We notably show that rescoring apolar ligands in hydrophobic pockets leads to frequent GRIM failures. In the second phase, 102 FXR agonists were ranked by decreasing affinity according to the Gibbs free energy of the corresponding GRIM-selected poses, computed by the HYDE scoring function. Interestingly, this fast and simple rescoring scheme provided the third most accurate ranking method among 57 contributions. Although the obtained ranking is still unsuitable for hit to lead optimization, the GRIM-HYDE scoring scheme is accurate and fast enough to post-process virtual screening data.

Molecular Docking Explains Atomic Interaction between Plant-originated Ligands and Oncogenic E7 Protein of High Risk Human Papillomavirus Type 16

Directory of Open Access Journals (Sweden)

Satish Kumar

2014-12-01

Full Text Available Cervical cancer caused by Human papillomavirus (HPV is one of the leading causes of cancer mortality in women worldwide, particularly in the developing countries. In the last few decades, various compounds from plant origin such as Curcumin, Epigallocatechin gallate (EGCG, Jaceosidin, Resveratrol etc. have been used as anti cancer therapeutic agents. Different studies have shown these plant-originated compounds are able to suppress HPV infection. The E6 and E7 oncoproteins of high-risk HPV play a key role in HPV related cancers. In this study, we explored these ligands from plants origin against E7 oncoprotein of high risk HPV 16, which is known to inactivate tumor suppressor pRb protein. A robust homology model of HPV 16 E7 was built to foresee the interaction mechanism of E7 oncoprotein with these ligands using structure-based drug designing approach. Docking studies demonstrate the interaction of these ligands with pRb binding site of E7 protein by residues Tyr52, Asn53, Val55, Phe57, Cys59, Ser63, Thr64, Thr72, Arg77, Glu80 and Asp81 and help restoration of pRb functioning. This in silico based atomic interaction between these ligands and E7 protein may assist in validating the plant-originated ligands as effective drugs against HPV.

Effects of electrostatic interactions on ligand dissociation kinetics

Science.gov (United States)

Erbaş, Aykut; de la Cruz, Monica Olvera; Marko, John F.

2018-02-01

We study unbinding of multivalent cationic ligands from oppositely charged polymeric binding sites sparsely grafted on a flat neutral substrate. Our molecular dynamics simulations are suggested by single-molecule studies of protein-DNA interactions. We consider univalent salt concentrations spanning roughly a 1000-fold range, together with various concentrations of excess ligands in solution. To reveal the ionic effects on unbinding kinetics of spontaneous and facilitated dissociation mechanisms, we treat electrostatic interactions both at a Debye-Hückel (DH) (or implicit ions, i.e., use of an electrostatic potential with a prescribed decay length) level and by the more precise approach of considering all ionic species explicitly in the simulations. We find that the DH approach systematically overestimates unbinding rates, relative to the calculations where all ion pairs are present explicitly in solution, although many aspects of the two types of calculation are qualitatively similar. For facilitated dissociation (FD) (acceleration of unbinding by free ligands in solution) explicit-ion simulations lead to unbinding at lower free-ligand concentrations. Our simulations predict a variety of FD regimes as a function of free-ligand and ion concentrations; a particularly interesting regime is at intermediate concentrations of ligands where nonelectrostatic binding strength controls FD. We conclude that explicit-ion electrostatic modeling is an essential component to quantitatively tackle problems in molecular ligand dissociation, including nucleic-acid-binding proteins.

Increased precision for analysis of protein-ligand dissociation constants determined from chemical shift titrations

Energy Technology Data Exchange (ETDEWEB)

Markin, Craig J.; Spyracopoulos, Leo, E-mail: leo.spyracopoulos@ualberta.ca [University of Alberta, Department of Biochemistry (Canada)

2012-06-15

NMR is ideally suited for the analysis of protein-protein and protein ligand interactions with dissociation constants ranging from {approx}2 {mu}M to {approx}1 mM, and with kinetics in the fast exchange regime on the NMR timescale. For the determination of dissociation constants (K{sub D}) of 1:1 protein-protein or protein-ligand interactions using NMR, the protein and ligand concentrations must necessarily be similar in magnitude to the K{sub D}, and nonlinear least squares analysis of chemical shift changes as a function of ligand concentration is employed to determine estimates for the parameters K{sub D} and the maximum chemical shift change ({Delta}{delta}{sub max}). During a typical NMR titration, the initial protein concentration, [P{sub 0}], is held nearly constant. For this condition, to determine the most accurate parameters for K{sub D} and {Delta}{delta}{sub max} from nonlinear least squares analyses requires initial protein concentrations that are {approx}0.5 Multiplication-Sign K{sub D}, and a maximum concentration for the ligand, or titrant, of {approx}10 Multiplication-Sign [P{sub 0}]. From a practical standpoint, these requirements are often difficult to achieve. Using Monte Carlo simulations, we demonstrate that co-variation of the ligand and protein concentrations during a titration leads to an increase in the precision of the fitted K{sub D} and {Delta}{delta}{sub max} values when [P{sub 0}] > K{sub D}. Importantly, judicious choice of protein and ligand concentrations for a given NMR titration, combined with nonlinear least squares analyses using two independent variables (ligand and protein concentrations) and two parameters (K{sub D} and {Delta}{delta}{sub max}) is a straightforward approach to increasing the accuracy of measured dissociation constants for 1:1 protein-ligand interactions.

Spectrophotometric method for determination of bifunctional macrocyclic ligands in macrocyclic ligand-protein conjugates

International Nuclear Information System (INIS)

Dadachova, E.; Chappell, L.L.; Brechbiel, M.W.

1999-01-01

A simple spectrophotometric assay for determination of bifunctional polyazacarboxylate-macrocyclic ligands of different sizes that are conjugated to proteins has been developed for: 12-membered macrocycle DOTA (2-[4-nitrobenzyl]-1, 4, 7, 10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) and analogs, the 15-membered PEPA macrocycle (2-[4-nitrobenzyl]-1,4,7,10,13-pentaazacyclopentadecane-N,N',N'',N''',N'''' -pentaacetic acid), and the large 18-membered macrocycle HEHA (1,4,7,10,13,16-hexaazacyclooctadecane-N,N',N'',N''',N''''-hexaacetic acid). The method is based on titration of the blue-colored 1:1 Pb(II)-Arsenazo III (AAIII) complex with the polyazacarboxylate macrocyclic ligand in the concentration range of 0-2.5 μM, wherein color change occurring upon transchelation of the Pb(II) from the AAIII to the polyazamacrocyclic ligand is monitored at 656 nm. The assay is performed at ambient temperature within 20 min without any interfering interaction between the protein and Pb(II)-AA(III) complex. Thus, this method also provides a ligand-to-protein ratio (L/P ratio) that reflects the effective number of ligands per protein molecule available to radiolabeling. The method is not suitable for 14-membered TETA macrocycle (2-[4-nitrobenzyl]-1, 4, 8, 11-tetraazacyclotetradecane N,N',N'',N'''-tetraacetic acid) because of low stability constant of Pb(II)-TETA complex. The method is rapid, simple and may be customized for other polyazacarboxylate macrocyclic ligands

NMRKIN: Simulating line shapes from two-dimensional spectra of proteins upon ligand binding

International Nuclear Information System (INIS)

Guenther, Ulrich L.; Schaffhausen, Brian

2002-01-01

The analysis of the shape of signals in NMR spectra is a powerful tool to study exchange and reaction kinetics. Line shapes in two-dimensional spectra of proteins recorded for titrations with ligands provide information about binding rates observed at individual residues. Here we describe a fast method to simulate a series of line shapes derived from two-dimensional spectra of a protein during a ligand titration. This procedure, which takes the mutual effects of two dimensions into account, has been implemented in MATLAB as an add-on to NMRLab (Guenther et al., 2000). In addition, more complex kinetic models, including sequential and parallel reactions, were simulated to demonstrate common features of more complex line shapes which could be encountered in protein-ligand interactions. As an example of this method, we describe its application to line shapes obtained for a titration of the p85 N-SH2 domain of PI3-kinase with a peptide derived from polyomavirus middle T antigen (MT)

Structural insights, protein-ligand interactions and spectroscopic characterization of isoformononetin

Science.gov (United States)

Srivastava, Anubha; Singh, Harshita; Mishra, Rashmi; Dev, Kapil; Tandon, Poonam; Maurya, Rakesh

2017-04-01

Isoformononetin, a methoxylated isoflavone present in medicinal plants, has non-estrogenic bone forming effect via differential mitogen-activated protein kinase (MAPK) signaling. Spectroscopic (FT-Raman, FT-IR, UV-vis and NMR spectra) and quantum chemical calculations using density functional theory (DFT) and 6-311++G(d,p) as a large basis set have been employed to study the structural and electronic properties of isoformononetin. A detailed conformational analysis is performed to determine the stability among conformers and the various possibilities of intramolecular hydrogen bonding formation. Molecular docking studies with different protein kinases were performed on isoformononetin and previously studied isoflavonoid, formononetin in order to understand their inhibitory nature and the effect of functional groups on osteogenic or osteoporosis associated proteins. It is found that the oxygen atoms of methoxy, hydroxyl groups attached to phenyl rings R1, R3 and carbonyl group attached to pyran ring R2, play a major role in binding with the protein kinases that is responsible for the osteoporosis; however, no hydrophobic interactions are observed between rings of ligand and protein. The electronic properties such as HOMO and LUMO energies were determined by time-dependent TD-DFT which predict that conformer II is a little bit more stable and chemically low reactive than conformer I of isoformononetin. To estimate the structure-activity relationship, the molecular electrostatic potential (MEP) surface map, and reactivity descriptors are calculated from the optimized geometry of the molecule. From these results, it is also found that isoformononetin is kinetically more stable, less toxic, weak electrophile and chemically less reactive than formononetin. The atoms in molecules and natural bond orbital analysis are applied for the detailed analysis of intra and intermolecular hydrogen bonding interactions.

AMMOS2: a web server for protein-ligand-water complexes refinement via molecular mechanics.

Science.gov (United States)

Labbé, Céline M; Pencheva, Tania; Jereva, Dessislava; Desvillechabrol, Dimitri; Becot, Jérôme; Villoutreix, Bruno O; Pajeva, Ilza; Miteva, Maria A

2017-07-03

AMMOS2 is an interactive web server for efficient computational refinement of protein-small organic molecule complexes. The AMMOS2 protocol employs atomic-level energy minimization of a large number of experimental or modeled protein-ligand complexes. The web server is based on the previously developed standalone software AMMOS (Automatic Molecular Mechanics Optimization for in silico Screening). AMMOS utilizes the physics-based force field AMMP sp4 and performs optimization of protein-ligand interactions at five levels of flexibility of the protein receptor. The new version 2 of AMMOS implemented in the AMMOS2 web server allows the users to include explicit water molecules and individual metal ions in the protein-ligand complexes during minimization. The web server provides comprehensive analysis of computed energies and interactive visualization of refined protein-ligand complexes. The ligands are ranked by the minimized binding energies allowing the users to perform additional analysis for drug discovery or chemical biology projects. The web server has been extensively tested on 21 diverse protein-ligand complexes. AMMOS2 minimization shows consistent improvement over the initial complex structures in terms of minimized protein-ligand binding energies and water positions optimization. The AMMOS2 web server is freely available without any registration requirement at the URL: http://drugmod.rpbs.univ-paris-diderot.fr/ammosHome.php. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

A sequence-based dynamic ensemble learning system for protein ligand-binding site prediction

KAUST Repository

Chen, Peng

2015-12-03

Background: Proteins have the fundamental ability to selectively bind to other molecules and perform specific functions through such interactions, such as protein-ligand binding. Accurate prediction of protein residues that physically bind to ligands is important for drug design and protein docking studies. Most of the successful protein-ligand binding predictions were based on known structures. However, structural information is not largely available in practice due to the huge gap between the number of known protein sequences and that of experimentally solved structures

A sequence-based dynamic ensemble learning system for protein ligand-binding site prediction

KAUST Repository

Chen, Peng; Hu, ShanShan; Zhang, Jun; Gao, Xin; Li, Jinyan; Xia, Junfeng; Wang, Bing

2015-01-01

Background: Proteins have the fundamental ability to selectively bind to other molecules and perform specific functions through such interactions, such as protein-ligand binding. Accurate prediction of protein residues that physically bind to ligands is important for drug design and protein docking studies. Most of the successful protein-ligand binding predictions were based on known structures. However, structural information is not largely available in practice due to the huge gap between the number of known protein sequences and that of experimentally solved structures

Cloud computing for protein-ligand binding site comparison.

Science.gov (United States)

Hung, Che-Lun; Hua, Guan-Jie

2013-01-01

The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery.

Ligand cluster-based protein network and ePlatton, a multi-target ligand finder.

Science.gov (United States)

Du, Yu; Shi, Tieliu

2016-01-01

Small molecules are information carriers that make cells aware of external changes and couple internal metabolic and signalling pathway systems with each other. In some specific physiological status, natural or artificial molecules are used to interact with selective biological targets to activate or inhibit their functions to achieve expected biological and physiological output. Millions of years of evolution have optimized biological processes and pathways and now the endocrine and immune system cannot work properly without some key small molecules. In the past thousands of years, the human race has managed to find many medicines against diseases by trail-and-error experience. In the recent decades, with the deepening understanding of life and the progress of molecular biology, researchers spare no effort to design molecules targeting one or two key enzymes and receptors related to corresponding diseases. But recent studies in pharmacogenomics have shown that polypharmacology may be necessary for the effects of drugs, which challenge the paradigm, 'one drug, one target, one disease'. Nowadays, cheminformatics and structural biology can help us reasonably take advantage of the polypharmacology to design next-generation promiscuous drugs and drug combination therapies. 234,591 protein-ligand interactions were extracted from ChEMBL. By the 2D structure similarity, 13,769 ligand emerged from 156,151 distinct ligands which were recognized by 1477 proteins. Ligand cluster- and sequence-based protein networks (LCBN, SBN) were constructed, compared and analysed. For assisting compound designing, exploring polypharmacology and finding possible drug combination, we integrated the pathway, disease, drug adverse reaction and the relationship of targets and ligand clusters into the web platform, ePlatton, which is available at http://www.megabionet.org/eplatton. Although there were some disagreements between the LCBN and SBN, communities in both networks were largely the same

Engineering bacterial surface displayed human norovirus capsid proteins: A novel system to explore interaction between norovirus and ligands

Directory of Open Access Journals (Sweden)

Mengya eNiu

2015-12-01

Full Text Available Human noroviruses (HuNoVs are major contributors to acute nonbacterial gastroenteritis outbreaks. Many aspects of HuNoVs are poorly understood due to both the current inability to culture HuNoVs, and the lack of efficient small animal models. Surrogates for HuNoVs, such as recombinant viral like particles (VLPs expressed in eukaryotic system or P particles expressed in prokaryotic system, have been used for studies in immunology and interaction between the virus and its receptors. However, it is difficult to use VLPs or P particles to collect or isolate potential ligands binding to these recombinant capsid proteins. In this study, a new strategy was used to collect HuNoVs binding ligands through the use of ice nucleation protein (INP to display recombinant capsid proteins of HuNoVs on bacterial surfaces. The viral protein-ligand complex could be easily separated by a low speed centrifugation step. This system was also used to explore interaction between recombinant capsid proteins of HuNoVs and their receptors. In this system, the VP1 capsid encoding gene (ORF2 and the protruding domain (P domain encoding gene (3’ terminal fragment of ORF2 of HuNoVs GI.1 and GII.4 were fused with 5’ terminal fragment of ice nucleation protein encoding gene (inaQn. The results demonstrated that the recombinant VP1 and P domains of HuNoVs were expressed and anchored on the surface of Escherichia coli BL21 cells after the bacteria were transformed with the corresponding plasmids. Both cell surface displayed VP1 and P domains could be recognized by HuNoVs specific antibodies and interact with the viral histo-blood group antigens receptors. In both cases, displayed P domains had better binding abilities than VP1. This new strategy of using displayed HuNoVs capsid proteins on the bacterial surface could be utilized to separate HuNoVs binding components from complex samples, to investigate interaction between the virus and its receptors, as well as to develop an

Calculation of protein-ligand binding affinities.

Science.gov (United States)

Gilson, Michael K; Zhou, Huan-Xiang

2007-01-01

Accurate methods of computing the affinity of a small molecule with a protein are needed to speed the discovery of new medications and biological probes. This paper reviews physics-based models of binding, beginning with a summary of the changes in potential energy, solvation energy, and configurational entropy that influence affinity, and a theoretical overview to frame the discussion of specific computational approaches. Important advances are reported in modeling protein-ligand energetics, such as the incorporation of electronic polarization and the use of quantum mechanical methods. Recent calculations suggest that changes in configurational entropy strongly oppose binding and must be included if accurate affinities are to be obtained. The linear interaction energy (LIE) and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) methods are analyzed, as are free energy pathway methods, which show promise and may be ready for more extensive testing. Ultimately, major improvements in modeling accuracy will likely require advances on multiple fronts, as well as continued validation against experiment.

Quantitation of species differences in albumin–ligand interactions for bovine, human and rat serum albumins using fluorescence spectroscopy: A test case with some Sudlow's site I ligands

International Nuclear Information System (INIS)

Poór, Miklós; Li, Yin; Matisz, Gergely; Kiss, László; Kunsági-Máté, Sándor; Kőszegi, Tamás

2014-01-01

Albumin, the most abundant plasma protein is an approximately 67 kDa sized water-soluble macromolecule. Since several drugs and xenobiotics circulate in the blood at least partially in albumin-bound form, albumin plays a key role in the pharmacokinetics/toxicokinetics of these chemicals. Most of the drugs and xenobiotics are Sudlow's site I ligands. In numerous studies, bovine serum albumin (BSA) is used for modeling albumin–ligand interactions and the results are extrapolated to human serum albumin (HSA). Furthermore, only limited information is available related to albumin–ligand interactions of different albumin species. Therefore, in our study, we have focused on the quantification of differences between bovine, human and rat serum albumin (RSA) using four Sudlow's site I ligands (luteolin, ochratoxin A, phenylbutazone and warfarin). Interactions were analyzed by fluorescence spectroscopy. Stability constants as well as competing capacities of the ligands were determined, and thermodynamic study was also performed. Our results highlight that there could be major differences between BSA, HSA and RSA in their ligand binding properties. Based on our observations we emphasize that in molecular aspects BSA behaves considerably differently from HSA or from albumins of other species therefore, it is strongly recommended to apply at least some confirmatory measurements when data obtained from other species are attempted to be extrapolated to HSA. -- Highlights: • Albumin–ligand interactions of human, bovine and rat albumins were studied. • Four Sudlow's site I ligands were tested by fluorescence spectroscopy. • Substantial differences were found in stability constants among albumin complexes. • Competing capacity of ligands showed major differences in the studied species. • Data obtained for BSA cannot be directly extrapolated to human albumin

Peptide–polymer ligands for a tandem WW-domain, an adaptive multivalent protein–protein interaction: lessons on the thermodynamic fitness of flexible ligands

Directory of Open Access Journals (Sweden)

Katharina Koschek

2015-05-01

Full Text Available Three polymers, poly(N-(2-hydroxypropylmethacrylamide (pHPMA, hyperbranched polyglycerol (hPG, and dextran were investigated as carriers for multivalent ligands targeting the adaptive tandem WW-domain of formin-binding protein (FBP21. Polymer carriers were conjugated with 3–9 copies of the proline-rich decapeptide GPPPRGPPPR-NH2 (P1. Binding of the obtained peptide–polymer conjugates to the tandem WW-domain was investigated employing isothermal titration calorimetry (ITC to determine the binding affinity, the enthalpic and entropic contributions to free binding energy, and the stoichiometry of binding for all peptide–polymer conjugates. Binding affinities of all multivalent ligands were in the µM range, strongly amplified compared to the monovalent ligand P1 with a KD > 1 mM. In addition, concise differences were observed, pHPMA and hPG carriers showed moderate affinity and bound 2.3–2.8 peptides per protein binding site resulting in the formation of aggregates. Dextran-based conjugates displayed affinities down to 1.2 µM, forming complexes with low stoichiometry, and no precipitation. Experimental results were compared with parameters obtained from molecular dynamics simulations in order to understand the observed differences between the three carrier materials. In summary, the more rigid and condensed peptide–polymer conjugates based on the dextran scaffold seem to be superior to induce multivalent binding and to increase affinity, while the more flexible and dendritic polymers, pHPMA and hPG are suitable to induce crosslinking upon binding.

A Global Map of Lipid-Binding Proteins and Their Ligandability in Cells.

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Niphakis, Micah J; Lum, Kenneth M; Cognetta, Armand B; Correia, Bruno E; Ichu, Taka-Aki; Olucha, Jose; Brown, Steven J; Kundu, Soumajit; Piscitelli, Fabiana; Rosen, Hugh; Cravatt, Benjamin F

2015-06-18

Lipids play central roles in physiology and disease, where their structural, metabolic, and signaling functions often arise from interactions with proteins. Here, we describe a set of lipid-based chemical proteomic probes and their global interaction map in mammalian cells. These interactions involve hundreds of proteins from diverse functional classes and frequently occur at sites of drug action. We determine the target profiles for several drugs across the lipid-interaction proteome, revealing that its ligandable content extends far beyond traditionally defined categories of druggable proteins. In further support of this finding, we describe a selective ligand for the lipid-binding protein nucleobindin-1 (NUCB1) and show that this compound perturbs the hydrolytic and oxidative metabolism of endocannabinoids in cells. The described chemical proteomic platform thus provides an integrated path to both discover and pharmacologically characterize a wide range of proteins that participate in lipid pathways in cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Effect of urea on protein-ligand association.

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Stepanian, Lora; Son, Ikbae; Chalikian, Tigran V

2017-12-01

We combine experimental and theoretical approaches to investigate the influence of a cosolvent on a ligand-protein association event. We apply fluorescence measurements to determining the affinity of the inhibitor tri-N-acetylglucosamine [(GlcNAc) 3 ] for lysozyme at urea concentrations ranging from 0 to 8M. Notwithstanding that, at room temperature and neutral pH, lysozyme retains its native conformation up to the solubility limit of urea, the affinity of (GlcNAc) 3 for the protein steadily decreases as the concentration of urea increases. We analyze the urea dependence of the binding free energy within the framework of a simplified statistical thermodynamics-based model that accounts for the excluded volume effect and direct solute-solvent interactions. The analysis reveals that the detrimental action of urea on the inhibitor-lysozyme binding originates from competition between the free energy contributions of the excluded volume effect and direct solute-solvent interactions. The free energy contribution of direct urea-solute interactions narrowly overcomes the excluded volume contribution thereby resulting in urea weakening the protein-ligand association. More broadly, the successful application of the simple model employed in this work points to the possibility of its use in quantifying the stabilizing/destabilizing action of individual cosolvents on biochemical folding and binding reactions. Copyright © 2016 Elsevier B.V. All rights reserved.

AutoSite: an automated approach for pseudo-ligands prediction—from ligand-binding sites identification to predicting key ligand atoms

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Ravindranath, Pradeep Anand; Sanner, Michel F.

2016-01-01

Motivation: The identification of ligand-binding sites from a protein structure facilitates computational drug design and optimization, and protein function assignment. We introduce AutoSite: an efficient software tool for identifying ligand-binding sites and predicting pseudo ligand corresponding to each binding site identified. Binding sites are reported as clusters of 3D points called fills in which every point is labelled as hydrophobic or as hydrogen bond donor or acceptor. From these fills AutoSite derives feature points: a set of putative positions of hydrophobic-, and hydrogen-bond forming ligand atoms. Results: We show that AutoSite identifies ligand-binding sites with higher accuracy than other leading methods, and produces fills that better matches the ligand shape and properties, than the fills obtained with a software program with similar capabilities, AutoLigand. In addition, we demonstrate that for the Astex Diverse Set, the feature points identify 79% of hydrophobic ligand atoms, and 81% and 62% of the hydrogen acceptor and donor hydrogen ligand atoms interacting with the receptor, and predict 81.2% of water molecules mediating interactions between ligand and receptor. Finally, we illustrate potential uses of the predicted feature points in the context of lead optimization in drug discovery projects. Availability and Implementation: http://adfr.scripps.edu/AutoDockFR/autosite.html Contact: sanner@scripps.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27354702

Accounting for Intraligand Interactions in Flexible Ligand Docking with a PMF-Based Scoring Function.

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Lizunov, A Y; Gonchar, A L; Zaitseva, N I; Zosimov, V V

2015-10-26

We analyzed the frequency with which intraligand contacts occurred in a set of 1300 protein-ligand complexes [ Plewczynski et al. J. Comput. Chem. 2011 , 32 , 742 - 755 .]. Our analysis showed that flexible ligands often form intraligand hydrophobic contacts, while intraligand hydrogen bonds are rare. The test set was also thoroughly investigated and classified. We suggest a universal method for enhancement of a scoring function based on a potential of mean force (PMF-based score) by adding a term accounting for intraligand interactions. The method was implemented via in-house developed program, utilizing an Algo_score scoring function [ Ramensky et al. Proteins: Struct., Funct., Genet. 2007 , 69 , 349 - 357 .] based on the Tarasov-Muryshev PMF [ Muryshev et al. J. Comput.-Aided Mol. Des. 2003 , 17 , 597 - 605 .]. The enhancement of the scoring function was shown to significantly improve the docking and scoring quality for flexible ligands in the test set of 1300 protein-ligand complexes [ Plewczynski et al. J. Comput. Chem. 2011 , 32 , 742 - 755 .]. We then investigated the correlation of the docking results with two parameters of intraligand interactions estimation. These parameters are the weight of intraligand interactions and the minimum number of bonds between the ligand atoms required to take their interaction into account.

Macromolecular competition titration method accessing thermodynamics of the unmodified macromolecule-ligand interactions through spectroscopic titrations of fluorescent analogs.

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Bujalowski, Wlodzimierz; Jezewska, Maria J

2011-01-01

Analysis of thermodynamically rigorous binding isotherms provides fundamental information about the energetics of the ligand-macromolecule interactions and often an invaluable insight about the structure of the formed complexes. The Macromolecular Competition Titration (MCT) method enables one to quantitatively obtain interaction parameters of protein-nucleic acid interactions, which may not be available by other methods, particularly for the unmodified long polymer lattices and specific nucleic acid substrates, if the binding is not accompanied by adequate spectroscopic signal changes. The method can be applied using different fluorescent nucleic acids or fluorophores, although the etheno-derivatives of nucleic acid are especially suitable as they are relatively easy to prepare, have significant blue fluorescence, their excitation band lies far from the protein absorption spectrum, and the modification eliminates the possibility of base pairing with other nucleic acids. The MCT method is not limited to the specific size of the reference nucleic acid. Particularly, a simple analysis of the competition titration experiments is described in which the fluorescent, short fragment of nucleic acid, spanning the exact site-size of the protein-nucleic acid complex, and binding with only a 1:1 stoichiometry to the protein, is used as a reference macromolecule. Although the MCT method is predominantly discussed as applied to studying protein-nucleic acid interactions, it can generally be applied to any ligand-macromolecule system by monitoring the association reaction using the spectroscopic signal originating from the reference macromolecule in the presence of the competing macromolecule, whose interaction parameters with the ligand are to be determined. Copyright © 2011 Elsevier Inc. All rights reserved.

LiGRO: a graphical user interface for protein-ligand molecular dynamics.

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Kagami, Luciano Porto; das Neves, Gustavo Machado; da Silva, Alan Wilter Sousa; Caceres, Rafael Andrade; Kawano, Daniel Fábio; Eifler-Lima, Vera Lucia

2017-10-04

To speed up the drug-discovery process, molecular dynamics (MD) calculations performed in GROMACS can be coupled to docking simulations for the post-screening analyses of large compound libraries. This requires generating the topology of the ligands in different software, some basic knowledge of Linux command lines, and a certain familiarity in handling the output files. LiGRO-the python-based graphical interface introduced here-was designed to overcome these protein-ligand parameterization challenges by allowing the graphical (non command line-based) control of GROMACS (MD and analysis), ACPYPE (ligand topology builder) and PLIP (protein-binder interactions monitor)-programs that can be used together to fully perform and analyze the outputs of complex MD simulations (including energy minimization and NVT/NPT equilibration). By allowing the calculation of linear interaction energies in a simple and quick fashion, LiGRO can be used in the drug-discovery pipeline to select compounds with a better protein-binding interaction profile. The design of LiGRO allows researchers to freely download and modify the software, with the source code being available under the terms of a GPLv3 license from http://www.ufrgs.br/lasomfarmacia/ligro/ .

Quantitation of species differences in albumin–ligand interactions for bovine, human and rat serum albumins using fluorescence spectroscopy: A test case with some Sudlow's site I ligands

Energy Technology Data Exchange (ETDEWEB)

Poór, Miklós [Institute of Laboratory Medicine, University of Pécs, Ifjúság u. 13, Pécs H-7624 (Hungary); Li, Yin; Matisz, Gergely [Department of General and Physical Chemistry, University of Pécs, Pécs H-7624 (Hungary); János Szentágothai Research Center, Pécs H-7624 (Hungary); Kiss, László [Department of General and Physical Chemistry, University of Pécs, Pécs H-7624 (Hungary); Kunsági-Máté, Sándor [Department of General and Physical Chemistry, University of Pécs, Pécs H-7624 (Hungary); János Szentágothai Research Center, Pécs H-7624 (Hungary); Kőszegi, Tamás, E-mail: koszegit@freemail.hu [Institute of Laboratory Medicine, University of Pécs, Ifjúság u. 13, Pécs H-7624 (Hungary)

2014-01-15

Albumin, the most abundant plasma protein is an approximately 67 kDa sized water-soluble macromolecule. Since several drugs and xenobiotics circulate in the blood at least partially in albumin-bound form, albumin plays a key role in the pharmacokinetics/toxicokinetics of these chemicals. Most of the drugs and xenobiotics are Sudlow's site I ligands. In numerous studies, bovine serum albumin (BSA) is used for modeling albumin–ligand interactions and the results are extrapolated to human serum albumin (HSA). Furthermore, only limited information is available related to albumin–ligand interactions of different albumin species. Therefore, in our study, we have focused on the quantification of differences between bovine, human and rat serum albumin (RSA) using four Sudlow's site I ligands (luteolin, ochratoxin A, phenylbutazone and warfarin). Interactions were analyzed by fluorescence spectroscopy. Stability constants as well as competing capacities of the ligands were determined, and thermodynamic study was also performed. Our results highlight that there could be major differences between BSA, HSA and RSA in their ligand binding properties. Based on our observations we emphasize that in molecular aspects BSA behaves considerably differently from HSA or from albumins of other species therefore, it is strongly recommended to apply at least some confirmatory measurements when data obtained from other species are attempted to be extrapolated to HSA. -- Highlights: • Albumin–ligand interactions of human, bovine and rat albumins were studied. • Four Sudlow's site I ligands were tested by fluorescence spectroscopy. • Substantial differences were found in stability constants among albumin complexes. • Competing capacity of ligands showed major differences in the studied species. • Data obtained for BSA cannot be directly extrapolated to human albumin.

KLIFS : a knowledge-based structural database to navigate kinase-ligand interaction space

NARCIS (Netherlands)

van Linden, O.P.J.; Kooistra, A.J.; Leurs, R.; de Esch, I.J.P.; de Graaf, C.

2013-01-01

Protein kinases regulate the majority of signal transduction pathways in cells and have become important targets for the development of designer drugs. We present a systematic analysis of kinase-ligand interactions in all regions of the catalytic cleft of all 1252 human kinase-ligand cocrystal

Role of ligand-ligand vs. core-core interactions in gold nanoclusters.

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Milowska, Karolina Z; Stolarczyk, Jacek K

2016-05-14

The controlled assembly of ligand-coated gold nanoclusters (NCs) into larger structures paves the way for new applications ranging from electronics to nanomedicine. Here, we demonstrate through rigorous density functional theory (DFT) calculations employing novel functionals accounting for van der Waals forces that the ligand-ligand interactions determine whether stable assemblies can be formed. The study of NCs with different core sizes, symmetry forms, ligand lengths, mutual crystal orientations, and in the presence of a solvent suggests that core-to-core van der Waals interactions play a lesser role in the assembly. The dominant interactions originate from combination of steric effects, augmented by ligand bundling on NC facets, and related to them changes in electronic properties induced by neighbouring NCs. We also show that, in contrast to standard colloidal theory approach, DFT correctly reproduces the surprising experimental trends in the strength of the inter-particle interaction observed when varying the length of the ligands. The results underpin the importance of understanding NC interactions in designing gold NCs for a specific function.

Monitoring lysin motif-ligand interactions via tryptophan analog fluorescence spectroscopy

NARCIS (Netherlands)

Petrovic, Dejan M.; Leenhouts, Kees; van Roosmalen, Maarten L.; KleinJan, Fenneke; Broos, Jaap

2012-01-01

The lysin motif (LysM) is a peptidoglycan binding protein domain found in a wide range of prokaryotes and eukaryotes. Various techniques have been used to study the LysM-ligand interaction, but a sensitive spectroscopic method to directly monitor this interaction has not been reported. Here a

Study of new interactions of glitazone's stereoisomers and the endogenous ligand 15d-PGJ2 on six different PPAR gamma proteins.

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Álvarez-Almazán, Samuel; Bello, Martiniano; Tamay-Cach, Feliciano; Martínez-Archundia, Marlet; Alemán-González-Duhart, Diana; Correa-Basurto, José; Mendieta-Wejebe, Jessica Elena

2017-10-15

Diabetes mellitus is a chronic disease characterized by hyperglycemia, insulin resistance and hyperlipidemia. Glitazones or thiazolidinediones (TZD) are drugs that act as insulin-sensitizing agents whose molecular target is the peroxisome proliferator-activated receptor gamma (PPARγ). The euglycemic action of TZD has been linked with the induction of type 4 glucose transporter. However, it has been shown that the effect of TZD depends on the specific stereoisomer that interacts with PPARγ. Therefore, this work is focused on exploring the interactions and geometry adopted by glitazone's stereoisomers and one endogenous ligand on different conformations of the six crystals of the PPARγ protein using molecular docking and molecular dynamics (MD) simulations accompanied by the MMGBSA approach. Specifically, the 2,4-thiazolidinedione ring, pioglitazone (PIO), rosiglitazone (ROSI) and troglitazone (TRO) stereoisomers (exogenous ligands), as well as the endogenous ligand 15d-PGJ2, were evaluated. The six crystallographic structures of PPARγ are available at Protein Data Bank as the PDB entries 2PRG, 4PRG, 3T03, 1I7I, 1FM6, and 4EMA. According to the results, a boomerang shape and a particular location of ligands were found with low variations according to the protein conformations. The 15d-PGJ2, TZD, PIO, ROSI and (S,S)-TRO enantiomers were mostly stabilized by twenty hydrophobic residues: Phe226, Pro227, Leu228, Ile281, Phe282, Cys285, Ala292, Ile296, Ile326, Tyr327, Met329, Leu330, Leu333, Met334, Val339, Ile341, Met348, Leu353, Phe363 and Met364. Most hydrogen bond interactions were found between the polar groups of ligands with Arg288, Ser289, Lys367, Gln286, His323, Glu343 and His449 residues. An energetic analysis revealed binding free energy trends that supported known experimental findings of other authors describing better binding properties for PIO, ROSI and (S,S)-TRO than for 15d-PGJ2 and the TZD ring. Copyright © 2017 Elsevier Inc. All rights reserved.

Sampling and energy evaluation challenges in ligand binding protein design.

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Dou, Jiayi; Doyle, Lindsey; Jr Greisen, Per; Schena, Alberto; Park, Hahnbeom; Johnsson, Kai; Stoddard, Barry L; Baker, David

2017-12-01

The steroid hormone 17α-hydroxylprogesterone (17-OHP) is a biomarker for congenital adrenal hyperplasia and hence there is considerable interest in development of sensors for this compound. We used computational protein design to generate protein models with binding sites for 17-OHP containing an extended, nonpolar, shape-complementary binding pocket for the four-ring core of the compound, and hydrogen bonding residues at the base of the pocket to interact with carbonyl and hydroxyl groups at the more polar end of the ligand. Eight of 16 designed proteins experimentally tested bind 17-OHP with micromolar affinity. A co-crystal structure of one of the designs revealed that 17-OHP is rotated 180° around a pseudo-two-fold axis in the compound and displays multiple binding modes within the pocket, while still interacting with all of the designed residues in the engineered site. Subsequent rounds of mutagenesis and binding selection improved the ligand affinity to nanomolar range, while appearing to constrain the ligand to a single bound conformation that maintains the same "flipped" orientation relative to the original design. We trace the discrepancy in the design calculations to two sources: first, a failure to model subtle backbone changes which alter the distribution of sidechain rotameric states and second, an underestimation of the energetic cost of desolvating the carbonyl and hydroxyl groups of the ligand. The difference between design model and crystal structure thus arises from both sampling limitations and energy function inaccuracies that are exacerbated by the near two-fold symmetry of the molecule. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

Correcting binding parameters for interacting ligand-lattice systems

Science.gov (United States)

Hervy, Jordan; Bicout, Dominique J.

2017-07-01

Binding of ligands to macromolecules is central to many functional and regulatory biological processes. Key parameters characterizing ligand-macromolecule interactions are the stoichiometry, inducing the number of ligands per macromolecule binding site, and the dissociation constant, quantifying the ligand-binding site affinity. Both these parameters can be obtained from analyses of classical saturation experiments using the standard binding equation that offers the great advantage of mathematical simplicity but becomes an approximation for situations of interest when a ligand binds and covers more than one single binding site on the macromolecule. Using the framework of car-parking problem with latticelike macromolecules where each ligand can cover simultaneously several consecutive binding sites, we showed that employing the standard analysis leads to underestimation of binding parameters, i.e., ligands appear larger than they actually are and their affinity is also greater than it is. Therefore, we have derived expressions allowing to determine the ligand size and true binding parameters (stoichiometry and dissociation constant) as a function of apparent binding parameters retrieved from standard saturation experiments.

Tyrosine Kinase Ligand-Receptor Pair Prediction by Using Support Vector Machine

Directory of Open Access Journals (Sweden)

Masayuki Yarimizu

2015-01-01

Full Text Available Receptor tyrosine kinases are essential proteins involved in cellular differentiation and proliferation in vivo and are heavily involved in allergic diseases, diabetes, and onset/proliferation of cancerous cells. Identifying the interacting partner of this protein, a growth factor ligand, will provide a deeper understanding of cellular proliferation/differentiation and other cell processes. In this study, we developed a method for predicting tyrosine kinase ligand-receptor pairs from their amino acid sequences. We collected tyrosine kinase ligand-receptor pairs from the Database of Interacting Proteins (DIP and UniProtKB, filtered them by removing sequence redundancy, and used them as a dataset for machine learning and assessment of predictive performance. Our prediction method is based on support vector machines (SVMs, and we evaluated several input features suitable for tyrosine kinase for machine learning and compared and analyzed the results. Using sequence pattern information and domain information extracted from sequences as input features, we obtained 0.996 of the area under the receiver operating characteristic curve. This accuracy is higher than that obtained from general protein-protein interaction pair predictions.

Novel Technology for Protein-Protein Interaction-based Targeted Drug Discovery

Directory of Open Access Journals (Sweden)

Jung Me Hwang

2011-12-01

Full Text Available We have developed a simple but highly efficient in-cell protein-protein interaction (PPI discovery system based on the translocation properties of protein kinase C- and its C1a domain in live cells. This system allows the visual detection of trimeric and dimeric protein interactions including cytosolic, nuclear, and/or membrane proteins with their cognate ligands. In addition, this system can be used to identify pharmacological small compounds that inhibit specific PPIs. These properties make this PPI system an attractive tool for screening drug candidates and mapping the protein interactome.

Protein-ligand interactions investigated by thermal shift assays (TSA) and dual polarization interferometry (DPI).

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Grøftehauge, Morten K; Hajizadeh, Nelly R; Swann, Marcus J; Pohl, Ehmke

2015-01-01

Over the last decades, a wide range of biophysical techniques investigating protein-ligand interactions have become indispensable tools to complement high-resolution crystal structure determinations. Current approaches in solution range from high-throughput-capable methods such as thermal shift assays (TSA) to highly accurate techniques including microscale thermophoresis (MST) and isothermal titration calorimetry (ITC) that can provide a full thermodynamic description of binding events. Surface-based methods such as surface plasmon resonance (SPR) and dual polarization interferometry (DPI) allow real-time measurements and can provide kinetic parameters as well as binding constants. DPI provides additional spatial information about the binding event. Here, an account is presented of new developments and recent applications of TSA and DPI connected to crystallography.

Consistent two-dimensional visualization of protein-ligand complex series

Directory of Open Access Journals (Sweden)

Stierand Katrin

2011-06-01

Full Text Available Abstract Background The comparative two-dimensional graphical representation of protein-ligand complex series featuring different ligands bound to the same active site offers a quick insight in their binding mode differences. In comparison to arbitrary orientations of the residue molecules in the individual complex depictions a consistent placement improves the legibility and comparability within the series. The automatic generation of such consistent layouts offers the possibility to apply it to large data sets originating from computer-aided drug design methods. Results We developed a new approach, which automatically generates a consistent layout of interacting residues for a given series of complexes. Based on the structural three-dimensional input information, a global two-dimensional layout for all residues of the complex ensemble is computed. The algorithm incorporates the three-dimensional adjacencies of the active site residues in order to find an universally valid circular arrangement of the residues around the ligand. Subsequent to a two-dimensional ligand superimposition step, a global placement for each residue is derived from the set of already placed ligands. The method generates high-quality layouts, showing mostly overlap-free solutions with molecules which are displayed as structure diagrams providing interaction information in atomic detail. Application examples document an improved legibility compared to series of diagrams whose layouts are calculated independently from each other. Conclusions The presented method extends the field of complex series visualizations. A series of molecules binding to the same protein active site is drawn in a graphically consistent way. Compared to existing approaches these drawings substantially simplify the visual analysis of large compound series.

Nanoparticle-Based Receptors Mimic Protein-Ligand Recognition.

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Riccardi, Laura; Gabrielli, Luca; Sun, Xiaohuan; De Biasi, Federico; Rastrelli, Federico; Mancin, Fabrizio; De Vivo, Marco

2017-07-13

The self-assembly of a monolayer of ligands on the surface of noble-metal nanoparticles dictates the fundamental nanoparticle's behavior and its functionality. In this combined computational-experimental study, we analyze the structure, organization, and dynamics of functionalized coating thiols in monolayer-protected gold nanoparticles (AuNPs). We explain how functionalized coating thiols self-organize through a delicate and somehow counterintuitive balance of interactions within the monolayer itself and with the solvent. We further describe how the nature and plasticity of these interactions modulate nanoparticle-based chemosensing. Importantly, we found that self-organization of coating thiols can induce the formation of binding pockets in AuNPs. These transient cavities can accommodate small molecules, mimicking protein-ligand recognition, which could explain the selectivity and sensitivity observed for different organic analytes in NMR chemosensing experiments. Thus, our findings advocate for the rational design of tailored coating groups to form specific recognition binding sites on monolayer-protected AuNPs.

Enhance the performance of current scoring functions with the aid of 3D protein-ligand interaction fingerprints.

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Liu, Jie; Su, Minyi; Liu, Zhihai; Li, Jie; Li, Yan; Wang, Renxiao

2017-07-18

In structure-based drug design, binding affinity prediction remains as a challenging goal for current scoring functions. Development of target-biased scoring functions provides a new possibility for tackling this problem, but this approach is also associated with certain technical difficulties. We previously reported the Knowledge-Guided Scoring (KGS) method as an alternative approach (BMC Bioinformatics, 2010, 11, 193-208). The key idea is to compute the binding affinity of a given protein-ligand complex based on the known binding data of an appropriate reference complex, so the error in binding affinity prediction can be reduced effectively. In this study, we have developed an upgraded version, i.e. KGS2, by employing 3D protein-ligand interaction fingerprints in reference selection. KGS2 was evaluated in combination with four scoring functions (X-Score, ChemPLP, ASP, and GoldScore) on five drug targets (HIV-1 protease, carbonic anhydrase 2, beta-secretase 1, beta-trypsin, and checkpoint kinase 1). In the in situ scoring test, considerable improvements were observed in most cases after application of KGS2. Besides, the performance of KGS2 was always better than KGS in all cases. In the more challenging molecular docking test, application of KGS2 also led to improved structure-activity relationship in some cases. KGS2 can be applied as a convenient "add-on" to current scoring functions without the need to re-engineer them, and its application is not limited to certain target proteins as customized scoring functions. As an interpolation method, its accuracy in principle can be improved further with the increasing knowledge of protein-ligand complex structures and binding affinity data. We expect that KGS2 will become a practical tool for enhancing the performance of current scoring functions in binding affinity prediction. The KGS2 software is available upon contacting the authors.

ProBiS-ligands: a web server for prediction of ligands by examination of protein binding sites.

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Konc, Janez; Janežič, Dušanka

2014-07-01

The ProBiS-ligands web server predicts binding of ligands to a protein structure. Starting with a protein structure or binding site, ProBiS-ligands first identifies template proteins in the Protein Data Bank that share similar binding sites. Based on the superimpositions of the query protein and the similar binding sites found, the server then transposes the ligand structures from those sites to the query protein. Such ligand prediction supports many activities, e.g. drug repurposing. The ProBiS-ligands web server, an extension of the ProBiS web server, is open and free to all users at http://probis.cmm.ki.si/ligands. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Pharmacophore searching: A potential solution for correcting unknown ligands (UNK) labelling errors in Protein Data Bank (PDB'S).

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Ibrahim, Musadiq; Lapthorn, Adrian Jonathan; Ibrahim, Mohammad

2017-08-01

The Protein Data Bank (PDB) is the single most important repository of structural data for proteins and other biologically relevant molecules. Therefore, it is critically important to keep the PDB data, error-free as much as possible. In this study, we have critically examined PDB structures of 292 protein molecules which have been deposited in the repository along with potentially incorrect ligands labelled as Unknown ligands (UNK). Pharmacophores were generated for all the protein structures by using Discovery Studio Visualizer (DSV) and Accelrys, Catalyst ® . The generated pharmacophores were subjected to the database search containing the reported ligand. Ligands obtained through Pharmacophore searching were then checked for fitting the observed electron density map by using Coot ® . The predicted ligands obtained via Pharmacophore searching fitted well with the observed electron density map, in comparison to the ligands reported in the PDB's. Based on our study we have learned that till may 2016, among 292 submitted structures in the PDB, at least 20 structures have ligands with a clear electron density but have been incorrectly labelled as unknown ligands (UNK). We have demonstrated that Pharmacophore searching and Coot ® can provide potential help to find suitable known ligands for these protein structures, the former for ligand search and the latter for electron density analysis. The use of these two techniques can facilitate the quick and reliable labelling of ligands where the electron density map serves as a reference. Copyright © 2017 Elsevier Inc. All rights reserved.

Post hoc support vector machine learning for impedimetric biosensors based on weak protein-ligand interactions.

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Rong, Y; Padron, A V; Hagerty, K J; Nelson, N; Chi, S; Keyhani, N O; Katz, J; Datta, S P A; Gomes, C; McLamore, E S

2018-04-30

Impedimetric biosensors for measuring small molecules based on weak/transient interactions between bioreceptors and target analytes are a challenge for detection electronics, particularly in field studies or in the analysis of complex matrices. Protein-ligand binding sensors have enormous potential for biosensing, but achieving accuracy in complex solutions is a major challenge. There is a need for simple post hoc analytical tools that are not computationally expensive, yet provide near real time feedback on data derived from impedance spectra. Here, we show the use of a simple, open source support vector machine learning algorithm for analyzing impedimetric data in lieu of using equivalent circuit analysis. We demonstrate two different protein-based biosensors to show that the tool can be used for various applications. We conclude with a mobile phone-based demonstration focused on the measurement of acetone, an important biomarker related to the onset of diabetic ketoacidosis. In all conditions tested, the open source classifier was capable of performing as well as, or better, than the equivalent circuit analysis for characterizing weak/transient interactions between a model ligand (acetone) and a small chemosensory protein derived from the tsetse fly. In addition, the tool has a low computational requirement, facilitating use for mobile acquisition systems such as mobile phones. The protocol is deployed through Jupyter notebook (an open source computing environment available for mobile phone, tablet or computer use) and the code was written in Python. For each of the applications, we provide step-by-step instructions in English, Spanish, Mandarin and Portuguese to facilitate widespread use. All codes were based on scikit-learn, an open source software machine learning library in the Python language, and were processed in Jupyter notebook, an open-source web application for Python. The tool can easily be integrated with the mobile biosensor equipment for rapid

Crystallization of protein–ligand complexes

International Nuclear Information System (INIS)

Hassell, Anne M.; An, Gang; Bledsoe, Randy K.; Bynum, Jane M.; Carter, H. Luke III; Deng, Su-Jun J.; Gampe, Robert T.; Grisard, Tamara E.; Madauss, Kevin P.; Nolte, Robert T.; Rocque, Warren J.; Wang, Liping; Weaver, Kurt L.; Williams, Shawn P.; Wisely, G. Bruce; Xu, Robert; Shewchuk, Lisa M.

2007-01-01

Methods presented for growing protein–ligand complexes fall into the categories of co-expression of the protein with the ligands of interest, use of the ligands during protein purification, cocrystallization and soaking the ligands into existing crystals. Obtaining diffraction-quality crystals has long been a bottleneck in solving the three-dimensional structures of proteins. Often proteins may be stabilized when they are complexed with a substrate, nucleic acid, cofactor or small molecule. These ligands, on the other hand, have the potential to induce significant conformational changes to the protein and ab initio screening may be required to find a new crystal form. This paper presents an overview of strategies in the following areas for obtaining crystals of protein–ligand complexes: (i) co-expression of the protein with the ligands of interest, (ii) use of the ligands during protein purification, (iii) cocrystallization and (iv) soaks

The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein*

Science.gov (United States)

Townsend, Philip D.; Rodgers, Thomas L.; Glover, Laura C.; Korhonen, Heidi J.; Richards, Shane A.; Colwell, Lucy J.; Pohl, Ehmke; Wilson, Mark R.; Hodgson, David R. W.; McLeish, Tom C. B.; Cann, Martin J.

2015-01-01

Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring. PMID:26187469

Evaluation of Docking Target Functions by the Comprehensive Investigation of Protein-Ligand Energy Minima.

Science.gov (United States)

Oferkin, Igor V; Katkova, Ekaterina V; Sulimov, Alexey V; Kutov, Danil C; Sobolev, Sergey I; Voevodin, Vladimir V; Sulimov, Vladimir B

2015-01-01

The adequate choice of the docking target function impacts the accuracy of the ligand positioning as well as the accuracy of the protein-ligand binding energy calculation. To evaluate a docking target function we compared positions of its minima with the experimentally known pose of the ligand in the protein active site. We evaluated five docking target functions based on either the MMFF94 force field or the PM7 quantum-chemical method with or without implicit solvent models: PCM, COSMO, and SGB. Each function was tested on the same set of 16 protein-ligand complexes. For exhaustive low-energy minima search the novel MPI parallelized docking program FLM and large supercomputer resources were used. Protein-ligand binding energies calculated using low-energy minima were compared with experimental values. It was demonstrated that the docking target function on the base of the MMFF94 force field in vacuo can be used for discovery of native or near native ligand positions by finding the low-energy local minima spectrum of the target function. The importance of solute-solvent interaction for the correct ligand positioning is demonstrated. It is shown that docking accuracy can be improved by replacement of the MMFF94 force field by the new semiempirical quantum-chemical PM7 method.

Evaluation of Docking Target Functions by the Comprehensive Investigation of Protein-Ligand Energy Minima

Directory of Open Access Journals (Sweden)

Igor V. Oferkin

2015-01-01

Full Text Available The adequate choice of the docking target function impacts the accuracy of the ligand positioning as well as the accuracy of the protein-ligand binding energy calculation. To evaluate a docking target function we compared positions of its minima with the experimentally known pose of the ligand in the protein active site. We evaluated five docking target functions based on either the MMFF94 force field or the PM7 quantum-chemical method with or without implicit solvent models: PCM, COSMO, and SGB. Each function was tested on the same set of 16 protein-ligand complexes. For exhaustive low-energy minima search the novel MPI parallelized docking program FLM and large supercomputer resources were used. Protein-ligand binding energies calculated using low-energy minima were compared with experimental values. It was demonstrated that the docking target function on the base of the MMFF94 force field in vacuo can be used for discovery of native or near native ligand positions by finding the low-energy local minima spectrum of the target function. The importance of solute-solvent interaction for the correct ligand positioning is demonstrated. It is shown that docking accuracy can be improved by replacement of the MMFF94 force field by the new semiempirical quantum-chemical PM7 method.

Free-energy relationships in ion channels activated by voltage and ligand

Science.gov (United States)

Chowdhury, Sandipan

2013-01-01

Many ion channels are modulated by multiple stimuli, which allow them to integrate a variety of cellular signals and precisely respond to physiological needs. Understanding how these different signaling pathways interact has been a challenge in part because of the complexity of underlying models. In this study, we analyzed the energetic relationships in polymodal ion channels using linkage principles. We first show that in proteins dually modulated by voltage and ligand, the net free-energy change can be obtained by measuring the charge-voltage (Q-V) relationship in zero ligand condition and the ligand binding curve at highly depolarizing membrane voltages. Next, we show that the voltage-dependent changes in ligand occupancy of the protein can be directly obtained by measuring the Q-V curves at multiple ligand concentrations. When a single reference ligand binding curve is available, this relationship allows us to reconstruct ligand binding curves at different voltages. More significantly, we establish that the shift of the Q-V curve between zero and saturating ligand concentration is a direct estimate of the interaction energy between the ligand- and voltage-dependent pathway. These free-energy relationships were tested by numerical simulations of a detailed gating model of the BK channel. Furthermore, as a proof of principle, we estimate the interaction energy between the ligand binding and voltage-dependent pathways for HCN2 channels whose ligand binding curves at various voltages are available. These emerging principles will be useful for high-throughput mutagenesis studies aimed at identifying interaction pathways between various regulatory domains in a polymodal ion channel. PMID:23250866

Structure-based design of ligands for protein basic domains: Application to the HIV-1 Tat protein

Science.gov (United States)

Filikov, Anton V.; James, Thomas L.

1998-05-01

A methodology has been developed for designing ligands to bind a flexible basic protein domain where the structure of the domain is essentially known. It is based on an empirical binding free energy function developed for highly charged complexes and on Monte Carlo simulations in internal coordinates with both the ligand and the receptor being flexible. HIV-1 encodes a transactivating regulatory protein called Tat. Binding of the basic domain of Tat to TAR RNA is required for efficient transcription of the viral genome. The structure of a biologically active peptide containing the Tat basic RNA-binding domain is available from NMR studies. The goal of the current project is to design a ligand which will bind to that basic domain and potentially inhibit the TAR-Tat interaction. The basic domain contains six arginine and two lysine residues. Our strategy was to design a ligand for arginine first and then a superligand for the basic domain by joining arginine ligands with a linker. Several possible arginine ligands were obtained by searching the Available Chemicals Directory with DOCK 3.5 software. Phytic acid, which can potentially bind multiple arginines, was chosen as a building block for the superligand. Calorimetric binding studies of several compounds to methylguanidine and Arg-/Lys-containing peptides were performed. The data were used to develop an empirical binding free energy function for prediction of affinity of the ligands for the Tat basic domain. Modeling of the conformations of the complexes with both the superligand and the basic domain being flexible has been carried out via Biased Probability Monte Carlo (BPMC) simulations in internal coordinates (ICM 2.6 suite of programs). The simulations used parameters to ensure correct folding, i.e., consistent with the experimental NMR structure of a 25-residue Tat peptide, from a random starting conformation. Superligands for the basic domain were designed by joining together two molecules of phytic acid with

Ligand and membrane-binding behavior of the phosphatidylinositol transfer proteins PITPα and PITPβ.

Science.gov (United States)

Baptist, Matilda; Panagabko, Candace; Cockcroft, Shamshad; Atkinson, Jeffrey

2016-12-01

Phosphatidylinositol transfer proteins (PITPs) are believed to be lipid transfer proteins because of their ability to transfer either phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane compartments, in vitro. However, the detailed mechanism of this transfer process is not fully established. To further understand the transfer mechanism of PITPs we examined the interaction of PITPs with membranes using dual polarization interferometry (DPI), which measures protein binding affinity on a flat immobilized lipid surface. In addition, a fluorescence resonance energy transfer (FRET)-based assay was also employed to monitor how quickly PITPs transfer their ligands to lipid vesicles. DPI analysis revealed that PITPβ had a higher affinity to membranes compared with PITPα. Furthermore, the FRET-based transfer assay revealed that PITPβ has a higher ligand transfer rate compared with PITPα. However, both PITPα and PITPβ demonstrated a preference for highly curved membrane surfaces during ligand transfer. In other words, ligand transfer rate was higher when the accepting vesicles were highly curved.

Sampling protein motion and solvent effect during ligand binding

Science.gov (United States)

Limongelli, Vittorio; Marinelli, Luciana; Cosconati, Sandro; La Motta, Concettina; Sartini, Stefania; Mugnaini, Laura; Da Settimo, Federico; Novellino, Ettore; Parrinello, Michele

2012-01-01

An exhaustive description of the molecular recognition mechanism between a ligand and its biological target is of great value because it provides the opportunity for an exogenous control of the related process. Very often this aim can be pursued using high resolution structures of the complex in combination with inexpensive computational protocols such as docking algorithms. Unfortunately, in many other cases a number of factors, like protein flexibility or solvent effects, increase the degree of complexity of ligand/protein interaction and these standard techniques are no longer sufficient to describe the binding event. We have experienced and tested these limits in the present study in which we have developed and revealed the mechanism of binding of a new series of potent inhibitors of Adenosine Deaminase. We have first performed a large number of docking calculations, which unfortunately failed to yield reliable results due to the dynamical character of the enzyme and the complex role of the solvent. Thus, we have stepped up the computational strategy using a protocol based on metadynamics. Our approach has allowed dealing with protein motion and solvation during ligand binding and finally identifying the lowest energy binding modes of the most potent compound of the series, 4-decyl-pyrazolo[1,5-a]pyrimidin-7-one. PMID:22238423

Evolution of a G protein-coupled receptor response by mutations in regulatory network interactions

DEFF Research Database (Denmark)

Di Roberto, Raphaël B; Chang, Belinda; Trusina, Ala

2016-01-01

All cellular functions depend on the concerted action of multiple proteins organized in complex networks. To understand how selection acts on protein networks, we used the yeast mating receptor Ste2, a pheromone-activated G protein-coupled receptor, as a model system. In Saccharomyces cerevisiae......, Ste2 is a hub in a network of interactions controlling both signal transduction and signal suppression. Through laboratory evolution, we obtained 21 mutant receptors sensitive to the pheromone of a related yeast species and investigated the molecular mechanisms behind this newfound sensitivity. While...... demonstrate that a new receptor-ligand pair can evolve through network-altering mutations independently of receptor-ligand binding, and suggest a potential role for such mutations in disease....

Models of protein-ligand crystal structures: trust, but verify.

Science.gov (United States)

Deller, Marc C; Rupp, Bernhard

2015-09-01

X-ray crystallography provides the most accurate models of protein-ligand structures. These models serve as the foundation of many computational methods including structure prediction, molecular modelling, and structure-based drug design. The success of these computational methods ultimately depends on the quality of the underlying protein-ligand models. X-ray crystallography offers the unparalleled advantage of a clear mathematical formalism relating the experimental data to the protein-ligand model. In the case of X-ray crystallography, the primary experimental evidence is the electron density of the molecules forming the crystal. The first step in the generation of an accurate and precise crystallographic model is the interpretation of the electron density of the crystal, typically carried out by construction of an atomic model. The atomic model must then be validated for fit to the experimental electron density and also for agreement with prior expectations of stereochemistry. Stringent validation of protein-ligand models has become possible as a result of the mandatory deposition of primary diffraction data, and many computational tools are now available to aid in the validation process. Validation of protein-ligand complexes has revealed some instances of overenthusiastic interpretation of ligand density. Fundamental concepts and metrics of protein-ligand quality validation are discussed and we highlight software tools to assist in this process. It is essential that end users select high quality protein-ligand models for their computational and biological studies, and we provide an overview of how this can be achieved.

Pharmacological Profile of Nociceptin/Orphanin FQ Receptors Interacting with G-Proteins and β-Arrestins 2.

Directory of Open Access Journals (Sweden)

D Malfacini

Full Text Available Nociceptin/orphanin FQ (N/OFQ controls several biological functions by selectively activating an opioid like receptor named N/OFQ peptide receptor (NOP. Biased agonism is emerging as an important and therapeutically relevant pharmacological concept in the field of G protein coupled receptors including opioids. To evaluate the relevance of this phenomenon in the NOP receptor, we used a bioluminescence resonance energy transfer technology to measure the interactions of the NOP receptor with either G proteins or β-arrestin 2 in the absence and in presence of increasing concentration of ligands. A large panel of receptor ligands was investigated by comparing their ability to promote or block NOP/G protein and NOP/arrestin interactions. In this study we report a systematic analysis of the functional selectivity of NOP receptor ligands. NOP/G protein interactions (investigated in cell membranes allowed a precise estimation of both ligand potency and efficacy yielding data highly consistent with the known pharmacological profile of this receptor. The same panel of ligands displayed marked differences in the ability to promote NOP/β-arrestin 2 interactions (evaluated in whole cells. In particular, full agonists displayed a general lower potency and for some ligands an inverted rank order of potency was noted. Most partial agonists behaved as pure competitive antagonists of receptor/arrestin interaction. Antagonists displayed similar values of potency for NOP/Gβ1 or NOP/β-arrestin 2 interaction. Using N/OFQ as reference ligand we computed the bias factors of NOP ligands and a number of agonists with greater efficacy at G protein coupling were identified.

The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein.

Science.gov (United States)

Townsend, Philip D; Rodgers, Thomas L; Glover, Laura C; Korhonen, Heidi J; Richards, Shane A; Colwell, Lucy J; Pohl, Ehmke; Wilson, Mark R; Hodgson, David R W; McLeish, Tom C B; Cann, Martin J

2015-09-04

Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure-wise discrimination of cytosine, thymine, and uracil by proteins in terms of their nonbonded interactions.

Science.gov (United States)

Usha, S; Selvaraj, S

2014-01-01

The molecular recognition and discrimination of very similar ligand moieties by proteins are important subjects in protein-ligand interaction studies. Specificity in the recognition of molecules is determined by the arrangement of protein and ligand atoms in space. The three pyrimidine bases, viz. cytosine, thymine, and uracil, are structurally similar, but the proteins that bind to them are able to discriminate them and form interactions. Since nonbonded interactions are responsible for molecular recognition processes in biological systems, our work attempts to understand some of the underlying principles of such recognition of pyrimidine molecular structures by proteins. The preferences of the amino acid residues to contact the pyrimidine bases in terms of nonbonded interactions; amino acid residue-ligand atom preferences; main chain and side chain atom contributions of amino acid residues; and solvent-accessible surface area of ligand atoms when forming complexes are analyzed. Our analysis shows that the amino acid residues, tyrosine and phenyl alanine, are highly involved in the pyrimidine interactions. Arginine prefers contacts with the cytosine base. The similarities and differences that exist between the interactions of the amino acid residues with each of the three pyrimidine base atoms in our analysis provide insights that can be exploited in designing specific inhibitors competitive to the ligands.

Mechanistic pathways of recognition of a solvent-inaccessible cavity of protein by a ligand

Science.gov (United States)

Mondal, Jagannath; Pandit, Subhendu; Dandekar, Bhupendra; Vallurupalli, Pramodh

One of the puzzling questions in the realm of protein-ligand recognition is how a solvent-inaccessible hydrophobic cavity of a protein gets recognized by a ligand. We address the topic by simulating, for the first time, the complete binding process of benzene from aqueous media to the well-known buried cavity of L99A T4 Lysozyme at an atomistic resolution. Our multiple unbiased microsecond-long trajectories, which were completely blind to the location of target binding site, are able to unequivocally identify the kinetic pathways along which benzene molecule meanders across the solvent and protein and ultimately spontaneously recognizes the deeply buried cavity of L99A T4 Lysozyme at an accurate precision. Our simulation, combined with analysis based on markov state model and free energy calculation, reveals that there are more than one distinct ligand binding pathways. Intriguingly, each of the identified pathways involves the transient opening of a channel of the protein prior to ligand binding. The work will also decipher rich mechanistic details on unbinding kinetics of the ligand as obtained from enhanced sampling techniques.

Size versus polarizability in protein-ligand interactions: binding of noble gases within engineered cavities in phage T4 lysozyme.

Science.gov (United States)

Quillin, M L; Breyer, W A; Griswold, I J; Matthews, B W

2000-09-29

To investigate the relative importance of size and polarizability in ligand binding within proteins, we have determined the crystal structures of pseudo wild-type and cavity-containing mutant phage T4 lysozymes in the presence of argon, krypton, and xenon. These proteins provide a representative sample of predominantly apolar cavities of varying size and shape. Even though the volumes of these cavities range up to the equivalent of five xenon atoms, the noble gases bind preferentially at highly localized sites that appear to be defined by constrictions in the walls of the cavities, coupled with the relatively large radii of the noble gases. The cavities within pseudo wild-type and L121A lysozymes each bind only a single atom of noble gas, while the cavities within mutants L133A and F153A have two independent binding sites, and the L99A cavity has three interacting sites. The binding of noble gases within two double mutants was studied to characterize the additivity of binding at such sites. In general, when a cavity in a protein is created by a "large-to-small" substitution, the surrounding residues relax somewhat to reduce the volume of the cavity. The binding of xenon and, to a lesser degree, krypton and argon, tend to expand the volume of the cavity and to return it closer to what it would have been had no relaxation occurred. In nearly all cases, the extent of binding of the noble gases follows the trend xenon>krypton>argon. Pressure titrations of the L99A mutant have confirmed that the crystallographic occupancies accurately reflect fractional saturation of the binding sites. The trend in noble gas affinity can be understood in terms of the effects of size and polarizability on the intermolecular potential. The plasticity of the protein matrix permits repulsion due to increased ligand size to be more than compensated for by attraction due to increased ligand polarizability. These results have implications for the mechanism of general anesthesia, the migration

A simple detection method for low-affinity membrane protein interactions by baculoviral display.

Directory of Open Access Journals (Sweden)

Toshiko Sakihama

Full Text Available BACKGROUND: Membrane protein interactions play an important role in cell-to-cell recognition in various biological activities such as in the immune or neural system. Nevertheless, there has remained the major obstacle of expression of the membrane proteins in their active form. Recently, we and other investigators found that functional membrane proteins express on baculovirus particles (budded virus, BV. In this study, we applied this BV display system to detect interaction between membrane proteins important for cell-to-cell interaction in immune system. METHODOLOGY/PRINCIPAL FINDINGS: We infected Sf9 cells with recombinant baculovirus encoding the T cell membrane protein CD2 or its ligand CD58 and recovered the BV. We detected specific interaction between CD2-displaying BV and CD58-displaying BV by an enzyme-linked immunosorbent assay (ELISA. Using this system, we also detected specific interaction between two other membrane receptor-ligand pairs, CD40-CD40 ligand (CD40L, and glucocorticoid-induced TNFR family-related protein (GITR-GITR ligand (GITRL. Furthermore, we observed specific binding of BV displaying CD58, CD40L, or GITRL to cells naturally expressing their respective receptors by flowcytometric analysis using anti-baculoviral gp64 antibody. Finally we isolated CD2 cDNA from a cDNA expression library by magnetic separation using CD58-displaying BV and anti-gp64 antibody. CONCLUSIONS: We found the BV display system worked effectively in the detection of the interaction of membrane proteins. Since various membrane proteins and their oligomeric complexes can be displayed on BV in the native form, this BV display system should prove highly useful in the search for natural ligands or to develop screening systems for therapeutic antibodies and/or compounds.

Prediction of Protein-Protein Interactions Related to Protein Complexes Based on Protein Interaction Networks

Directory of Open Access Journals (Sweden)

Peng Liu

2015-01-01

Full Text Available A method for predicting protein-protein interactions based on detected protein complexes is proposed to repair deficient interactions derived from high-throughput biological experiments. Protein complexes are pruned and decomposed into small parts based on the adaptive k-cores method to predict protein-protein interactions associated with the complexes. The proposed method is adaptive to protein complexes with different structure, number, and size of nodes in a protein-protein interaction network. Based on different complex sets detected by various algorithms, we can obtain different prediction sets of protein-protein interactions. The reliability of the predicted interaction sets is proved by using estimations with statistical tests and direct confirmation of the biological data. In comparison with the approaches which predict the interactions based on the cliques, the overlap of the predictions is small. Similarly, the overlaps among the predicted sets of interactions derived from various complex sets are also small. Thus, every predicted set of interactions may complement and improve the quality of the original network data. Meanwhile, the predictions from the proposed method replenish protein-protein interactions associated with protein complexes using only the network topology.

Transfer of the human NKG2D ligands UL16 binding proteins (ULBP) 1-3 is related to lytic granule release and leads to ligand retransfer and killing of ULBP-recipient natural killer cells.

Science.gov (United States)

López-Cobo, Sheila; Romera-Cárdenas, Gema; García-Cuesta, Eva M; Reyburn, Hugh T; Valés-Gómez, Mar

2015-09-01

After immune interactions, membrane fragments can be transferred between cells. This fast transfer of molecules is transient and shows selectivity for certain proteins; however, the constraints underlying acquisition of a protein are unknown. To characterize the mechanism and functional consequences of this process in natural killer (NK) cells, we have compared the transfer of different NKG2D ligands. We show that human NKG2D ligands can be acquired by NK cells with different efficiencies. The main findings are that NKG2D ligand transfer is related to immune activation and receptor-ligand interaction and that NK cells acquire these proteins during interactions with target cells that lead to degranulation. Our results further demonstrate that NK cells that have acquired NKG2D ligands can stimulate activation of autologous NK cells. Surprisingly, NK cells can also re-transfer the acquired molecule to autologous effector cells during this immune recognition that leads to their death. These data demonstrate that transfer of molecules occurs as a consequence of immune recognition and imply that this process might play a role in homeostatic tuning-down of the immune response or be used as marker of interaction. © 2015 John Wiley & Sons Ltd.

Aromatic interactions impact ligand binding and function at serotonin 5-HT2C G protein-coupled receptors: receptor homology modelling, ligand docking, and molecular dynamics results validated by experimental studies

Science.gov (United States)

Córdova-Sintjago, Tania; Villa, Nancy; Fang, Lijuan; Booth, Raymond G.

2014-02-01

The serotonin (5-hydroxytryptamine, 5-HT) 5-HT2 G protein-coupled receptor (GPCR) family consists of types 2A, 2B, and 2C that share ∼75% transmembrane (TM) sequence identity. Agonists for 5-HT2C receptors are under development for psychoses; whereas, at 5-HT2A receptors, antipsychotic effects are associated with antagonists - in fact, 5-HT2A agonists can cause hallucinations and 5-HT2B agonists cause cardiotoxicity. It is known that 5-HT2A TM6 residues W6.48, F6.51, and F6.52 impact ligand binding and function; however, ligand interactions with these residues at the 5-HT2C receptor have not been reported. To predict and validate molecular determinants for 5-HT2C-specific activation, results from receptor homology modelling, ligand docking, and molecular dynamics simulation studies were compared with experimental results for ligand binding and function at wild type and W6.48A, F6.51A, and F6.52A point-mutated 5-HT2C receptors.

Dynamical Binding Modes Determine Agonistic and Antagonistic Ligand Effects in the Prostate-Specific G-Protein Coupled Receptor (PSGR).

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Wolf, Steffen; Jovancevic, Nikolina; Gelis, Lian; Pietsch, Sebastian; Hatt, Hanns; Gerwert, Klaus

2017-11-22

We analysed the ligand-based activation mechanism of the prostate-specific G-protein coupled receptor (PSGR), which is an olfactory receptor that mediates cellular growth in prostate cancer cells. Furthermore, it is an olfactory receptor with a known chemically near identic antagonist/agonist pair, α- and β-ionone. Using a combined theoretical and experimental approach, we propose that this receptor is activated by a ligand-induced rearrangement of a protein-internal hydrogen bond network. Surprisingly, this rearrangement is not induced by interaction of the ligand with the network, but by dynamic van der Waals contacts of the ligand with the involved amino acid side chains, altering their conformations and intraprotein connectivity. Ligand recognition in this GPCR is therefore highly stereo selective, but seemingly lacks any ligand recognition via polar contacts. A putative olfactory receptor-based drug design scheme will have to take this unique mode of protein/ligand action into account.

[Supercomputer investigation of the protein-ligand system low-energy minima].

Science.gov (United States)

Oferkin, I V; Sulimov, A V; Katkova, E V; Kutov, D K; Grigoriev, F V; Kondakova, O A; Sulimov, V B

2015-01-01

The accuracy of the protein-ligand binding energy calculations and ligand positioning is strongly influenced by the choice of the docking target function. This work demonstrates the evaluation of the five different target functions used in docking: functions based on MMFF94 force field and functions based on PM7 quantum-chemical method accounting or without accounting the implicit solvent model (PCM, COSMO or SGB). For these purposes the ligand positions corresponding to the minima of the target function and the experimentally known ligand positions in the protein active site (crystal ligand positions) were compared. Each function was examined on the same test-set of 16 protein-ligand complexes. The new parallelized docking program FLM based on Monte Carlo search algorithm was developed to perform the comprehensive low-energy minima search and to calculate the protein-ligand binding energy. This study demonstrates that the docking target function based on the MMFF94 force field can be used to detect the crystal or near crystal positions of the ligand by the finding the low-energy local minima spectrum of the target function. The importance of solvent accounting in the docking process for the accurate ligand positioning is also shown. The accuracy of the ligand positioning as well as the correlation between the calculated and experimentally determined protein-ligand binding energies are improved when the MMFF94 force field is substituted by the new PM7 method with implicit solvent accounting.

Ligand Binding Domain Protein in Tetracycline-Inducible Expression

African Journals Online (AJOL)

Purpose: To investigate tetracycline-inducible expression system for producing clinically usable, highquality liver X receptor ligand-binding domain recombinant protein. Methods: In this study, we have expressed and purified the recombinant liver X receptor β-ligand binding domain proteins in E. coli using a tetracycline ...

A Protein Data Bank survey reveals shortening of intermolecular hydrogen bonds in ligand-protein complexes when a halogenated ligand is an H-bond donor.

Science.gov (United States)

Poznański, Jarosław; Poznańska, Anna; Shugar, David

2014-01-01

Halogen bonding in ligand-protein complexes is currently widely exploited, e.g. in drug design or supramolecular chemistry. But little attention has been directed to other effects that may result from replacement of a hydrogen by a strongly electronegative halogen. Analysis of almost 30000 hydrogen bonds between protein and ligand demonstrates that the length of a hydrogen bond depends on the type of donor-acceptor pair. Interestingly, lengths of hydrogen bonds between a protein and a halogenated ligand are visibly shorter than those estimated for the same family of proteins in complexes with non-halogenated ligands. Taking into account the effect of halogenation on hydrogen bonding is thus important when evaluating structural and/or energetic parameters of ligand-protein complexes. All these observations are consistent with the concept that halogenation increases the acidity of the proximal amino/imino/hydroxyl groups and thus makes them better, i.e. stronger, H-bond donors.

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

Science.gov (United States)

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

2015-09-11

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

Dendrimer-protein interactions versus dendrimer-based nanomedicine.

Science.gov (United States)

Shcharbin, Dzmitry; Shcharbina, Natallia; Dzmitruk, Volha; Pedziwiatr-Werbicka, Elzbieta; Ionov, Maksim; Mignani, Serge; de la Mata, F Javier; Gómez, Rafael; Muñoz-Fernández, Maria Angeles; Majoral, Jean-Pierre; Bryszewska, Maria

2017-04-01

Dendrimers are hyperbranched polymers belonging to the huge class of nanomedical devices. Their wide application in biology and medicine requires understanding of the fundamental mechanisms of their interactions with biological systems. Summarizing, electrostatic force plays the predominant role in dendrimer-protein interactions, especially with charged dendrimers. Other kinds of interactions have been proven, such as H-bonding, van der Waals forces, and even hydrophobic interactions. These interactions depend on the characteristics of both participants: flexibility and surface charge of a dendrimer, rigidity of protein structure and the localization of charged amino acids at its surface. pH and ionic strength of solutions can significantly modulate interactions. Ligands and cofactors attached to a protein can also change dendrimer-protein interactions. Binding of dendrimers to a protein can change its secondary structure, conformation, intramolecular mobility and functional activity. However, this strongly depends on rigidity versus flexibility of a protein's structure. In addition, the potential applications of dendrimers to nanomedicine are reviwed related to dendrimer-protein interactions. Copyright © 2017 Elsevier B.V. All rights reserved.

Fast automated placement of polar hydrogen atoms in protein-ligand complexes

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Lippert Tobias

2009-08-01

Full Text Available Abstract Background Hydrogen bonds play a major role in the stabilization of protein-ligand complexes. The ability of a functional group to form them depends on the position of its hydrogen atoms. An accurate knowledge of the positions of hydrogen atoms in proteins is therefore important to correctly identify hydrogen bonds and their properties. The high mobility of hydrogen atoms introduces several degrees of freedom: Tautomeric states, where a hydrogen atom alters its binding partner, torsional changes where the position of the hydrogen atom is rotated around the last heavy-atom bond in a residue, and protonation states, where the number of hydrogen atoms at a functional group may change. Also, side-chain flips in glutamine and asparagine and histidine residues, which are common crystallographic ambiguities must be identified before structure-based calculations can be conducted. Results We have implemented a method to determine the most probable hydrogen atom positions in a given protein-ligand complex. Optimality of hydrogen bond geometries is determined by an empirical scoring function which is used in molecular docking. This allows to evaluate protein-ligand interactions with an established model. Also, our method allows to resolve common crystallographic ambiguities such as as flipped amide groups and histidine residues. To ensure high speed, we make use of a dynamic programming approach. Conclusion Our results were checked against selected high-resolution structures from an external dataset, for which the positions of the hydrogen atoms have been validated manually. The quality of our results is comparable to that of other programs, with the advantage of being fast enough to be applied on-the-fly for interactive usage or during score evaluation.

Photo-cross-linked small-molecule microarrays as chemical genomic tools for dissecting protein-ligand interactions.

Science.gov (United States)

Kanoh, Naoki; Asami, Aya; Kawatani, Makoto; Honda, Kaori; Kumashiro, Saori; Takayama, Hiroshi; Simizu, Siro; Amemiya, Tomoyuki; Kondoh, Yasumitsu; Hatakeyama, Satoru; Tsuganezawa, Keiko; Utata, Rei; Tanaka, Akiko; Yokoyama, Shigeyuki; Tashiro, Hideo; Osada, Hiroyuki

2006-12-18

We have developed a unique photo-cross-linking approach for immobilizing a variety of small molecules in a functional-group-independent manner. Our approach depends on the reactivity of the carbene species generated from trifluoromethylaryldiazirine upon UV irradiation. It was demonstrated in model experiments that the photogenerated carbenes were able to react with every small molecule tested, and they produced multiple conjugates in most cases. It was also found in on-array immobilization experiments that various small molecules were immobilized, and the immobilized small molecules retained their ability to interact with their binding proteins. With this approach, photo-cross-linked microarrays of about 2000 natural products and drugs were constructed. This photo-cross-linked microarray format was found to be useful not merely for ligand screening but also to study the structure-activity relationship, that is, the relationship between the structural motif (or pharmacophore) found in small molecules and its binding affinity toward a protein, by taking advantage of the nonselective nature of the photo-cross-linking process.

Preparation of a novel dual-function strong cation exchange/hydrophobic interaction chromatography stationary phase for protein separation.

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Zhao, Kailou; Yang, Li; Wang, Xuejiao; Bai, Quan; Yang, Fan; Wang, Fei

2012-08-30

We have explored a novel dual-function stationary phase which combines both strong cation exchange (SCX) and hydrophobic interaction chromatography (HIC) characteristics. The novel dual-function stationary phase is based on porous and spherical silica gel functionalized with ligand containing sulfonic and benzyl groups capable of electrostatic and hydrophobic interaction functionalities, which displays HIC character in a high salt concentration, and IEC character in a low salt concentration in mobile phase employed. As a result, it can be employed to separate proteins with SCX and HIC modes, respectively. The resolution and selectivity of the dual-function stationary phase were evaluated under both HIC and SCX modes with standard proteins and can be comparable to that of conventional IEC and HIC columns. More than 96% of mass and bioactivity recoveries of proteins can be achieved in both HIC and SCX modes, respectively. The results indicated that the novel dual-function column could replace two individual SCX and HIC columns for protein separation. Mixed retention mechanism of proteins on this dual-function column based on stoichiometric displacement theory (SDT) in LC was investigated to find the optimal balance of the magnitude of electrostatic and hydrophobic interactions between protein and the ligand on the silica surface in order to obtain high resolution and selectivity for protein separation. In addition, the effects of the hydrophobicity of the ligand of the dual-function packings and pH of the mobile phase used on protein separation were also investigated in detail. The results show that the ligand with suitable hydrophobicity to match the electrostatic interaction is very important to prepare the dual-function stationary phase, and a better resolution and selectivity can be obtained at pH 6.5 in SCX mode. Therefore, the dual-function column can replace two individual SCX and HIC columns for protein separation and be used to set up two-dimensional liquid

A Protein Data Bank survey reveals shortening of intermolecular hydrogen bonds in ligand-protein complexes when a halogenated ligand is an H-bond donor.

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Jarosław Poznański

Full Text Available Halogen bonding in ligand-protein complexes is currently widely exploited, e.g. in drug design or supramolecular chemistry. But little attention has been directed to other effects that may result from replacement of a hydrogen by a strongly electronegative halogen. Analysis of almost 30000 hydrogen bonds between protein and ligand demonstrates that the length of a hydrogen bond depends on the type of donor-acceptor pair. Interestingly, lengths of hydrogen bonds between a protein and a halogenated ligand are visibly shorter than those estimated for the same family of proteins in complexes with non-halogenated ligands. Taking into account the effect of halogenation on hydrogen bonding is thus important when evaluating structural and/or energetic parameters of ligand-protein complexes. All these observations are consistent with the concept that halogenation increases the acidity of the proximal amino/imino/hydroxyl groups and thus makes them better, i.e. stronger, H-bond donors.

iPfam: a database of protein family and domain interactions found in the Protein Data Bank.

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Finn, Robert D; Miller, Benjamin L; Clements, Jody; Bateman, Alex

2014-01-01

The database iPfam, available at http://ipfam.org, catalogues Pfam domain interactions based on known 3D structures that are found in the Protein Data Bank, providing interaction data at the molecular level. Previously, the iPfam domain-domain interaction data was integrated within the Pfam database and website, but it has now been migrated to a separate database. This allows for independent development, improving data access and giving clearer separation between the protein family and interactions datasets. In addition to domain-domain interactions, iPfam has been expanded to include interaction data for domain bound small molecule ligands. Functional annotations are provided from source databases, supplemented by the incorporation of Wikipedia articles where available. iPfam (version 1.0) contains >9500 domain-domain and 15 500 domain-ligand interactions. The new website provides access to this data in a variety of ways, including interactive visualizations of the interaction data.

IgG-Fc-mediated effector functions: molecular definition of interaction sites for effector ligands and the role of glycosylation.

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Jefferis, R; Lund, J; Pound, J D

1998-06-01

The Fc region of human IgG expresses interaction sites for many effector ligands. In this review the topographical distributions of ten of these sites are discussed in relation to functional requirement. It is apparent that interaction sites localised to the inter-CH2-CH3 domain region of the Fc allow for functional divalency, whereas sites localised to the hinge proximal region of the CH2 domain are functionally monovalent, with expression of the latter sites being particularly dependent on glycosylation. All x-ray crystal structures for Fc and Fc-ligand complexes report that the protein structure of the hinge proximal region of the CH2 domain is "disordered", suggesting "internal mobility". We propose a model in which such "internal mobility" results in the generation of a dynamic equilibrium between multiple conformers, certain of which express interaction sites specific to individual ligands. The emerging understanding of the influence of oligosaccharide/protein interactions on protein conformation and biological function of IgG antibodies suggests a potential to generate novel glycoforms of antibody molecules having unique profiles of effector functions.

Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction.

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Eble, Johannes A

2018-02-15

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial parameter to compare different ligands, e.g., competitive inhibitors, protein isoforms and mutants, for their binding strength to a binding partner. Dissociation constants are determined by plotting concentrations of bound versus free ligand as binding curves. In contrast, titration curves, in which a signal that is proportional to the concentration of bound ligand is plotted against the total concentration of added ligand, are much easier to record. The signal can be detected spectroscopically and by enzyme-linked immunosorbent assay (ELISA). This is exemplified in a protocol for a titration ELISA that measures the binding of the snake venom-derived rhodocetin to its immobilized target domain of α2β1 integrin. Titration ELISAs are versatile and widely used. Any pair of interacting proteins can be used as immobilized receptor and soluble ligand, provided that both proteins are pure, and their concentrations are known. The difficulty so far has been to determine the dissociation constant from a titration curve. In this study, a mathematical function underlying titration curves is introduced. Without any error-prone graphical estimation of a saturation yield, this algorithm allows processing of the raw data (signal intensities at different concentrations of added ligand) directly by mathematical evaluation via non-linear regression. Thus, several titration curves can be recorded simultaneously and transformed into a set of characteristic parameters, among them the dissociation constant and the concentration of binding-active receptor, and they can be evaluated statistically. When combined with this algorithm, titration ELISAs gain the advantage of directly presenting the dissociation constant. Therefore, they may be used more efficiently in the future.

IChem: A Versatile Toolkit for Detecting, Comparing, and Predicting Protein-Ligand Interactions.

Science.gov (United States)

Da Silva, Franck; Desaphy, Jeremy; Rognan, Didier

2018-03-20

Structure-based ligand design requires an exact description of the topology of molecular entities under scrutiny. IChem is a software package that reflects the many contributions of our research group in this area over the last decade. It facilitates and automates many tasks (e.g., ligand/cofactor atom typing, identification of key water molecules) usually left to the modeler's choice. It therefore permits the detection of molecular interactions between two molecules in a very precise and flexible manner. Moreover, IChem enables the conversion of intricate three-dimensional (3D) molecular objects into simple representations (fingerprints, graphs) that facilitate knowledge acquisition at very high throughput. The toolkit is an ideal companion for setting up and performing many structure-based design computations. © 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Protein-protein interactions: an application of Tus-Ter mediated protein microarray system.

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Sitaraman, Kalavathy; Chatterjee, Deb K

2011-01-01

In this chapter, we present a novel, cost-effective microarray strategy that utilizes expression-ready plasmid DNAs to generate protein arrays on-demand and its use to validate protein-protein interactions. These expression plasmids were constructed in such a way so as to serve a dual purpose of synthesizing the protein of interest as well as capturing the synthesized protein. The microarray system is based on the high affinity binding of Escherichia coli "Tus" protein to "Ter," a 20 bp DNA sequence involved in the regulation of DNA replication. The protein expression is carried out in a cell-free protein synthesis system, with rabbit reticulocyte lysates, and the target proteins are detected either by labeled incorporated tag specific or by gene-specific antibodies. This microarray system has been successfully used for the detection of protein-protein interaction because both the target protein and the query protein can be transcribed and translated simultaneously in the microarray slides. The utility of this system for detecting protein-protein interaction is demonstrated by a few well-known examples: Jun/Fos, FRB/FKBP12, p53/MDM2, and CDK4/p16. In all these cases, the presence of protein complexes resulted in the localization of fluorophores at the specific sites of the immobilized target plasmids. Interestingly, during our interactions studies we also detected a previously unknown interaction between CDK2 and p16. Thus, this Tus-Ter based system of protein microarray can be used for the validation of known protein interactions as well as for identifying new protein-protein interactions. In addition, it can be used to examine and identify targets of nucleic acid-protein, ligand-receptor, enzyme-substrate, and drug-protein interactions.

Insights into an original pocket-ligand pair classification: a promising tool for ligand profile prediction.

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Stéphanie Pérot

Full Text Available Pockets are today at the cornerstones of modern drug discovery projects and at the crossroad of several research fields, from structural biology to mathematical modeling. Being able to predict if a small molecule could bind to one or more protein targets or if a protein could bind to some given ligands is very useful for drug discovery endeavors, anticipation of binding to off- and anti-targets. To date, several studies explore such questions from chemogenomic approach to reverse docking methods. Most of these studies have been performed either from the viewpoint of ligands or targets. However it seems valuable to use information from both ligands and target binding pockets. Hence, we present a multivariate approach relating ligand properties with protein pocket properties from the analysis of known ligand-protein interactions. We explored and optimized the pocket-ligand pair space by combining pocket and ligand descriptors using Principal Component Analysis and developed a classification engine on this paired space, revealing five main clusters of pocket-ligand pairs sharing specific and similar structural or physico-chemical properties. These pocket-ligand pair clusters highlight correspondences between pocket and ligand topological and physico-chemical properties and capture relevant information with respect to protein-ligand interactions. Based on these pocket-ligand correspondences, a protocol of prediction of clusters sharing similarity in terms of recognition characteristics is developed for a given pocket-ligand complex and gives high performances. It is then extended to cluster prediction for a given pocket in order to acquire knowledge about its expected ligand profile or to cluster prediction for a given ligand in order to acquire knowledge about its expected pocket profile. This prediction approach shows promising results and could contribute to predict some ligand properties critical for binding to a given pocket, and conversely

Insights into an original pocket-ligand pair classification: a promising tool for ligand profile prediction.

Science.gov (United States)

Pérot, Stéphanie; Regad, Leslie; Reynès, Christelle; Spérandio, Olivier; Miteva, Maria A; Villoutreix, Bruno O; Camproux, Anne-Claude

2013-01-01

Pockets are today at the cornerstones of modern drug discovery projects and at the crossroad of several research fields, from structural biology to mathematical modeling. Being able to predict if a small molecule could bind to one or more protein targets or if a protein could bind to some given ligands is very useful for drug discovery endeavors, anticipation of binding to off- and anti-targets. To date, several studies explore such questions from chemogenomic approach to reverse docking methods. Most of these studies have been performed either from the viewpoint of ligands or targets. However it seems valuable to use information from both ligands and target binding pockets. Hence, we present a multivariate approach relating ligand properties with protein pocket properties from the analysis of known ligand-protein interactions. We explored and optimized the pocket-ligand pair space by combining pocket and ligand descriptors using Principal Component Analysis and developed a classification engine on this paired space, revealing five main clusters of pocket-ligand pairs sharing specific and similar structural or physico-chemical properties. These pocket-ligand pair clusters highlight correspondences between pocket and ligand topological and physico-chemical properties and capture relevant information with respect to protein-ligand interactions. Based on these pocket-ligand correspondences, a protocol of prediction of clusters sharing similarity in terms of recognition characteristics is developed for a given pocket-ligand complex and gives high performances. It is then extended to cluster prediction for a given pocket in order to acquire knowledge about its expected ligand profile or to cluster prediction for a given ligand in order to acquire knowledge about its expected pocket profile. This prediction approach shows promising results and could contribute to predict some ligand properties critical for binding to a given pocket, and conversely, some key pocket

(S)Pinning down protein interactions by NMR

DEFF Research Database (Denmark)

Teilum, Kaare; Kunze, Micha Ben Achim; Erlendsson, Simon

2017-01-01

Protein molecules are highly diverse communication platforms and their interaction repertoire stretches from atoms over small molecules such as sugars and lipids to macromolecules. An important route to understanding molecular communication is to quantitatively describe their interactions...... all types of protein reactions, which can span orders of magnitudes in affinities, reaction rates and lifetimes of states. As the more versatile technique, solution NMR spectroscopy offers a remarkable catalogue of methods that can be successfully applied to the quantitative as well as qualitative...... descriptions of protein interactions. In this review we provide an easy-access approach to NMR for the non-NMR specialist and describe how and when solution state NMR spectroscopy is the method of choice for addressing protein ligand interaction. We describe very briefly the theoretical background...

Leptospiral outer membrane protein microarray, a novel approach to identification of host ligand-binding proteins.

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Pinne, Marija; Matsunaga, James; Haake, David A

2012-11-01

Leptospirosis is a zoonosis with worldwide distribution caused by pathogenic spirochetes belonging to the genus Leptospira. The leptospiral life cycle involves transmission via freshwater and colonization of the renal tubules of their reservoir hosts. Infection requires adherence to cell surfaces and extracellular matrix components of host tissues. These host-pathogen interactions involve outer membrane proteins (OMPs) expressed on the bacterial surface. In this study, we developed an Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 OMP microarray containing all predicted lipoproteins and transmembrane OMPs. A total of 401 leptospiral genes or their fragments were transcribed and translated in vitro and printed on nitrocellulose-coated glass slides. We investigated the potential of this protein microarray to screen for interactions between leptospiral OMPs and fibronectin (Fn). This approach resulted in the identification of the recently described fibronectin-binding protein, LIC10258 (MFn8, Lsa66), and 14 novel Fn-binding proteins, denoted Microarray Fn-binding proteins (MFns). We confirmed Fn binding of purified recombinant LIC11612 (MFn1), LIC10714 (MFn2), LIC11051 (MFn6), LIC11436 (MFn7), LIC10258 (MFn8, Lsa66), and LIC10537 (MFn9) by far-Western blot assays. Moreover, we obtained specific antibodies to MFn1, MFn7, MFn8 (Lsa66), and MFn9 and demonstrated that MFn1, MFn7, and MFn9 are expressed and surface exposed under in vitro growth conditions. Further, we demonstrated that MFn1, MFn4 (LIC12631, Sph2), and MFn7 enable leptospires to bind fibronectin when expressed in the saprophyte, Leptospira biflexa. Protein microarrays are valuable tools for high-throughput identification of novel host ligand-binding proteins that have the potential to play key roles in the virulence mechanisms of pathogens.

Identification of putative agouti-related protein(87-132)-melanocortin-4 receptor interactions by homology molecular modeling and validation using chimeric peptide ligands.

Science.gov (United States)

Wilczynski, Andrzej; Wang, Xiang S; Joseph, Christine G; Xiang, Zhimin; Bauzo, Rayna M; Scott, Joseph W; Sorensen, Nicholas B; Shaw, Amanda M; Millard, William J; Richards, Nigel G; Haskell-Luevano, Carrie

2004-04-22

Agouti-related protein (AGRP) is one of only two naturally known antagonists of G-protein-coupled receptors (GPCRs) identified to date. Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis. Alpha-melanocyte stimulating hormone (alpha-MSH) is one of the known endogenous agonists for these melanocortin receptors. Insight into putative interactions between the antagonist AGRP amino acids with the melanocortin-4 receptor (MC4R) may be important for the design of unique ligands for the treatment of obesity related diseases and is currently lacking in the literature. A three-dimensional homology molecular model of the mouse MC4 receptor complex with the hAGRP(87-132) ligand docked into the receptor has been developed to identify putative antagonist ligand-receptor interactions. Key putative AGRP-MC4R interactions include the Arg111 of hAGRP(87-132) interacting in a negatively charged pocket located in a cavity formed by transmembrane spanning (TM) helices 1, 2, 3, and 7, capped by the acidic first extracellular loop (EL1) and specifically with the conserved melanocortin receptor residues mMC4R Glu92 (TM2), mMC4R Asp114 (TM3), and mMC4R Asp118 (TM3). Additionally, Phe112 and Phe113 of hAGRP(87-132) putatively interact with an aromatic hydrophobic pocket formed by the mMC4 receptor residues Phe176 (TM4), Phe193 (TM5), Phe253 (TM6), and Phe254 (TM6). To validate the AGRP-mMC4R model complex presented herein from a ligand perspective, we generated nine chimeric peptide ligands based on a modified antagonist template of the hAGRP(109-118) (Tyr-c[Asp-Arg-Phe-Phe-Asn-Ala-Phe-Dpr]-Tyr-NH(2)). In these chimeric ligands, the antagonist AGRP Arg-Phe-Phe residues were replaced by the melanocortin agonist His/D-Phe-Arg-Trp amino acids. These peptides resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3-5Rs). The most notable results include the identification of a novel subnanomolar melanocortin peptide

Exploring the role of water in molecular recognition: predicting protein ligandability using a combinatorial search of surface hydration sites

Science.gov (United States)

Vukovic, Sinisa; Brennan, Paul E.; Huggins, David J.

2016-09-01

The interaction between any two biological molecules must compete with their interaction with water molecules. This makes water the most important molecule in medicine, as it controls the interactions of every therapeutic with its target. A small molecule binding to a protein is able to recognize a unique binding site on a protein by displacing bound water molecules from specific hydration sites. Quantifying the interactions of these water molecules allows us to estimate the potential of the protein to bind a small molecule. This is referred to as ligandability. In the study, we describe a method to predict ligandability by performing a search of all possible combinations of hydration sites on protein surfaces. We predict ligandability as the summed binding free energy for each of the constituent hydration sites, computed using inhomogeneous fluid solvation theory. We compared the predicted ligandability with the maximum observed binding affinity for 20 proteins in the human bromodomain family. Based on this comparison, it was determined that effective inhibitors have been developed for the majority of bromodomains, in the range from 10 to 100 nM. However, we predict that more potent inhibitors can be developed for the bromodomains BPTF and BRD7 with relative ease, but that further efforts to develop inhibitors for ATAD2 will be extremely challenging. We have also made predictions for the 14 bromodomains with no reported small molecule K d values by isothermal titration calorimetry. The calculations predict that PBRM1(1) will be a challenging target, while others such as TAF1L(2), PBRM1(4) and TAF1(2), should be highly ligandable. As an outcome of this work, we assembled a database of experimental maximal K d that can serve as a community resource assisting medicinal chemistry efforts focused on BRDs. Effective prediction of ligandability would be a very useful tool in the drug discovery process.

Exploring the role of water in molecular recognition: predicting protein ligandability using a combinatorial search of surface hydration sites.

Science.gov (United States)

Vukovic, Sinisa; Brennan, Paul E; Huggins, David J

2016-09-01

The interaction between any two biological molecules must compete with their interaction with water molecules. This makes water the most important molecule in medicine, as it controls the interactions of every therapeutic with its target. A small molecule binding to a protein is able to recognize a unique binding site on a protein by displacing bound water molecules from specific hydration sites. Quantifying the interactions of these water molecules allows us to estimate the potential of the protein to bind a small molecule. This is referred to as ligandability. In the study, we describe a method to predict ligandability by performing a search of all possible combinations of hydration sites on protein surfaces. We predict ligandability as the summed binding free energy for each of the constituent hydration sites, computed using inhomogeneous fluid solvation theory. We compared the predicted ligandability with the maximum observed binding affinity for 20 proteins in the human bromodomain family. Based on this comparison, it was determined that effective inhibitors have been developed for the majority of bromodomains, in the range from 10 to 100 nM. However, we predict that more potent inhibitors can be developed for the bromodomains BPTF and BRD7 with relative ease, but that further efforts to develop inhibitors for ATAD2 will be extremely challenging. We have also made predictions for the 14 bromodomains with no reported small molecule K d values by isothermal titration calorimetry. The calculations predict that PBRM1(1) will be a challenging target, while others such as TAF1L(2), PBRM1(4) and TAF1(2), should be highly ligandable. As an outcome of this work, we assembled a database of experimental maximal K d that can serve as a community resource assisting medicinal chemistry efforts focused on BRDs. Effective prediction of ligandability would be a very useful tool in the drug discovery process.

Ligand recognition and domain structure of Vps10p, a vacuolar protein sorting receptor in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Jørgensen, M U; Emr, S D; Winther, Jakob R.

1999-01-01

Vp10p is a receptor that sorts several different vacuolar proteins by cycling between a late Golgi compartment and the endosome. The cytoplasmic tail of Vps10p is necessary for the recycling, whereas the lumenal domain is predicted to interact with the soluble ligands. We have studied ligand bind...

Ligand-promoted protein folding by biased kinetic partitioning.

Science.gov (United States)

Hingorani, Karan S; Metcalf, Matthew C; Deming, Derrick T; Garman, Scott C; Powers, Evan T; Gierasch, Lila M

2017-04-01

Protein folding in cells occurs in the presence of high concentrations of endogenous binding partners, and exogenous binding partners have been exploited as pharmacological chaperones. A combined mathematical modeling and experimental approach shows that a ligand improves the folding of a destabilized protein by biasing the kinetic partitioning between folding and alternative fates (aggregation or degradation). Computationally predicted inhibition of test protein aggregation and degradation as a function of ligand concentration are validated by experiments in two disparate cellular systems.

Dissecting Orthosteric Contacts for a Reverse-Fragment-Based Ligand Design.

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Chandramohan, Arun; Tulsian, Nikhil K; Anand, Ganesh S

2017-08-01

Orthosteric sites on proteins are formed typically from noncontiguous interacting sites in three-dimensional space where the composite binding interaction of a biological ligand is mediated by multiple synergistic interactions of its constituent functional groups. Through these multiple interactions, ligands stabilize both the ligand binding site and the local secondary structure. However, relative energetic contributions of the individual contacts in these protein-ligand interactions are difficult to resolve. Deconvolution of the contributions of these various functional groups in natural inhibitors/ligand would greatly aid in iterative fragment-based drug discovery (FBDD). In this study, we describe an approach of progressive unfolding of a target protein using a gradient of denaturant urea to reveal the individual energetic contributions of various ligand-functional groups to the affinity of the entire ligand. Through calibrated unfolding of two protein-ligand systems: cAMP-bound regulatory subunit of Protein Kinase A (RIα) and IBMX-bound phosphodiesterase8 (PDE8), monitored by amide hydrogen-deuterium exchange mass spectrometry, we show progressive disruption of individual orthosteric contacts in the ligand binding sites, allowing us to rank the energetic contributions of these individual interactions. In the two cAMP-binding sites of RIα, exocyclic phosphate oxygens of cAMP were identified to mediate stronger interactions than ribose 2'-OH in both the RIα-cAMP binding interfaces. Further, we have also ranked the relative contributions of the different functional groups of IBMX based on their interactions with the orthosteric residues of PDE8. This strategy for deconstruction of individual binding sites and identification of the strongest functional group interaction in enzyme orthosteric sites offers a rational starting point for FBDD.

The stability and formation of native proteins from unfolded monomers is increased through interactions with unrelated proteins.

Directory of Open Access Journals (Sweden)

Claudia Rodríguez-Almazán

Full Text Available The intracellular concentration of protein may be as high as 400 mg per ml; thus it seems inevitable that within the cell, numerous protein-protein contacts are constantly occurring. A basic biochemical principle states that the equilibrium of an association reaction can be shifted by ligand binding. This indicates that if within the cell many protein-protein interactions are indeed taking place, some fundamental characteristics of proteins would necessarily differ from those observed in traditional biochemical systems. Accordingly, we measured the effect of eight different proteins on the formation of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM from guanidinium chloride unfolded monomers. The eight proteins at concentrations of micrograms per ml induced an important increase on active dimer formation. Studies on the mechanism of this phenomenon showed that the proteins stabilize the dimeric structure of TbTIM, and that this is the driving force that promotes the formation of active dimers. Similar data were obtained with TIM from three other species. The heat changes that occur when TbTIM is mixed with lysozyme were determined by isothermal titration calorimetry; the results provided direct evidence of the weak interaction between apparently unrelated proteins. The data, therefore, are strongly suggestive that the numerous protein-protein interactions that occur in the intracellular space are an additional control factor in the formation and stability of proteins.

Protein-ligand interfaces are polarized: discovery of a strong trend for intermolecular hydrogen bonds to favor donors on the protein side with implications for predicting and designing ligand complexes.

Science.gov (United States)

Raschka, Sebastian; Wolf, Alex J; Bemister-Buffington, Joseph; Kuhn, Leslie A

2018-04-01

Understanding how proteins encode ligand specificity is fascinating and similar in importance to deciphering the genetic code. For protein-ligand recognition, the combination of an almost infinite variety of interfacial shapes and patterns of chemical groups makes the problem especially challenging. Here we analyze data across non-homologous proteins in complex with small biological ligands to address observations made in our inhibitor discovery projects: that proteins favor donating H-bonds to ligands and avoid using groups with both H-bond donor and acceptor capacity. The resulting clear and significant chemical group matching preferences elucidate the code for protein-native ligand binding, similar to the dominant patterns found in nucleic acid base-pairing. On average, 90% of the keto and carboxylate oxygens occurring in the biological ligands formed direct H-bonds to the protein. A two-fold preference was found for protein atoms to act as H-bond donors and ligand atoms to act as acceptors, and 76% of all intermolecular H-bonds involved an amine donor. Together, the tight chemical and geometric constraints associated with satisfying donor groups generate a hydrogen-bonding lock that can be matched only by ligands bearing the right acceptor-rich key. Measuring an index of H-bond preference based on the observed chemical trends proved sufficient to predict other protein-ligand complexes and can be used to guide molecular design. The resulting Hbind and Protein Recognition Index software packages are being made available for rigorously defining intermolecular H-bonds and measuring the extent to which H-bonding patterns in a given complex match the preference key.

Protein-ligand interfaces are polarized: discovery of a strong trend for intermolecular hydrogen bonds to favor donors on the protein side with implications for predicting and designing ligand complexes

Science.gov (United States)

Raschka, Sebastian; Wolf, Alex J.; Bemister-Buffington, Joseph; Kuhn, Leslie A.

2018-02-01

Understanding how proteins encode ligand specificity is fascinating and similar in importance to deciphering the genetic code. For protein-ligand recognition, the combination of an almost infinite variety of interfacial shapes and patterns of chemical groups makes the problem especially challenging. Here we analyze data across non-homologous proteins in complex with small biological ligands to address observations made in our inhibitor discovery projects: that proteins favor donating H-bonds to ligands and avoid using groups with both H-bond donor and acceptor capacity. The resulting clear and significant chemical group matching preferences elucidate the code for protein-native ligand binding, similar to the dominant patterns found in nucleic acid base-pairing. On average, 90% of the keto and carboxylate oxygens occurring in the biological ligands formed direct H-bonds to the protein. A two-fold preference was found for protein atoms to act as H-bond donors and ligand atoms to act as acceptors, and 76% of all intermolecular H-bonds involved an amine donor. Together, the tight chemical and geometric constraints associated with satisfying donor groups generate a hydrogen-bonding lock that can be matched only by ligands bearing the right acceptor-rich key. Measuring an index of H-bond preference based on the observed chemical trends proved sufficient to predict other protein-ligand complexes and can be used to guide molecular design. The resulting Hbind and Protein Recognition Index software packages are being made available for rigorously defining intermolecular H-bonds and measuring the extent to which H-bonding patterns in a given complex match the preference key.

Identification of NAD interacting residues in proteins

Directory of Open Access Journals (Sweden)

Raghava Gajendra PS

2010-03-01

Full Text Available Abstract Background Small molecular cofactors or ligands play a crucial role in the proper functioning of cells. Accurate annotation of their target proteins and binding sites is required for the complete understanding of reaction mechanisms. Nicotinamide adenine dinucleotide (NAD+ or NAD is one of the most commonly used organic cofactors in living cells, which plays a critical role in cellular metabolism, storage and regulatory processes. In the past, several NAD binding proteins (NADBP have been reported in the literature, which are responsible for a wide-range of activities in the cell. Attempts have been made to derive a rule for the binding of NAD+ to its target proteins. However, so far an efficient model could not be derived due to the time consuming process of structure determination, and limitations of similarity based approaches. Thus a sequence and non-similarity based method is needed to characterize the NAD binding sites to help in the annotation. In this study attempts have been made to predict NAD binding proteins and their interacting residues (NIRs from amino acid sequence using bioinformatics tools. Results We extracted 1556 proteins chains from 555 NAD binding proteins whose structure is available in Protein Data Bank. Then we removed all redundant protein chains and finally obtained 195 non-redundant NAD binding protein chains, where no two chains have more than 40% sequence identity. In this study all models were developed and evaluated using five-fold cross validation technique on the above dataset of 195 NAD binding proteins. While certain type of residues are preferred (e.g. Gly, Tyr, Thr, His in NAD interaction, residues like Ala, Glu, Leu, Lys are not preferred. A support vector machine (SVM based method has been developed using various window lengths of amino acid sequence for predicting NAD interacting residues and obtained maximum Matthew's correlation coefficient (MCC 0.47 with accuracy 74.13% at window length 17

A high pressure study of calmodulin-ligand interactions using small-angle X-ray and elastic incoherent neutron scattering.

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Cinar, Süleyman; Al-Ayoubi, Samy; Sternemann, Christian; Peters, Judith; Winter, Roland; Czeslik, Claus

2018-01-31

Calmodulin (CaM) is a Ca 2+ sensor and mediates Ca 2+ signaling through binding of numerous target ligands. The binding of ligands by Ca 2+ -saturated CaM (holo-CaM) is governed by attractive hydrophobic and electrostatic interactions that are weakened under high pressure in aqueous solutions. Moreover, the potential formation of void volumes upon ligand binding creates a further source of pressure sensitivity. Hence, high pressure is a suitable thermodynamic variable to probe protein-ligand interactions. In this study, we compare the binding of two different ligands to holo-CaM as a function of pressure by using X-ray and neutron scattering techniques. The two ligands are the farnesylated hypervariable region (HVR) of the K-Ras4B protein, which is a natural binding partner of holo-CaM, and the antagonist trifluoperazine (TFP), which is known to inhibit holo-CaM activity. From small-angle X-ray scattering experiments performed up to 3000 bar, we observe a pressure-induced partial unfolding of the free holo-CaM in the absence of ligands, where the two lobes of the dumbbell-shaped protein are slightly swelled. In contrast, upon binding TFP, holo-CaM forms a closed globular conformation, which is pressure stable at least up to 3000 bar. The HVR of K-Ras4B shows a different binding behavior, and the data suggest the dissociation of the holo-CaM/HVR complex under high pressure, probably due to a less dense protein contact of the HVR as compared to TFP. The elastic incoherent neutron scattering experiments corroborate these findings. Below 2000 bar, pressure induces enhanced atomic fluctuations in both holo-CaM/ligand complexes, but those of the holo-CaM/HVR complex seem to be larger. Thus, the inhibition of holo-CaM by TFP is supported by a low-volume ligand binding, albeit this is not associated with a rigidification of the complex structure on the sub-ns Å-scale.

Tryptophan tags and de novo designed complementary affinity ligands for the expression and purification of recombinant proteins.

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Pina, Ana Sofia; Carvalho, Sara; Dias, Ana Margarida G C; Guilherme, Márcia; Pereira, Alice S; Caraça, Luciana T; Coroadinha, Ana Sofia; Lowe, Christopher R; Roque, A Cecília A

2016-11-11

A common strategy for the production and purification of recombinant proteins is to fuse a tag to the protein terminal residues and employ a "tag-specific" ligand for fusion protein capture and purification. In this work, we explored the effect of two tryptophan-based tags, NWNWNW and WFWFWF, on the expression and purification of Green Fluorescence Protein (GFP) used as a model fusion protein. The titers obtained with the expression of these fusion proteins in soluble form were 0.11mgml -1 and 0.48mgml -1 for WFWFWF and NWNWNW, respectively. A combinatorial library comprising 64 ligands based on the Ugi reaction was prepared and screened for binding GFP-tagged and non-tagged proteins. Complementary ligands A2C2 and A3C1 were selected for the effective capture of NWNWNW and WFWFWF tagged proteins, respectively, in soluble forms. These affinity pairs displayed 10 6 M -1 affinity constants and Qmax values of 19.11±2.60ugg -1 and 79.39ugg -1 for the systems WFWFWF AND NWNWNW, respectively. GFP fused to the WFWFWF affinity tag was also produced as inclusion bodies, and a refolding-on column strategy was explored using the ligand A4C8, selected from the combinatorial library of ligands but in presence of denaturant agents. Copyright © 2016 Elsevier B.V. All rights reserved.

Interaction of molybdenum with blood serum proteins in vitro

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Bibr, B.; Kselikova, M.; Lener, J.

1985-01-01

The interaction of pentavalent and hexavalent 99 Mo compounds with rat and human serum was monitored in vitro by paper electrophoresis after incubation for one hour at 37 0 C. Hexavalent 99 Mo is not capable of interaction and, via sulfur ligands, forms unstable and unspecific bonds to the whole spectrum of serum proteins, in particular to albumin. Pentavalent 99 Mo binds strongly to alpha-2-macroglobulin in a ratio of 2 : 1; according to the nature of the ligand, it forms somewhat unstable bonds to albumin, beta-1-globulin and gamma-2-globulin. (author)

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

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Murphy, Patrick J. M.; Stone, Orrin J.; Anderson, Michelle E.

2011-01-01

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification. HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media

Molecular recognition in a diverse set of protein-ligand interactions studied with molecular dynamics simulations and end-point free energy calculations.

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Wang, Bo; Li, Liwei; Hurley, Thomas D; Meroueh, Samy O

2013-10-28

End-point free energy calculations using MM-GBSA and MM-PBSA provide a detailed understanding of molecular recognition in protein-ligand interactions. The binding free energy can be used to rank-order protein-ligand structures in virtual screening for compound or target identification. Here, we carry out free energy calculations for a diverse set of 11 proteins bound to 14 small molecules using extensive explicit-solvent MD simulations. The structure of these complexes was previously solved by crystallography and their binding studied with isothermal titration calorimetry (ITC) data enabling direct comparison to the MM-GBSA and MM-PBSA calculations. Four MM-GBSA and three MM-PBSA calculations reproduced the ITC free energy within 1 kcal·mol(-1) highlighting the challenges in reproducing the absolute free energy from end-point free energy calculations. MM-GBSA exhibited better rank-ordering with a Spearman ρ of 0.68 compared to 0.40 for MM-PBSA with dielectric constant (ε = 1). An increase in ε resulted in significantly better rank-ordering for MM-PBSA (ρ = 0.91 for ε = 10), but larger ε significantly reduced the contributions of electrostatics, suggesting that the improvement is due to the nonpolar and entropy components, rather than a better representation of the electrostatics. The SVRKB scoring function applied to MD snapshots resulted in excellent rank-ordering (ρ = 0.81). Calculations of the configurational entropy using normal-mode analysis led to free energies that correlated significantly better to the ITC free energy than the MD-based quasi-harmonic approach, but the computed entropies showed no correlation with the ITC entropy. When the adaptation energy is taken into consideration by running separate simulations for complex, apo, and ligand (MM-PBSAADAPT), there is less agreement with the ITC data for the individual free energies, but remarkably good rank-ordering is observed (ρ = 0.89). Interestingly, filtering MD snapshots by prescoring

Interaction between holo transferrin and HSA-PPIX complex in the presence of lomefloxacin: An evaluation of PPIX aggregation in protein-protein interactions

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Sattar, Zohreh; Iranfar, Hediye; Asoodeh, Ahmad; Saberi, Mohammad Reza; Mazhari, Mahboobeh; Chamani, Jamshidkhan

2012-11-01

Human serum albumin (HSA) and holo transferrin (TF) are two serum carrier proteins that are able to interact with each other, thereby altering their binding behavior toward their ligands. During the course of this study, the interaction between HSA-PPIX and TF, in the presence and absence of lomefloxacin (LMF), was for the first time investigated using different spectroscopic and molecular modeling techniques. Fluorescence spectroscopy experiments were performed in order to study conformational changes of proteins. The RLS technique was utilized to investigate the effect of LMF on J-aggregation of PPIX, which is the first report of its kind. Our findings present clear-cut evidence for the alteration of interactions between HSA and TF in the presence of PPIX and changes in drug-binding to HSA and HSA-PPIX complex upon interaction with TF. Moreover, molecular modeling studies suggested that the binding site for LMF became switched in the presence of PPIX, and that LMF bound to the site IIA of HSA. The obtained results should give new insight into research in this field and may cast some light on the dynamics of drugs in biological systems.

Recent Progress in Treating Protein–Ligand Interactions with Quantum-Mechanical Methods

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Nusret Duygu Yilmazer

2016-05-01

Full Text Available We review the first successes and failures of a “new wave” of quantum chemistry-based approaches to the treatment of protein/ligand interactions. These approaches share the use of “enhanced”, dispersion (D, and/or hydrogen-bond (H corrected density functional theory (DFT or semi-empirical quantum mechanical (SQM methods, in combination with ensemble weighting techniques of some form to capture entropic effects. Benchmark and model system calculations in comparison to high-level theoretical as well as experimental references have shown that both DFT-D (dispersion-corrected density functional theory and SQM-DH (dispersion and hydrogen bond-corrected semi-empirical quantum mechanical perform much more accurately than older DFT and SQM approaches and also standard docking methods. In addition, DFT-D might soon become and SQM-DH already is fast enough to compute a large number of binding modes of comparably large protein/ligand complexes, thus allowing for a more accurate assessment of entropic effects.

Automatic generation of bioinformatics tools for predicting protein-ligand binding sites.

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Komiyama, Yusuke; Banno, Masaki; Ueki, Kokoro; Saad, Gul; Shimizu, Kentaro

2016-03-15

Predictive tools that model protein-ligand binding on demand are needed to promote ligand research in an innovative drug-design environment. However, it takes considerable time and effort to develop predictive tools that can be applied to individual ligands. An automated production pipeline that can rapidly and efficiently develop user-friendly protein-ligand binding predictive tools would be useful. We developed a system for automatically generating protein-ligand binding predictions. Implementation of this system in a pipeline of Semantic Web technique-based web tools will allow users to specify a ligand and receive the tool within 0.5-1 day. We demonstrated high prediction accuracy for three machine learning algorithms and eight ligands. The source code and web application are freely available for download at http://utprot.net They are implemented in Python and supported on Linux. shimizu@bi.a.u-tokyo.ac.jp Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press.

Contribution of Adsorbed Protein Films to Nanoscopic Vibrations Exhibited by Bacteria Adhering through Ligand-Receptor Bonds.

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Song, Lei; Sjollema, Jelmer; Norde, Willem; Busscher, Henk J; van der Mei, Henny C

2015-09-29

Bacteria adhering to surfaces exhibit nanoscopic vibrations that depend on the viscoelasticity of the bond. The quantification of the nanoscopic vibrations of bacteria adhering to surfaces provides new opportunities to better understand the properties of the bond through which bacteria adhere and the mechanisms by which they resist detachment. Often, however, bacteria do not adhere to bare surfaces but to adsorbed protein films, on which adhesion involves highly specific ligand-receptor binding next to nonspecific DLVO interaction forces. Here we determine the contribution of adsorbed salivary protein and fibronectin films to vibrations exhibited by adhering streptococci and staphylococci, respectively. The streptococcal strain used has the ability to adhere to adsorbed salivary proteins films through antigen I/II ligand-receptor binding, while the staphylococcal strain used adheres to adsorbed fibronectin films through a proteinaceous ligand-receptor bond. In the absence of ligand-receptor binding, electrostatic interactions had a large impact on vibration amplitudes of adhering bacteria on glass. On an adsorbed salivary protein film, vibration amplitudes of adhering streptococci depended on the film softness as determined by QCM-D and were reduced after film fixation using glutaraldehyde. On a relatively stiff fibronectin film, cross-linking the film in glutaraldehyde hardly reduced its softness, and accordingly fibronectin film softness did not contribute to vibration amplitudes of adhering staphylococci. However, fixation of the staphylococcus-fibronectin bond further decreased vibration amplitudes, while fixation of the streptococcus bond hardly impacted vibration amplitudes. Summarizing, this study shows that both the softness of adsorbed protein films and the properties of the bond between an adhering bacterium and an adsorbed protein film play an important role in bacterial vibration amplitudes. These nanoscopic vibrations reflect the viscoelasticity of the

Ligand exchange chromatography of free amino acids and proteins on porous microparticulate zirconium oxide

International Nuclear Information System (INIS)

Blackwell, J.A.; Carr, P.W.

1992-01-01

The Lewis acid sites present on the underlying zirconium oxide particles are responsible for the unusual elution sequence for amino acids on copper loaded, phosphated zirconium oxide supports reported in an earlier study. To more thoroughly examine the effect of these strong Lewis acid sites in this paper. The authors have studied ligand exchange chromatography on copper loaded zirconium oxide particles. It is shown here that carboxylate functional groups on amino acid solutes strongly interact with surface Lewis acid sites. Addition of competing hard Lewis bases to the eluent attenuates these specific interactions. The result is a chromatographic system with high selectivity which is also suitable for ligand exchange chromatography of proteins

Ligand-regulated peptide aptamers.

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Miller, Russell A

2009-01-01

The peptide aptamer approach employs high-throughput selection to identify members of a randomized peptide library displayed from a scaffold protein by virtue of their interaction with a target molecule. Extending this approach, we have developed a peptide aptamer scaffold protein that can impart small-molecule control over the aptamer-target interaction. This ligand-regulated peptide (LiRP) scaffold, consisting of the protein domains FKBP12, FRB, and GST, binds to the cell-permeable small-molecule rapamycin and the binding of this molecule can prevent the interaction of the randomizable linker region connecting FKBP12 with FRB. Here we present a detailed protocol for the creation of a peptide aptamer plasmid library, selection of peptide aptamers using the LiRP scaffold in a yeast two-hybrid system, and the screening of those peptide aptamers for a ligand-regulated interaction.

Glucagon-like peptide-1 receptor ligand interactions: structural cross talk between ligands and the extracellular domain.

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Graham M West

Full Text Available Activation of the glucagon-like peptide-1 receptor (GLP-1R in pancreatic β-cells potentiates insulin production and is a current therapeutic target for the treatment of type 2 diabetes mellitus (T2DM. Like other class B G protein-coupled receptors (GPCRs, the GLP-1R contains an N-terminal extracellular ligand binding domain. N-terminal truncations on the peptide agonist generate antagonists capable of binding to the extracellular domain, but not capable of activating full length receptor. The main objective of this study was to use Hydrogen/deuterium exchange (HDX to identify how the amide hydrogen bonding network of peptide ligands and the extracellular domain of GLP-1R (nGLP-1R were altered by binding interactions and to then use this platform to validate direct binding events for putative GLP-1R small molecule ligands. The HDX studies presented here for two glucagon-like peptide-1 receptor (GLP-1R peptide ligands indicates that the antagonist exendin-4[9-39] is significantly destabilized in the presence of nonionic detergents as compared to the agonist exendin-4. Furthermore, HDX can detect stabilization of exendin-4 and exendin-4[9-39] hydrogen bonding networks at the N-terminal helix [Val19 to Lys27] upon binding to the N-terminal extracellular domain of GLP-1R (nGLP-1R. In addition we show hydrogen bonding network stabilization on nGLP-1R in response to ligand binding, and validate direct binding events with the extracellular domain of the receptor for putative GLP-1R small molecule ligands.

Characterization of the Raf kinase inhibitory protein (RKIP) binding pocket: NMR-based screening identifies small-molecule ligands.

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Shemon, Anne N; Heil, Gary L; Granovsky, Alexey E; Clark, Mathew M; McElheny, Dan; Chimon, Alexander; Rosner, Marsha R; Koide, Shohei

2010-05-05

Raf kinase inhibitory protein (RKIP), also known as phoshaptidylethanolamine binding protein (PEBP), has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE). In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized. In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity. This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential.

Characterization of the Raf kinase inhibitory protein (RKIP binding pocket: NMR-based screening identifies small-molecule ligands.

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Anne N Shemon

2010-05-01

Full Text Available Raf kinase inhibitory protein (RKIP, also known as phoshaptidylethanolamine binding protein (PEBP, has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE. In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized.In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity.This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential.

Molecular interactions of agonist and inverse agonist ligands at serotonin 5-HT2C G protein-coupled receptors: computational ligand docking and molecular dynamics studies validated by experimental mutagenesis results

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Córdova-Sintjago, Tania C.; Liu, Yue; Booth, Raymond G.

2015-02-01

To understand molecular determinants for ligand activation of the serotonin 5-HT2C G protein-coupled receptor (GPCR), a drug target for obesity and neuropsychiatric disorders, a 5-HT2C homology model was built according to an adrenergic β2 GPCR (β2AR) structure and validated using a 5-HT2B GPCR crystal structure. The models were equilibrated in a simulated phosphatidyl choline membrane for ligand docking and molecular dynamics studies. Ligands included (2S, 4R)-(-)-trans-4-(3'-bromo- and trifluoro-phenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalene-2-amine (3'-Br-PAT and 3'-CF3-PAT), a 5-HT2C agonist and inverse agonist, respectively. Distinct interactions of 3'-Br-PAT and 3'-CF3-PAT at the wild-type (WT) 5-HT2C receptor model were observed and experimental 5-HT2C receptor mutagenesis studies were undertaken to validate the modelling results. For example, the inverse agonist 3'-CF3-PAT docked deeper in the WT 5-HT2C binding pocket and altered the orientation of transmembrane helices (TM) 6 in comparison to the agonist 3'-Br-PAT, suggesting that changes in TM orientation that result from ligand binding impact function. For both PATs, mutation of 5-HT2C residues S3.36, T3.37, and F5.47 to alanine resulted in significantly decreased affinity, as predicted from modelling results. It was concluded that upon PAT binding, 5-HT2C residues T3.37 and F5.47 in TMs 3 and 5, respectively, engage in inter-helical interactions with TMs 4 and 6, respectively. The movement of TMs 5 and 6 upon agonist and inverse agonist ligand binding observed in the 5-HT2C receptor modelling studies was similar to movements reported for the activation and deactivation of the β2AR, suggesting common mechanisms among aminergic neurotransmitter GPCRs.

A Saccharomyces cerevisiae assay system to investigate ligand/AdipoR1 interactions that lead to cellular signaling.

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Mustapha Aouida

Full Text Available Adiponectin is a mammalian hormone that exerts anti-diabetic, anti-cancer and cardioprotective effects through interaction with its major ubiquitously expressed plasma membrane localized receptors, AdipoR1 and AdipoR2. Here, we report a Saccharomyces cerevisiae based method for investigating agonist-AdipoR interactions that is amenable for high-throughput scale-up and can be used to study both AdipoRs separately. Agonist-AdipoR1 interactions are detected using a split firefly luciferase assay based on reconstitution of firefly luciferase (Luc activity due to juxtaposition of its N- and C-terminal fragments, NLuc and CLuc, by ligand induced interaction of the chimeric proteins CLuc-AdipoR1 and APPL1-NLuc (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif 1-NLuc in a S. cerevisiae strain lacking the yeast homolog of AdipoRs (Izh2p. The assay monitors the earliest known step in the adiponectin-AdipoR anti-diabetic signaling cascade. We demonstrate that reconstituted Luc activity can be detected in colonies or cells using a CCD camera and quantified in cell suspensions using a microplate reader. AdipoR1-APPL1 interaction occurs in absence of ligand but can be stimulated specifically by agonists such as adiponectin and the tobacco protein osmotin that was shown to have AdipoR-dependent adiponectin-like biological activity in mammalian cells. To further validate this assay, we have modeled the three dimensional structures of receptor-ligand complexes of membrane-embedded AdipoR1 with cyclic peptides derived from osmotin or osmotin-like plant proteins. We demonstrate that the calculated AdipoR1-peptide binding energies correlate with the peptides' ability to behave as AdipoR1 agonists in the split luciferase assay. Further, we demonstrate agonist-AdipoR dependent activation of protein kinase A (PKA signaling and AMP activated protein kinase (AMPK phosphorylation in S. cerevisiae, which are

A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions

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Hostetler, Jessica B.; Sharma, Sumana; Bartholdson, S. Josefin; Wright, Gavin J.; Fairhurst, Rick M.; Rayner, Julian C.

2015-01-01

Background A vaccine targeting Plasmodium vivax will be an essential component of any comprehensive malaria elimination program, but major gaps in our understanding of P. vivax biology, including the protein-protein interactions that mediate merozoite invasion of reticulocytes, hinder the search for candidate antigens. Only one ligand-receptor interaction has been identified, that between P. vivax Duffy Binding Protein (PvDBP) and the erythrocyte Duffy Antigen Receptor for Chemokines (DARC), and strain-specific immune responses to PvDBP make it a complex vaccine target. To broaden the repertoire of potential P. vivax merozoite-stage vaccine targets, we exploited a recent breakthrough in expressing full-length ectodomains of Plasmodium proteins in a functionally-active form in mammalian cells and initiated a large-scale study of P. vivax merozoite proteins that are potentially involved in reticulocyte binding and invasion. Methodology/Principal Findings We selected 39 P. vivax proteins that are predicted to localize to the merozoite surface or invasive secretory organelles, some of which show homology to P. falciparum vaccine candidates. Of these, we were able to express 37 full-length protein ectodomains in a mammalian expression system, which has been previously used to express P. falciparum invasion ligands such as PfRH5. To establish whether the expressed proteins were correctly folded, we assessed whether they were recognized by antibodies from Cambodian patients with acute vivax malaria. IgG from these samples showed at least a two-fold change in reactivity over naïve controls in 27 of 34 antigens tested, and the majority showed heat-labile IgG immunoreactivity, suggesting the presence of conformation-sensitive epitopes and native tertiary protein structures. Using a method specifically designed to detect low-affinity, extracellular protein-protein interactions, we confirmed a predicted interaction between P. vivax 6-cysteine proteins P12 and P41, further

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

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Yu-Ru Zhang

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

Accuracy and precision of protein–ligand interaction kinetics determined from chemical shift titrations

International Nuclear Information System (INIS)

Markin, Craig J.; Spyracopoulos, Leo

2012-01-01

NMR-monitored chemical shift titrations for the study of weak protein–ligand interactions represent a rich source of information regarding thermodynamic parameters such as dissociation constants (K D ) in the micro- to millimolar range, populations for the free and ligand-bound states, and the kinetics of interconversion between states, which are typically within the fast exchange regime on the NMR timescale. We recently developed two chemical shift titration methods wherein co-variation of the total protein and ligand concentrations gives increased precision for the K D value of a 1:1 protein–ligand interaction (Markin and Spyracopoulos in J Biomol NMR 53: 125–138, 2012). In this study, we demonstrate that classical line shape analysis applied to a single set of 1 H– 15 N 2D HSQC NMR spectra acquired using precise protein–ligand chemical shift titration methods we developed, produces accurate and precise kinetic parameters such as the off-rate (k off ). For experimentally determined kinetics in the fast exchange regime on the NMR timescale, k off ∼ 3,000 s −1 in this work, the accuracy of classical line shape analysis was determined to be better than 5 % by conducting quantum mechanical NMR simulations of the chemical shift titration methods with the magnetic resonance toolkit GAMMA. Using Monte Carlo simulations, the experimental precision for k off from line shape analysis of NMR spectra was determined to be 13 %, in agreement with the theoretical precision of 12 % from line shape analysis of the GAMMA simulations in the presence of noise and protein concentration errors. In addition, GAMMA simulations were employed to demonstrate that line shape analysis has the potential to provide reasonably accurate and precise k off values over a wide range, from 100 to 15,000 s −1 . The validity of line shape analysis for k off values approaching intermediate exchange (∼100 s −1 ), may be facilitated by more accurate K D measurements from NMR

Intramolecular interaction influences binding of the Flax L5 and L6 resistance proteins to their AvrL567 ligands.

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Michael Ravensdale

Full Text Available L locus resistance (R proteins are nucleotide binding (NB-ARC leucine-rich repeat (LRR proteins from flax (Linum usitatissimum that provide race-specific resistance to the causal agent of flax rust disease, Melampsora lini. L5 and L6 are two alleles of the L locus that directly recognize variants of the fungal effector AvrL567. In this study, we have investigated the molecular details of this recognition by site-directed mutagenesis of AvrL567 and construction of chimeric L proteins. Single, double and triple mutations of polymorphic residues in a variety of AvrL567 variants showed additive effects on recognition strength, suggesting that multiple contact points are involved in recognition. Domain-swap experiments between L5 and L6 show that specificity differences are determined by their corresponding LRR regions. Most positively selected amino acid sites occur in the N- and C-terminal LRR units, and polymorphisms in the first seven and last four LRR units contribute to recognition specificity of L5 and L6 respectively. This further confirms that multiple, additive contact points occur between AvrL567 variants and either L5 or L6. However, we also observed that recognition of AvrL567 is affected by co-operative polymorphisms between both adjacent and distant domains of the R protein, including the TIR, ARC and LRR domains, implying that these residues are involved in intramolecular interactions to optimize detection of the pathogen and defense signal activation. We suggest a model where Avr ligand interaction directly competes with intramolecular interactions to cause activation of the R protein.

Ligand size is a major determinant of specificity in periplasmic oxyanion-binding proteins: the 1.2 A resolution crystal structure of Azotobacter vinelandii ModA.

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Lawson, D M; Williams, C E; Mitchenall, L A; Pau, R N

1998-12-15

. Periplasmic receptors constitute a diverse class of binding proteins that differ widely in size, sequence and ligand specificity. Nevertheless, almost all of them display a common beta/alpha folding motif and have similar tertiary structures consisting of two globular domains. The ligand is bound at the bottom of a deep cleft, which lies at the interface between these two domains. The oxyanion-binding proteins are notable in that they can discriminate between very similar ligands. . Azotobacter vinelandii is unusual in that it possesses two periplasmic molybdate-binding proteins. The crystal structure of one of these with bound ligand has been determined at 1.2 A resolution. It superficially resembles the structure of sulphate-binding protein (SBP) from Salmonella typhimurium and uses a similar constellation of hydrogen-bonding interactions to bind its ligand. However, the detailed interactions are distinct from those of SBP and the more closely related molybdate-binding protein of Escherichia coli. . Despite differences in the residues involved in binding, the volumes of the binding pockets in the A. vinelandii and E. coli molybdate-binding proteins are similar and are significantly larger than that of SBP. We conclude that the discrimination between molybdate and sulphate shown by these binding proteins is largely dependent upon small differences in the sizes of these two oxyanions.

Computing the binding affinity of a ligand buried deep inside a protein with the hybrid steered molecular dynamics

International Nuclear Information System (INIS)

Villarreal, Oscar D.; Yu, Lili; Rodriguez, Roberto A.; Chen, Liao Y.

2017-01-01

Computing the ligand-protein binding affinity (or the Gibbs free energy) with chemical accuracy has long been a challenge for which many methods/approaches have been developed and refined with various successful applications. False positives and, even more harmful, false negatives have been and still are a common occurrence in practical applications. Inevitable in all approaches are the errors in the force field parameters we obtain from quantum mechanical computation and/or empirical fittings for the intra- and inter-molecular interactions. These errors propagate to the final results of the computed binding affinities even if we were able to perfectly implement the statistical mechanics of all the processes relevant to a given problem. And they are actually amplified to various degrees even in the mature, sophisticated computational approaches. In particular, the free energy perturbation (alchemical) approaches amplify the errors in the force field parameters because they rely on extracting the small differences between similarly large numbers. In this paper, we develop a hybrid steered molecular dynamics (hSMD) approach to the difficult binding problems of a ligand buried deep inside a protein. Sampling the transition along a physical (not alchemical) dissociation path of opening up the binding cavity- -pulling out the ligand- -closing back the cavity, we can avoid the problem of error amplifications by not relying on small differences between similar numbers. We tested this new form of hSMD on retinol inside cellular retinol-binding protein 1 and three cases of a ligand (a benzylacetate, a 2-nitrothiophene, and a benzene) inside a T4 lysozyme L99A/M102Q(H) double mutant. In all cases, we obtained binding free energies in close agreement with the experimentally measured values. This indicates that the force field parameters we employed are accurate and that hSMD (a brute force, unsophisticated approach) is free from the problem of error amplification suffered by

Evaluation of the novel algorithm of flexible ligand docking with moveable target-protein atoms.

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Sulimov, Alexey V; Zheltkov, Dmitry A; Oferkin, Igor V; Kutov, Danil C; Katkova, Ekaterina V; Tyrtyshnikov, Eugene E; Sulimov, Vladimir B

2017-01-01

We present the novel docking algorithm based on the Tensor Train decomposition and the TT-Cross global optimization. The algorithm is applied to the docking problem with flexible ligand and moveable protein atoms. The energy of the protein-ligand complex is calculated in the frame of the MMFF94 force field in vacuum. The grid of precalculated energy potentials of probe ligand atoms in the field of the target protein atoms is not used. The energy of the protein-ligand complex for any given configuration is computed directly with the MMFF94 force field without any fitting parameters. The conformation space of the system coordinates is formed by translations and rotations of the ligand as a whole, by the ligand torsions and also by Cartesian coordinates of the selected target protein atoms. Mobility of protein and ligand atoms is taken into account in the docking process simultaneously and equally. The algorithm is realized in the novel parallel docking SOL-P program and results of its performance for a set of 30 protein-ligand complexes are presented. Dependence of the docking positioning accuracy is investigated as a function of parameters of the docking algorithm and the number of protein moveable atoms. It is shown that mobility of the protein atoms improves docking positioning accuracy. The SOL-P program is able to perform docking of a flexible ligand into the active site of the target protein with several dozens of protein moveable atoms: the native crystallized ligand pose is correctly found as the global energy minimum in the search space with 157 dimensions using 4700 CPU ∗ h at the Lomonosov supercomputer.

Relationship between hot spot residues and ligand binding hot spots in protein-protein interfaces.

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Zerbe, Brandon S; Hall, David R; Vajda, Sandor; Whitty, Adrian; Kozakov, Dima

2012-08-27

In the context of protein-protein interactions, the term "hot spot" refers to a residue or cluster of residues that makes a major contribution to the binding free energy, as determined by alanine scanning mutagenesis. In contrast, in pharmaceutical research, a hot spot is a site on a target protein that has high propensity for ligand binding and hence is potentially important for drug discovery. Here we examine the relationship between these two hot spot concepts by comparing alanine scanning data for a set of 15 proteins with results from mapping the protein surfaces for sites that can bind fragment-sized small molecules. We find the two types of hot spots are largely complementary; the residues protruding into hot spot regions identified by computational mapping or experimental fragment screening are almost always themselves hot spot residues as defined by alanine scanning experiments. Conversely, a residue that is found by alanine scanning to contribute little to binding rarely interacts with hot spot regions on the partner protein identified by fragment mapping. In spite of the strong correlation between the two hot spot concepts, they fundamentally differ, however. In particular, while identification of a hot spot by alanine scanning establishes the potential to generate substantial interaction energy with a binding partner, there are additional topological requirements to be a hot spot for small molecule binding. Hence, only a minority of hot spots identified by alanine scanning represent sites that are potentially useful for small inhibitor binding, and it is this subset that is identified by experimental or computational fragment screening.

DARC 2.0: Improved Docking and Virtual Screening at Protein Interaction Sites.

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Ragul Gowthaman

Full Text Available Over the past decade, protein-protein interactions have emerged as attractive but challenging targets for therapeutic intervention using small molecules. Due to the relatively flat surfaces that typify protein interaction sites, modern virtual screening tools developed for optimal performance against "traditional" protein targets perform less well when applied instead at protein interaction sites. Previously, we described a docking method specifically catered to the shallow binding modes characteristic of small-molecule inhibitors of protein interaction sites. This method, called DARC (Docking Approach using Ray Casting, operates by comparing the topography of the protein surface when "viewed" from a vantage point inside the protein against the topography of a bound ligand when "viewed" from the same vantage point. Here, we present five key enhancements to DARC. First, we use multiple vantage points to more accurately determine protein-ligand surface complementarity. Second, we describe a new scheme for rapidly determining optimal weights in the DARC scoring function. Third, we incorporate sampling of ligand conformers "on-the-fly" during docking. Fourth, we move beyond simple shape complementarity and introduce a term in the scoring function to capture electrostatic complementarity. Finally, we adjust the control flow in our GPU implementation of DARC to achieve greater speedup of these calculations. At each step of this study, we evaluate the performance of DARC in a "pose recapitulation" experiment: predicting the binding mode of 25 inhibitors each solved in complex with its distinct target protein (a protein interaction site. Whereas the previous version of DARC docked only one of these inhibitors to within 2 Å RMSD of its position in the crystal structure, the newer version achieves this level of accuracy for 12 of the 25 complexes, corresponding to a statistically significant performance improvement (p < 0.001. Collectively then, we find

Essential multimeric enzymes in kinetoplastid parasites: A host of potentially druggable protein-protein interactions.

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Wachsmuth, Leah M; Johnson, Meredith G; Gavenonis, Jason

2017-06-01

Parasitic diseases caused by kinetoplastid parasites of the genera Trypanosoma and Leishmania are an urgent public health crisis in the developing world. These closely related species possess a number of multimeric enzymes in highly conserved pathways involved in vital functions, such as redox homeostasis and nucleotide synthesis. Computational alanine scanning of these protein-protein interfaces has revealed a host of potentially ligandable sites on several established and emerging anti-parasitic drug targets. Analysis of interfaces with multiple clustered hotspots has suggested several potentially inhibitable protein-protein interactions that may have been overlooked by previous large-scale analyses focusing solely on secondary structure. These protein-protein interactions provide a promising lead for the development of new peptide and macrocycle inhibitors of these enzymes.

eMatchSite: sequence order-independent structure alignments of ligand binding pockets in protein models.

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Michal Brylinski

2014-09-01

Full Text Available Detecting similarities between ligand binding sites in the absence of global homology between target proteins has been recognized as one of the critical components of modern drug discovery. Local binding site alignments can be constructed using sequence order-independent techniques, however, to achieve a high accuracy, many current algorithms for binding site comparison require high-quality experimental protein structures, preferably in the bound conformational state. This, in turn, complicates proteome scale applications, where only various quality structure models are available for the majority of gene products. To improve the state-of-the-art, we developed eMatchSite, a new method for constructing sequence order-independent alignments of ligand binding sites in protein models. Large-scale benchmarking calculations using adenine-binding pockets in crystal structures demonstrate that eMatchSite generates accurate alignments for almost three times more protein pairs than SOIPPA. More importantly, eMatchSite offers a high tolerance to structural distortions in ligand binding regions in protein models. For example, the percentage of correctly aligned pairs of adenine-binding sites in weakly homologous protein models is only 4-9% lower than those aligned using crystal structures. This represents a significant improvement over other algorithms, e.g. the performance of eMatchSite in recognizing similar binding sites is 6% and 13% higher than that of SiteEngine using high- and moderate-quality protein models, respectively. Constructing biologically correct alignments using predicted ligand binding sites in protein models opens up the possibility to investigate drug-protein interaction networks for complete proteomes with prospective systems-level applications in polypharmacology and rational drug repositioning. eMatchSite is freely available to the academic community as a web-server and a stand-alone software distribution at http://www.brylinski.org/ematchsite.

High pressure as a tool for investigating protein-ligand interactions

International Nuclear Information System (INIS)

Marchal, S; Lange, R; Tortora, P; Balny, C

2004-01-01

In recent years, the application of pressure on biological systems has gained increasing interest. Pressure-induced destabilization of electrostatic and hydrophobic interactions is currently exploited to study conformational protein stability and macromolecular assemblies of proteins. Due to links between severe human pathologies and ordered protein oligomerization into aggregates, which have become apparent, a better knowledge of the molecular and structural determinants that ensure the packing efficiency and stability of such complexes has taken on special importance. Here, we report the effect of pressure on the property of human ataxin-3 of aggregation. The results indicate the importance of its polyglutamine chain length in the stability and the tendency of the protein to form spheroids. Partial unfolding of the protein leading to solvent exposure of hydrophobic domains appears to be a prerequisite in the aggregation process of ataxin-3

Geomfinder: a multi-feature identifier of similar three-dimensional protein patterns: a ligand-independent approach.

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Núñez-Vivanco, Gabriel; Valdés-Jiménez, Alejandro; Besoaín, Felipe; Reyes-Parada, Miguel

2016-01-01

Since the structure of proteins is more conserved than the sequence, the identification of conserved three-dimensional (3D) patterns among a set of proteins, can be important for protein function prediction, protein clustering, drug discovery and the establishment of evolutionary relationships. Thus, several computational applications to identify, describe and compare 3D patterns (or motifs) have been developed. Often, these tools consider a 3D pattern as that described by the residues surrounding co-crystallized/docked ligands available from X-ray crystal structures or homology models. Nevertheless, many of the protein structures stored in public databases do not provide information about the location and characteristics of ligand binding sites and/or other important 3D patterns such as allosteric sites, enzyme-cofactor interaction motifs, etc. This makes necessary the development of new ligand-independent methods to search and compare 3D patterns in all available protein structures. Here we introduce Geomfinder, an intuitive, flexible, alignment-free and ligand-independent web server for detailed estimation of similarities between all pairs of 3D patterns detected in any two given protein structures. We used around 1100 protein structures to form pairs of proteins which were assessed with Geomfinder. In these analyses each protein was considered in only one pair (e.g. in a subset of 100 different proteins, 50 pairs of proteins can be defined). Thus: (a) Geomfinder detected identical pairs of 3D patterns in a series of monoamine oxidase-B structures, which corresponded to the effectively similar ligand binding sites at these proteins; (b) we identified structural similarities among pairs of protein structures which are targets of compounds such as acarbose, benzamidine, adenosine triphosphate and pyridoxal phosphate; these similar 3D patterns are not detected using sequence-based methods; (c) the detailed evaluation of three specific cases showed the versatility

The alpha-fetoprotein (AFP) third domain: a search for AFP interaction sites of cell cycle proteins.

Science.gov (United States)

Mizejewski, G J

2016-09-01

The carboxy-terminal third domain of alpha-fetoprotein (AFP-3D) is known to harbor binding and/or interaction sites for hydrophobic ligands, receptors, and binding proteins. Such reports have established that AFP-3D consists of amino acid (AA) sequence stretches on the AFP polypeptide that engages in protein-to-protein interactions with various ligands and receptors. Using a computer software program specifically designed for such interactions, the present report identified AA sequence fragments on AFP-3D that could potentially interact with a variety of cell cycle proteins. The cell cycle proteins identified were (1) cyclins, (2) cyclin-dependent kinases, (3) cell cycle-associated proteins (inhibitors, checkpoints, initiators), and (4) ubiquitin ligases. Following detection of the AFP-3D to cell cycle protein interaction sites, the computer-derived AFP localization AA sequences were compared and aligned with previously reported hydrophobic ligand and receptor interaction sites on AFP-3D. A literature survey of the association of cell cycle proteins with AFP showed both positive relationships and correlations. Previous reports of experimental AFP-derived peptides effects on various cell cycle proteins served to confirm and verify the present computer cell cycle protein identifications. Cell cycle protein interactions with AFP-CD peptides have been reported in cultured MCF-7 breast cancer cells subjected to mRNA microarray analysis. After 7 days in culture with MCF-7 cells, the AFP-derived peptides were shown to downregulate cyclin E, SKP2, checkpoint suppressors, cyclin-dependent kinases, and ubiquitin ligases that modulate cyclin E/CdK2 transition from the G1 to the S-phase of the cell cycle. Thus, the experimental data on AFP-CD interaction with cell cycle proteins were consistent with the "in silico" findings.

A novel protein-protein interaction in the RES (REtention and Splicing) complex.

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Tripsianes, Konstantinos; Friberg, Anders; Barrandon, Charlotte; Brooks, Mark; van Tilbeurgh, Herman; Seraphin, Bertrand; Sattler, Michael

2014-10-10

The retention and splicing (RES) complex is a conserved spliceosome-associated module that was shown to enhance splicing of a subset of transcripts and promote the nuclear retention of unspliced pre-mRNAs in yeast. The heterotrimeric RES complex is organized around the Snu17p protein that binds to both the Bud13p and Pml1p subunits. Snu17p exhibits an RRM domain that resembles a U2AF homology motif (UHM) and Bud13p harbors a Trp residue reminiscent of an UHM-ligand motif (ULM). It has therefore been proposed that the interaction between Snu17p and Bud13p resembles canonical UHM-ULM complexes. Here, we have used biochemical and NMR structural analysis to characterize the structure of the yeast Snu17p-Bud13p complex. Unlike known UHMs that sequester the Trp residue of the ULM ligand in a hydrophobic pocket, Snu17p and Bud13p utilize a large interaction surface formed around the two helices of the Snu17p domain. In total 18 residues of the Bud13p ligand wrap around the Snu17p helical surface in an U-turn-like arrangement. The invariant Trp(232) in Bud13p is located in the center of the turn, and contacts surface residues of Snu17p. The structural data are supported by mutational analysis and indicate that Snu17p provides an extended binding surface with Bud13p that is notably distinct from canonical UHM-ULM interactions. Our data highlight structural diversity in RRM-protein interactions, analogous to the one seen for nucleic acid interactions. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Towards ligand docking including explicit interface water molecules.

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Gordon Lemmon

Full Text Available Small molecule docking predicts the interaction of a small molecule ligand with a protein at atomic-detail accuracy including position and conformation the ligand but also conformational changes of the protein upon ligand binding. While successful in the majority of cases, docking algorithms including RosettaLigand fail in some cases to predict the correct protein/ligand complex structure. In this study we show that simultaneous docking of explicit interface water molecules greatly improves Rosetta's ability to distinguish correct from incorrect ligand poses. This result holds true for both protein-centric water docking wherein waters are located relative to the protein binding site and ligand-centric water docking wherein waters move with the ligand during docking. Protein-centric docking is used to model 99 HIV-1 protease/protease inhibitor structures. We find protease inhibitor placement improving at a ratio of 9:1 when one critical interface water molecule is included in the docking simulation. Ligand-centric docking is applied to 341 structures from the CSAR benchmark of diverse protein/ligand complexes [1]. Across this diverse dataset we see up to 56% recovery of failed docking studies, when waters are included in the docking simulation.

Oligomeric protein structure networks: insights into protein-protein interactions

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Brinda KV

2005-12-01

Full Text Available Abstract Background Protein-protein association is essential for a variety of cellular processes and hence a large number of investigations are being carried out to understand the principles of protein-protein interactions. In this study, oligomeric protein structures are viewed from a network perspective to obtain new insights into protein association. Structure graphs of proteins have been constructed from a non-redundant set of protein oligomer crystal structures by considering amino acid residues as nodes and the edges are based on the strength of the non-covalent interactions between the residues. The analysis of such networks has been carried out in terms of amino acid clusters and hubs (highly connected residues with special emphasis to protein interfaces. Results A variety of interactions such as hydrogen bond, salt bridges, aromatic and hydrophobic interactions, which occur at the interfaces are identified in a consolidated manner as amino acid clusters at the interface, from this study. Moreover, the characterization of the highly connected hub-forming residues at the interfaces and their comparison with the hubs from the non-interface regions and the non-hubs in the interface regions show that there is a predominance of charged interactions at the interfaces. Further, strong and weak interfaces are identified on the basis of the interaction strength between amino acid residues and the sizes of the interface clusters, which also show that many protein interfaces are stronger than their monomeric protein cores. The interface strengths evaluated based on the interface clusters and hubs also correlate well with experimentally determined dissociation constants for known complexes. Finally, the interface hubs identified using the present method correlate very well with experimentally determined hotspots in the interfaces of protein complexes obtained from the Alanine Scanning Energetics database (ASEdb. A few predictions of interface hot

Study of the interactions between a proline-rich protein and a flavan-3-ol by NMR: residual structures in the natively unfolded protein provides anchorage points for the ligands.

Science.gov (United States)

Pascal, Christine; Paté, Franck; Cheynier, Véronique; Delsuc, Marc-André

2009-09-01

Astringency is one of the major organoleptic properties of food and beverages that are made from plants, such as tea, chocolate, beer, or red wine. This sensation is thought to be due to interactions between tannins and salivary proline-rich proteins, which are natively unfolded proteins. A human salivary proline-rich protein, namely IB-5, was produced by the recombinant method. Its interactions with a model tannin, epigallocatechin gallate (EGCG), the major flavan-3-ol in green tea, were studied here. Circular dichroism experiments showed that IB-5 presents residual structures (PPII helices) when the ionic strength is close to that in saliva. In the presence of these residual structures, IB-5 undergoes an increase in structural content upon binding to EGCG. NMR data corroborated the presence of preformed structural elements within the protein prior to binding and a partial assignment was proposed, showing partial structuration. TOCSY experiments showed that amino acids that are involved in PPII helices are more likely to interact with EGCG than those in random coil regions, as if they were anchorage points for the ligand. The signal from IB-5 in the DOSY NMR spectrum revealed an increase in polydispersity upon addition of EGCG while the mean hydrodynamic radius remained unchanged. This strongly suggests the formation of IB-5/EGCG aggregates.

Mapping the Conformational Dynamics of E-selectin upon Interaction with its Ligands

KAUST Repository

Aleisa, Fajr A

2013-05-15

Selectins are key adhesion molecules responsible for initiating a multistep process that leads a cell out of the blood circulation and into a tissue or organ. The adhesion of cells (expressing ligands) to the endothelium (expressing the selectin i.e.,E-selectin) occurs through spatio-temporally regulated interactions that are mediated by multiple intra- and inter-cellular components. The mechanism of cell adhesion is investigated primarily using ensemble-based experiments, which provides indirect information about how individual molecules work in such a complex system. Recent developments in single-molecule (SM) fluorescence detection allow for the visualization of individual molecules with a good spatio-temporal resolution nanometer spatial resolution and millisecond time resolution). Furthermore, advanced SM fluorescence techniques such as Förster Resonance Energy Transfer (FRET) and super-resolution microscopy provide unique opportunities to obtain information about nanometer-scale conformational dynamics of proteins as well as nano-scale architectures of biological samples. Therefore, the state-of-the-art SM techniques are powerful tools for investigating complex biological system such as the mechanism of cell adhesion. In this project, several constructs of fluorescently labeled E-selectin will be used to study the conformational dynamics of E-selectin binding to its ligand(s) using SM-FRET and combination of SM-FRET and force microscopy. These studies will be beneficial to fully understand the mechanistic details of cell adhesion and migration of cells using the established model system of hematopoietic stem cells (HSCs) adhesion to the selectin expressing endothelial cells (such as the E-selectin expressing endothelial cells in the bone marrow).

Thermodynamic Characterization of Hydration Sites from Integral Equation-Derived Free Energy Densities: Application to Protein Binding Sites and Ligand Series.

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Güssregen, Stefan; Matter, Hans; Hessler, Gerhard; Lionta, Evanthia; Heil, Jochen; Kast, Stefan M

2017-07-24

Water molecules play an essential role for mediating interactions between ligands and protein binding sites. Displacement of specific water molecules can favorably modulate the free energy of binding of protein-ligand complexes. Here, the nature of water interactions in protein binding sites is investigated by 3D RISM (three-dimensional reference interaction site model) integral equation theory to understand and exploit local thermodynamic features of water molecules by ranking their possible displacement in structure-based design. Unlike molecular dynamics-based approaches, 3D RISM theory allows for fast and noise-free calculations using the same detailed level of solute-solvent interaction description. Here we correlate molecular water entities instead of mere site density maxima with local contributions to the solvation free energy using novel algorithms. Distinct water molecules and hydration sites are investigated in multiple protein-ligand X-ray structures, namely streptavidin, factor Xa, and factor VIIa, based on 3D RISM-derived free energy density fields. Our approach allows the semiquantitative assessment of whether a given structural water molecule can potentially be targeted for replacement in structure-based design. Finally, PLS-based regression models from free energy density fields used within a 3D-QSAR approach (CARMa - comparative analysis of 3D RISM Maps) are shown to be able to extract relevant information for the interpretation of structure-activity relationship (SAR) trends, as demonstrated for a series of serine protease inhibitors.

Prediction of vitamin interacting residues in a vitamin binding protein using evolutionary information

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Panwar Bharat

2013-02-01

Full Text Available Abstract Background The vitamins are important cofactors in various enzymatic-reactions. In past, many inhibitors have been designed against vitamin binding pockets in order to inhibit vitamin-protein interactions. Thus, it is important to identify vitamin interacting residues in a protein. It is possible to detect vitamin-binding pockets on a protein, if its tertiary structure is known. Unfortunately tertiary structures of limited proteins are available. Therefore, it is important to develop in-silico models for predicting vitamin interacting residues in protein from its primary structure. Results In this study, first we compared protein-interacting residues of vitamins with other ligands using Two Sample Logo (TSL. It was observed that ATP, GTP, NAD, FAD and mannose preferred {G,R,K,S,H}, {G,K,T,S,D,N}, {T,G,Y}, {G,Y,W} and {Y,D,W,N,E} residues respectively, whereas vitamins preferred {Y,F,S,W,T,G,H} residues for the interaction with proteins. Furthermore, compositional information of preferred and non-preferred residues along with patterns-specificity was also observed within different vitamin-classes. Vitamins A, B and B6 preferred {F,I,W,Y,L,V}, {S,Y,G,T,H,W,N,E} and {S,T,G,H,Y,N} interacting residues respectively. It suggested that protein-binding patterns of vitamins are different from other ligands, and motivated us to develop separate predictor for vitamins and their sub-classes. The four different prediction modules, (i vitamin interacting residues (VIRs, (ii vitamin-A interacting residues (VAIRs, (iii vitamin-B interacting residues (VBIRs and (iv pyridoxal-5-phosphate (vitamin B6 interacting residues (PLPIRs have been developed. We applied various classifiers of SVM, BayesNet, NaiveBayes, ComplementNaiveBayes, NaiveBayesMultinomial, RandomForest and IBk etc., as machine learning techniques, using binary and Position-Specific Scoring Matrix (PSSM features of protein sequences. Finally, we selected best performing SVM modules and

Prediction of vitamin interacting residues in a vitamin binding protein using evolutionary information.

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Panwar, Bharat; Gupta, Sudheer; Raghava, Gajendra P S

2013-02-07

The vitamins are important cofactors in various enzymatic-reactions. In past, many inhibitors have been designed against vitamin binding pockets in order to inhibit vitamin-protein interactions. Thus, it is important to identify vitamin interacting residues in a protein. It is possible to detect vitamin-binding pockets on a protein, if its tertiary structure is known. Unfortunately tertiary structures of limited proteins are available. Therefore, it is important to develop in-silico models for predicting vitamin interacting residues in protein from its primary structure. In this study, first we compared protein-interacting residues of vitamins with other ligands using Two Sample Logo (TSL). It was observed that ATP, GTP, NAD, FAD and mannose preferred {G,R,K,S,H}, {G,K,T,S,D,N}, {T,G,Y}, {G,Y,W} and {Y,D,W,N,E} residues respectively, whereas vitamins preferred {Y,F,S,W,T,G,H} residues for the interaction with proteins. Furthermore, compositional information of preferred and non-preferred residues along with patterns-specificity was also observed within different vitamin-classes. Vitamins A, B and B6 preferred {F,I,W,Y,L,V}, {S,Y,G,T,H,W,N,E} and {S,T,G,H,Y,N} interacting residues respectively. It suggested that protein-binding patterns of vitamins are different from other ligands, and motivated us to develop separate predictor for vitamins and their sub-classes. The four different prediction modules, (i) vitamin interacting residues (VIRs), (ii) vitamin-A interacting residues (VAIRs), (iii) vitamin-B interacting residues (VBIRs) and (iv) pyridoxal-5-phosphate (vitamin B6) interacting residues (PLPIRs) have been developed. We applied various classifiers of SVM, BayesNet, NaiveBayes, ComplementNaiveBayes, NaiveBayesMultinomial, RandomForest and IBk etc., as machine learning techniques, using binary and Position-Specific Scoring Matrix (PSSM) features of protein sequences. Finally, we selected best performing SVM modules and obtained highest MCC of 0.53, 0.48, 0.61, 0

Magnetic levitation as a platform for competitive protein-ligand binding assays.

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Shapiro, Nathan D; Soh, Siowling; Mirica, Katherine A; Whitesides, George M

2012-07-17

This paper describes a method based on magnetic levitation (MagLev) that is capable of indirectly measuring the binding of unlabeled ligands to unlabeled protein. We demonstrate this method by measuring the affinity of unlabeled bovine carbonic anhydrase (BCA) for a variety of ligands (most of which are benzene sulfonamide derivatives). This method utilizes porous gel beads that are functionalized with a common aryl sulfonamide ligand. The beads are incubated with BCA and allowed to reach an equilibrium state in which the majority of the immobilized ligands are bound to BCA. Since the beads are less dense than the protein, protein binding to the bead increases the overall density of the bead. This change in density can be monitored using MagLev. Transferring the beads to a solution containing no protein creates a situation where net protein efflux from the bead is thermodynamically favorable. The rate at which protein leaves the bead for the solution can be calculated from the rate at which the levitation height of the bead changes. If another small molecule ligand of BCA is dissolved in the solution, the rate of protein efflux is accelerated significantly. This paper develops a reaction-diffusion (RD) model to explain both this observation, and the physical-organic chemistry that underlies it. Using this model, we calculate the dissociation constants of several unlabeled ligands from BCA, using plots of levitation height versus time. Notably, although this method requires no electricity, and only a single piece of inexpensive equipment, it can measure accurately the binding of unlabeled proteins to small molecules over a wide range of dissociation constants (K(d) values within the range from ~10 nM to 100 μM are measured easily). Assays performed using this method generally can be completed within a relatively short time period (20 min-2 h). A deficiency of this system is that it is not, in its present form, applicable to proteins with molecular weight greater

A Saccharomyces cerevisiae Assay System to Investigate Ligand/AdipoR1 Interactions That Lead to Cellular Signaling

KAUST Repository

Aouida, Mustapha

2013-06-07

Adiponectin is a mammalian hormone that exerts anti-diabetic, anti-cancer and cardioprotective effects through interaction with its major ubiquitously expressed plasma membrane localized receptors, AdipoR1 and AdipoR2. Here, we report a Saccharomyces cerevisiae based method for investigating agonist-AdipoR interactions that is amenable for high-throughput scale-up and can be used to study both AdipoRs separately. Agonist-AdipoR1 interactions are detected using a split firefly luciferase assay based on reconstitution of firefly luciferase (Luc) activity due to juxtaposition of its N- and C-terminal fragments, NLuc and CLuc, by ligand induced interaction of the chimeric proteins CLuc-AdipoR1 and APPL1-NLuc (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif 1-NLuc) in a S. cerevisiae strain lacking the yeast homolog of AdipoRs (Izh2p). The assay monitors the earliest known step in the adiponectin-AdipoR anti-diabetic signaling cascade. We demonstrate that reconstituted Luc activity can be detected in colonies or cells using a CCD camera and quantified in cell suspensions using a microplate reader. AdipoR1-APPL1 interaction occurs in absence of ligand but can be stimulated specifically by agonists such as adiponectin and the tobacco protein osmotin that was shown to have AdipoR-dependent adiponectin-like biological activity in mammalian cells. To further validate this assay, we have modeled the three dimensional structures of receptor-ligand complexes of membrane-embedded AdipoR1 with cyclic peptides derived from osmotin or osmotin-like plant proteins. We demonstrate that the calculated AdipoR1-peptide binding energies correlate with the peptides\\' ability to behave as AdipoR1 agonists in the split luciferase assay. Further, we demonstrate agonist-AdipoR dependent activation of protein kinase A (PKA) signaling and AMP activated protein kinase (AMPK) phosphorylation in S. cerevisiae, which are homologous to

Differential TAM receptor-ligand-phospholipid interactions delimit differential TAM bioactivities.

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Lew, Erin D; Oh, Jennifer; Burrola, Patrick G; Lax, Irit; Zagórska, Anna; Través, Paqui G; Schlessinger, Joseph; Lemke, Greg

2014-09-29

The TAM receptor tyrosine kinases Tyro3, Axl, and Mer regulate key features of cellular physiology, yet the differential activities of the TAM ligands Gas6 and Protein S are poorly understood. We have used biochemical and genetic analyses to delineate the rules for TAM receptor-ligand engagement and find that the TAMs segregate into two groups based on ligand specificity, regulation by phosphatidylserine, and function. Tyro3 and Mer are activated by both ligands but only Gas6 activates Axl. Optimal TAM signaling requires coincident TAM ligand engagement of both its receptor and the phospholipid phosphatidylserine (PtdSer): Gas6 lacking its PtdSer-binding 'Gla domain' is significantly weakened as a Tyro3/Mer agonist and is inert as an Axl agonist, even though it binds to Axl with wild-type affinity. In two settings of TAM-dependent homeostatic phagocytosis, Mer plays a predominant role while Axl is dispensable, and activation of Mer by Protein S is sufficient to drive phagocytosis.

IQGAP1 is a novel CXCR2-interacting protein and essential component of the "chemosynapse".

Directory of Open Access Journals (Sweden)

Nicole F Neel

Full Text Available Chemotaxis is essential for a number of physiological processes including leukocyte recruitment. Chemokines initiate intracellular signaling pathways necessary for chemotaxis through binding seven transmembrane G protein-couple receptors. Little is known about the proteins that interact with the intracellular domains of chemokine receptors to initiate cellular signaling upon ligand binding. CXCR2 is a major chemokine receptor expressed on several cell types, including endothelial cells and neutrophils. We hypothesize that multiple proteins interact with the intracellular domains of CXCR2 upon ligand stimulation and these interactions comprise a "chemosynapse", and play important roles in transducing CXCR2 mediated signaling processes.In an effort to define the complex of proteins that assemble upon CXCR2 activation to relay signals from activated chemokine receptors, a proteomics approach was employed to identify proteins that co-associate with CXCR2 with or without ligand stimulation. The components of the CXCR2 "chemosynapse" are involved in processes ranging from intracellular trafficking to cytoskeletal modification. IQ motif containing GTPase activating protein 1 (IQGAP1 was among the novel proteins identified to interact directly with CXCR2. Herein, we demonstrate that CXCR2 co-localizes with IQGAP1 at the leading edge of polarized human neutrophils and CXCR2 expressing differentiated HL-60 cells. Moreover, amino acids 1-160 of IQGAP1 directly interact with the carboxyl-terminal domain of CXCR2 and stimulation with CXCL8 enhances IQGAP1 association with Cdc42.Our studies indicate that IQGAP1 is a novel essential component of the CXCR2 "chemosynapse".

DARC: Mapping Surface Topography by Ray-Casting for Effective Virtual Screening at Protein Interaction Sites.

Science.gov (United States)

Gowthaman, Ragul; Miller, Sven A; Rogers, Steven; Khowsathit, Jittasak; Lan, Lan; Bai, Nan; Johnson, David K; Liu, Chunjing; Xu, Liang; Anbanandam, Asokan; Aubé, Jeffrey; Roy, Anuradha; Karanicolas, John

2016-05-12

Protein-protein interactions represent an exciting and challenging target class for therapeutic intervention using small molecules. Protein interaction sites are often devoid of the deep surface pockets presented by "traditional" drug targets, and crystal structures reveal that inhibitors typically engage these sites using very shallow binding modes. As a consequence, modern virtual screening tools developed to identify inhibitors of traditional drug targets do not perform as well when they are instead deployed at protein interaction sites. To address the need for novel inhibitors of important protein interactions, here we introduce an alternate docking strategy specifically designed for this regime. Our method, termed DARC (Docking Approach using Ray-Casting), matches the topography of a surface pocket "observed" from within the protein to the topography "observed" when viewing a potential ligand from the same vantage point. We applied DARC to carry out a virtual screen against the protein interaction site of human antiapoptotic protein Mcl-1 and found that four of the top-scoring 21 compounds showed clear inhibition in a biochemical assay. The Ki values for these compounds ranged from 1.2 to 21 μM, and each had ligand efficiency comparable to promising small-molecule inhibitors of other protein-protein interactions. These hit compounds do not resemble the natural (protein) binding partner of Mcl-1, nor do they resemble any known inhibitors of Mcl-1. Our results thus demonstrate the utility of DARC for identifying novel inhibitors of protein-protein interactions.

Target-specific NMR detection of protein–ligand interactions with antibody-relayed {sup 15}N-group selective STD

Energy Technology Data Exchange (ETDEWEB)

Hetényi, Anasztázia [University of Szeged, Department of Medical Chemistry (Hungary); Hegedűs, Zsófia [University of Szeged, SZTE-MTA Lendület Foldamer Research Group, Institute of Pharmaceutical Analysis Department (Hungary); Fajka-Boja, Roberta; Monostori, Éva [Biological Research Center of the Hungarian Academy of Sciences, Lymphocyte Signal Transduction Laboratory, Institute of Genetics (Hungary); Kövér, Katalin E. [University of Debrecen, Department of Inorganic and Analytical Chemistry (Hungary); Martinek, Tamás A., E-mail: martinek@pharm.u-szeged.hu [University of Szeged, SZTE-MTA Lendület Foldamer Research Group, Institute of Pharmaceutical Analysis Department (Hungary)

2016-12-15

Fragment-based drug design has been successfully applied to challenging targets where the detection of the weak protein–ligand interactions is a key element. {sup 1}H saturation transfer difference (STD) NMR spectroscopy is a powerful technique for this work but it requires pure homogeneous proteins as targets. Monoclonal antibody (mAb)-relayed {sup 15}N-GS STD spectroscopy has been developed to resolve the problem of protein mixtures and impure proteins. A {sup 15}N-labelled target-specific mAb is selectively irradiated and the saturation is relayed through the target to the ligand. Tests on the anti-Gal-1 mAb/Gal-1/lactose system showed that the approach is experimentally feasible in a reasonable time frame. This method allows detection and identification of binding molecules directly from a protein mixture in a multicomponent system.

Understanding Protein-Protein Interactions Using Local Structural Features

DEFF Research Database (Denmark)

Planas-Iglesias, Joan; Bonet, Jaume; García-García, Javier

2013-01-01

Protein-protein interactions (PPIs) play a relevant role among the different functions of a cell. Identifying the PPI network of a given organism (interactome) is useful to shed light on the key molecular mechanisms within a biological system. In this work, we show the role of structural features...... interacting and non-interacting protein pairs to classify the structural features that sustain the binding (or non-binding) behavior. Our study indicates that not only the interacting region but also the rest of the protein surface are important for the interaction fate. The interpretation...... to score the likelihood of the interaction between two proteins and to develop a method for the prediction of PPIs. We have tested our method on several sets with unbalanced ratios of interactions and non-interactions to simulate real conditions, obtaining accuracies higher than 25% in the most unfavorable...

Analogies and surprising differences between recombinant nitric oxide synthase-like proteins from Staphylococcus aureus and Bacillus anthracis in their interactions with l-arginine analogs and iron ligands.

Science.gov (United States)

Salard, Isabelle; Mercey, Emilie; Rekka, Eleni; Boucher, Jean-Luc; Nioche, Pierre; Mikula, Ivan; Martasek, Pavel; Raman, C S; Mansuy, Daniel

2006-12-01

Genome sequencing has recently shown the presence of genes coding for NO-synthase (NOS)-like proteins in bacteria. The roles of these proteins remain unclear. The interactions of a series of l-arginine (l-arg) analogs and iron ligands with two recombinant NOS-like proteins from Staphylococcus aureus (saNOS) and Bacillus anthracis (baNOS) have been studied by UV-visible spectroscopy. SaNOS and baNOS in their ferric native state, as well as their complexes with l-arg analogs and with various ligands, exhibit spectral characteristics highly similar to the corresponding complexes of heme-thiolate proteins such as cytochromes P450 and NOSs. However, saNOS greatly differs from baNOS at the level of three main properties: (i) native saNOS mainly exists under an hexacoordinated low-spin ferric state whereas native baNOS is mainly high-spin, (ii) the addition of tetrahydrobiopterin (H4B) or H4B analogs leads to an increase of the affinity of l-arg for saNOS but not for baNOS, and (iii) saNOS Fe(II), contrary to baNOS, binds relatively bulky ligands such as nitrosoalkanes and tert-butylisocyanide. Thus, saNOS exhibits properties very similar to those of the oxygenase domain of inducible NOS (iNOS(oxy)) not containing H4B, as expected for a NOSoxy-like protein that does not contain H4B. By contrast, the properties of baNOS which look like those of H4B-containing iNOS(oxy) are unexpected for a NOS-like protein not containing H4B. The origin of these surprising properties of baNOS remains to be determined.

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

Energy Technology Data Exchange (ETDEWEB)

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

2017-03-06

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

Determination of ligand binding modes in weak protein–ligand complexes using sparse NMR data

Energy Technology Data Exchange (ETDEWEB)

Mohanty, Biswaranjan; Williams, Martin L.; Doak, Bradley C.; Vazirani, Mansha; Ilyichova, Olga [Monash University, Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences (Australia); Wang, Geqing [La Trobe University, La Trobe Institute for Molecular Bioscience (Australia); Bermel, Wolfgang [Bruker Biospin GmbH (Germany); Simpson, Jamie S.; Chalmers, David K. [Monash University, Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences (Australia); King, Glenn F. [The University of Queensland, Institute for Molecular Bioscience (Australia); Mobli, Mehdi, E-mail: m.mobli@uq.edu.au [The University of Queensland, Centre for Advanced Imaging (Australia); Scanlon, Martin J., E-mail: martin.scanlon@monash.edu [Monash University, Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences (Australia)

2016-11-15

We describe a general approach to determine the binding pose of small molecules in weakly bound protein–ligand complexes by deriving distance constraints between the ligand and methyl groups from all methyl-containing residues of the protein. We demonstrate that using a single sample, which can be prepared without the use of expensive precursors, it is possible to generate high-resolution data rapidly and obtain the resonance assignments of Ile, Leu, Val, Ala and Thr methyl groups using triple resonance scalar correlation data. The same sample may be used to obtain Met {sup ε}CH{sub 3} assignments using NOESY-based methods, although the superior sensitivity of NOESY using [U-{sup 13}C,{sup 15}N]-labeled protein makes the use of this second sample more efficient. We describe a structural model for a weakly binding ligand bound to its target protein, DsbA, derived from intermolecular methyl-to-ligand nuclear Overhauser enhancements, and demonstrate that the ability to assign all methyl resonances in the spectrum is essential to derive an accurate model of the structure. Once the methyl assignments have been obtained, this approach provides a rapid means to generate structural models for weakly bound protein–ligand complexes. Such weak complexes are often found at the beginning of programs of fragment based drug design and can be challenging to characterize using X-ray crystallography.

Binding Ligand Prediction for Proteins Using Partial Matching of Local Surface Patches

Directory of Open Access Journals (Sweden)

Lee Sael

2010-12-01

Full Text Available Functional elucidation of uncharacterized protein structures is an important task in bioinformatics. We report our new approach for structure-based function prediction which captures local surface features of ligand binding pockets. Function of proteins, specifically, binding ligands of proteins, can be predicted by finding similar local surface regions of known proteins. To enable partial comparison of binding sites in proteins, a weighted bipartite matching algorithm is used to match pairs of surface patches. The surface patches are encoded with the 3D Zernike descriptors. Unlike the existing methods which compare global characteristics of the protein fold or the global pocket shape, the local surface patch method can find functional similarity between non-homologous proteins and binding pockets for flexible ligand molecules. The proposed method improves prediction results over global pocket shape-based method which was previously developed by our group.

Binding ligand prediction for proteins using partial matching of local surface patches.

Science.gov (United States)

Sael, Lee; Kihara, Daisuke

2010-01-01

Functional elucidation of uncharacterized protein structures is an important task in bioinformatics. We report our new approach for structure-based function prediction which captures local surface features of ligand binding pockets. Function of proteins, specifically, binding ligands of proteins, can be predicted by finding similar local surface regions of known proteins. To enable partial comparison of binding sites in proteins, a weighted bipartite matching algorithm is used to match pairs of surface patches. The surface patches are encoded with the 3D Zernike descriptors. Unlike the existing methods which compare global characteristics of the protein fold or the global pocket shape, the local surface patch method can find functional similarity between non-homologous proteins and binding pockets for flexible ligand molecules. The proposed method improves prediction results over global pocket shape-based method which was previously developed by our group.

PSOVina: The hybrid particle swarm optimization algorithm for protein-ligand docking.

Science.gov (United States)

Ng, Marcus C K; Fong, Simon; Siu, Shirley W I

2015-06-01

Protein-ligand docking is an essential step in modern drug discovery process. The challenge here is to accurately predict and efficiently optimize the position and orientation of ligands in the binding pocket of a target protein. In this paper, we present a new method called PSOVina which combined the particle swarm optimization (PSO) algorithm with the efficient Broyden-Fletcher-Goldfarb-Shannon (BFGS) local search method adopted in AutoDock Vina to tackle the conformational search problem in docking. Using a diverse data set of 201 protein-ligand complexes from the PDBbind database and a full set of ligands and decoys for four representative targets from the directory of useful decoys (DUD) virtual screening data set, we assessed the docking performance of PSOVina in comparison to the original Vina program. Our results showed that PSOVina achieves a remarkable execution time reduction of 51-60% without compromising the prediction accuracies in the docking and virtual screening experiments. This improvement in time efficiency makes PSOVina a better choice of a docking tool in large-scale protein-ligand docking applications. Our work lays the foundation for the future development of swarm-based algorithms in molecular docking programs. PSOVina is freely available to non-commercial users at http://cbbio.cis.umac.mo .

Understanding the Selectivity Mechanism of the Human Asialoglycoprotein Receptor (ASGP-R toward Gal- and Man- type Ligands for Predicting Interactions with Exogenous Sugars

Directory of Open Access Journals (Sweden)

Emo Chiellini

2007-01-01

Full Text Available A practical approach for addressing the computer simulation of protein-carbohydrate interactions is described here. An articulated computational protocol was setup and validated by checking its ability to predict experimental data, available in theliterature, and concerning the selectivity shown by the Carbohydrate Recognition Domain(CRD of the human asialoglycoprotein receptor (ASGP-R toward Gal-type ligands. Somerequired features responsible for the interactions were identified. Subsequently the sameprotocol was applied to monomer sugar molecules that constitute the building blocks foralginates and ulvans. Such sugar polymers may supply a low-cost source of rare sugars witha potential impact on several industrial applications, from pharmaceutical to fine chemicalindustry. An example of their applicative exploitation could be given by their use indeveloping biomaterial with adhesion properties toward hepatocytes, through interactionwith the ASGP-R. Such a receptor has been already proposed as a target for exogenousmolecules, specifically in the case of hepatocytes, for diagnostic and therapeutic purposes.The DOCK5.2 program was used to search optimal locations of the above ligands of interestinto CRD binding site and to roughly estimate interaction energies. Finally, the binding ∆G oftheoretical protein-ligand complexes was estimated by using the DelPhi program in which thesolvation free energy is accounted for with a continuum solvent model, by solving the Poisson-Boltzmann equation. The structure analysis of the obtained complexes and their ∆G values suggest that one of the sugar monomers of interest shows the desired characteristics.

A molecular dynamics investigation of CDK8/CycC and ligand binding: conformational flexibility and implication in drug discovery

Science.gov (United States)

Cholko, Timothy; Chen, Wei; Tang, Zhiye; Chang, Chia-en A.

2018-05-01

Abnormal activity of cyclin-dependent kinase 8 (CDK8) along with its partner protein cyclin C (CycC) is a common feature of many diseases including colorectal cancer. Using molecular dynamics (MD) simulations, this study determined the dynamics of the CDK8-CycC system and we obtained detailed breakdowns of binding energy contributions for four type-I and five type-II CDK8 inhibitors. We revealed system motions and conformational changes that will affect ligand binding, confirmed the essentialness of CycC for inclusion in future computational studies, and provide guidance in development of CDK8 binders. We employed unbiased all-atom MD simulations for 500 ns on twelve CDK8-CycC systems, including apoproteins and protein-ligand complexes, then performed principal component analysis (PCA) and measured the RMSF of key regions to identify protein dynamics. Binding pocket volume analysis identified conformational changes that accompany ligand binding. Next, H-bond analysis, residue-wise interaction calculations, and MM/PBSA were performed to characterize protein-ligand interactions and find the binding energy. We discovered that CycC is vital for maintaining a proper conformation of CDK8 to facilitate ligand binding and that the system exhibits motion that should be carefully considered in future computational work. Surprisingly, we found that motion of the activation loop did not affect ligand binding. Type-I and type-II ligand binding is driven by van der Waals interactions, but electrostatic energy and entropic penalties affect type-II binding as well. Binding of both ligand types affects protein flexibility. Based on this we provide suggestions for development of tighter-binding CDK8 inhibitors and offer insight that can aid future computational studies.

Ligand-free, protein-bound technetium-99m. Evidence for tumour localisation

International Nuclear Information System (INIS)

Jakovljevic, A.C.; Pojer, P.M.

1984-11-01

An hypothesis that cations accumulate in tumours independent of ligand is tested. A preparation of technetium-99m known to be ligand-free (that is, the technetium is protein bound and no other ligand is injected) has been shown to accumulate in a T-cell lymphoma

DOCLASP - Docking ligands to target proteins using spatial and electrostatic congruence extracted from a known holoenzyme and applying simple geometrical transformations.

Science.gov (United States)

Chakraborty, Sandeep

2014-01-01

The ability to accurately and effectively predict the interaction between proteins and small drug-like compounds has long intrigued researchers for pedagogic, humanitarian and economic reasons. Protein docking methods (AutoDock, GOLD, DOCK, FlexX and Glide to name a few) rank a large number of possible conformations of protein-ligand complexes using fast algorithms. Previously, it has been shown that structural congruence leading to the same enzymatic function necessitates the congruence of electrostatic properties (CLASP). The current work presents a methodology for docking a ligand into a target protein, provided that there is at least one known holoenzyme with ligand bound - DOCLASP (Docking using CLASP). The contact points of the ligand in the holoenzyme defines a motif, which is used to query the target enzyme using CLASP. If there are significant matches, the holoenzyme and the target protein are superimposed based on congruent atoms. The same linear and rotational transformations are also applied to the ligand, thus creating a unified coordinate framework having the holoenzyme, the ligand and the target enzyme. In the current work, the dipeptidyl peptidase-IV inhibitor vildagliptin was docked to the PI-PLC structure complexed with myo-inositol using DOCLASP. Also, corroboration of the docking of phenylthiourea to the modelled structure of polyphenol oxidase (JrPPO1) from walnut is provided based on the subsequently solved structure of JrPPO1 (PDBid:5CE9). Analysis of the binding of the antitrypanosomial drug suramin to nine non-homologous proteins in the PDB database shows a diverse set of binding motifs, and multiple binding sites in the phospholipase A2-likeproteins from the Bothrops genus of pitvipers. The conformational changes in the suramin molecule on binding highlights the challenges in docking flexible ligands into an already 'plastic' binding site. Thus, DOCLASP presents a method for 'soft docking' ligands to proteins with low computational

Optimizing the protein switch: altering nuclear import and export signals, and ligand binding domain

Science.gov (United States)

Kakar, Mudit; Davis, James R.; Kern, Steve E.; Lim, Carol S.

2007-01-01

Ligand regulated localization controllable protein constructs were optimized in this study. Several constructs were made from a classical nuclear export signal (HIV-rev, MAPKK, or progesterone receptor) in combination with a SV40 T-antigen type nuclear import signal. Different ligand binding domains (LBDs from glucocorticoid receptor or progesterone receptor) were also tested for their ability to impart control over localization of proteins. This study was designed to create constructs which are cytoplasmic in the absence of ligand and nuclear in the presence of ligand, and also to regulate the amount of protein translocating to the nucleus on ligand induction. The balance between the strengths of import and export signals was critical for overall localization of proteins. The amount of protein entering the nucleus was also affected by the dose of ligand (10-100nM). However, the overall import characteristics were determined by the strengths of localization signals and the inherent localization properties of the LBD used. This study established that the amount of protein present in a particular compartment can be regulated by the use of localization signals of various strengths. These optimized localization controllable protein constructs can be used to correct for diseases due to aberrant localization of proteins. PMID:17574289

Interaction between alkaline earth cations and oxo-ligands. DFT study of the affinity of the Ca2+ cation for carbonyl ligands.

Science.gov (United States)

da Costa, Leonardo Moreira; Carneiro, José Walkimar de Mesquita; Romeiro, Gilberto Alves; Paes, Lilian Weitzel Coelho

2011-02-01

The affinity of the Ca(2+) ion for a set of substituted carbonyl ligands was analyzed with both the DFT (B3LYP/6-31+G(d)) and semi-empirical (PM6) methods. Two types of ligands were studied: a set of monosubstituted [O=CH(R)] and a set of disubstituted ligands [O=C(R)(2)] (R=H, F, Cl, Br, OH, OCH(3), CH(3), CN, NH(2) and NO(2)), with R either directly bound to the carbonyl carbon atom or to the para position of a phenyl ring. The interaction energy was calculated to quantify the affinity of the Ca(2+) cation for the ligands. Geometric and electronic parameters were correlated with the intensity of the metal-ligand interaction. The electronic nature of the substituent is the main parameter that determines the interaction energy. Donor groups make the interaction energy more negative (stabilizing the complex formed), while acceptor groups make the interaction energy less negative (destabilizing the complex formed).

The utilization of BSA-modified chip on the investigation of ligand ...

African Journals Online (AJOL)

Administrator

2009-12-15

Dec 15, 2009 ... investigation of ligand/protein interaction with surface plasma resonance ... for immobilizing proteins or low-molecular-weight ligands to dextran ..... contamination in dynamic aqueous environments using optical sensors. Anal.

Crystal-contact engineering to obtain a crystal form of the Kelch domain of human Keap1 suitable for ligand-soaking experiments

International Nuclear Information System (INIS)

Hörer, Stefan; Reinert, Dirk; Ostmann, Katja; Hoevels, Yvette; Nar, Herbert

2013-01-01

A mutant of the Kelch domain of the human Keap1 protein has been designed in order to enable the soaking of small-molecule ligands. The apo structure of this mutant is reported at 1.98 Å resolution and the suitability of the crystal system has been demonstrated by the structure of the mutated Keap1 Kelch domain in complex with a cyclic peptide derived from Nrf2. Keap1 is a substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex and plays an important role in the cellular response to oxidative stress. It binds Nrf2 with its Kelch domain and thus triggers the ubiquitinylation and degradation of Nrf2. Oxidative stress prevents the degradation of Nrf2 and leads to the activation of cytoprotective genes. Therefore, Keap1 is an attractive drug target in inflammatory diseases. The support of a medicinal chemistry effort by structural research requires a robust crystallization system in which the crystals are preferably suited for performing soaking experiments. This facilitates the generation of protein–ligand complexes in a routine and high-throughput manner. The structure of human Keap1 has been described previously. In this crystal form, however, the binding site for Nrf2 was blocked by a crystal contact. This interaction was analysed and mutations were introduced to disrupt this crystal contact. One double mutation (E540A/E542A) crystallized in a new crystal form in which the binding site for Nrf2 was not blocked and was accessible to small-molecule ligands. The crystal structures of the apo form of the mutated Keap1 Kelch domain (1.98 Å resolution) and of the complex with an Nrf2-derived peptide obtained by soaking (2.20 Å resolution) are reported

Energetics of the protein-DNA-water interaction

Directory of Open Access Journals (Sweden)

Marabotti Anna

2007-01-01

Full Text Available Abstract Background To understand the energetics of the interaction between protein and DNA we analyzed 39 crystallographically characterized complexes with the HINT (Hydropathic INTeractions computational model. HINT is an empirical free energy force field based on solvent partitioning of small molecules between water and 1-octanol. Our previous studies on protein-ligand complexes demonstrated that free energy predictions were significantly improved by taking into account the energetic contribution of water molecules that form at least one hydrogen bond with each interacting species. Results An initial correlation between the calculated HINT scores and the experimentally determined binding free energies in the protein-DNA system exhibited a relatively poor r2 of 0.21 and standard error of ± 1.71 kcal mol-1. However, the inclusion of 261 waters that bridge protein and DNA improved the HINT score-free energy correlation to an r2 of 0.56 and standard error of ± 1.28 kcal mol-1. Analysis of the water role and energy contributions indicate that 46% of the bridging waters act as linkers between amino acids and nucleotide bases at the protein-DNA interface, while the remaining 54% are largely involved in screening unfavorable electrostatic contacts. Conclusion This study quantifies the key energetic role of bridging waters in protein-DNA associations. In addition, the relevant role of hydrophobic interactions and entropy in driving protein-DNA association is indicated by analyses of interaction character showing that, together, the favorable polar and unfavorable polar/hydrophobic-polar interactions (i.e., desolvation mostly cancel.

SCOWLP: a web-based database for detailed characterization and visualization of protein interfaces

Directory of Open Access Journals (Sweden)

Schroeder Michael

2006-03-01

Full Text Available Abstract Background Currently there is a strong need for methods that help to obtain an accurate description of protein interfaces in order to be able to understand the principles that govern molecular recognition and protein function. Many of the recent efforts to computationally identify and characterize protein networks extract protein interaction information at atomic resolution from the PDB. However, they pay none or little attention to small protein ligands and solvent. They are key components and mediators of protein interactions and fundamental for a complete description of protein interfaces. Interactome profiling requires the development of computational tools to extract and analyze protein-protein, protein-ligand and detailed solvent interaction information from the PDB in an automatic and comparative fashion. Adding this information to the existing one on protein-protein interactions will allow us to better understand protein interaction networks and protein function. Description SCOWLP (Structural Characterization Of Water, Ligands and Proteins is a user-friendly and publicly accessible web-based relational database for detailed characterization and visualization of the PDB protein interfaces. The SCOWLP database includes proteins, peptidic-ligands and interface water molecules as descriptors of protein interfaces. It contains currently 74,907 protein interfaces and 2,093,976 residue-residue interactions formed by 60,664 structural units (protein domains and peptidic-ligands and their interacting solvent. The SCOWLP web-server allows detailed structural analysis and comparisons of protein interfaces at atomic level by text query of PDB codes and/or by navigating a SCOP-based tree. It includes a visualization tool to interactively display the interfaces and label interacting residues and interface solvent by atomic physicochemical properties. SCOWLP is automatically updated with every SCOP release. Conclusion SCOWLP enriches

Application of virus-like particles (VLP) to NMR characterization of viral membrane protein interactions

Energy Technology Data Exchange (ETDEWEB)

Antanasijevic, Aleksandar; Kingsley, Carolyn [University of Illinois at Chicago, Department of Biochemistry and Molecular Genetics (United States); Basu, Arnab; Bowlin, Terry L. [Microbiotix Inc. (United States); Rong, Lijun [University of Illinois at Chicago, Department of Microbiology and Immunology (United States); Caffrey, Michael, E-mail: caffrey@uic.edu [University of Illinois at Chicago, Department of Biochemistry and Molecular Genetics (United States)

2016-03-15

The membrane proteins of viruses play critical roles in the virus life cycle and are attractive targets for therapeutic intervention. Virus-like particles (VLP) present the possibility to study the biochemical and biophysical properties of viral membrane proteins in their native environment. Specifically, the VLP constructs contain the entire protein sequence and are comprised of native membrane components including lipids, cholesterol, carbohydrates and cellular proteins. In this study we prepare VLP containing full-length hemagglutinin (HA) or neuraminidase (NA) from influenza and characterize their interactions with small molecule inhibitors. Using HA-VLP, we first show that VLP samples prepared using the standard sucrose gradient purification scheme contain significant amounts of serum proteins, which exhibit high potential for non-specific interactions, thereby complicating NMR studies of ligand-target interactions. We then show that the serum contaminants may be largely removed with the addition of a gel filtration chromatography step. Next, using HA-VLP we demonstrate that WaterLOGSY NMR is significantly more sensitive than Saturation Transfer Difference (STD) NMR for the study of ligand interactions with membrane bound targets. In addition, we compare the ligand orientation to HA embedded in VLP with that of recombinant HA by STD NMR. In a subsequent step, using NA-VLP we characterize the kinetic and binding properties of substrate analogs and inhibitors of NA, including study of the H274Y-NA mutant, which leads to wide spread resistance to current influenza antivirals. In summary, our work suggests that VLP have high potential to become standard tools in biochemical and biophysical studies of viral membrane proteins, particularly when VLP are highly purified and combined with control VLP containing native membrane proteins.

Crystal structure of LGR4-Rspo1 complex: insights into the divergent mechanisms of ligand recognition by leucine-rich repeat G-protein-coupled receptors (LGRs).

Science.gov (United States)

Xu, Jin-Gen; Huang, Chunfeng; Yang, Zhengfeng; Jin, Mengmeng; Fu, Panhan; Zhang, Ni; Luo, Jian; Li, Dali; Liu, Mingyao; Zhou, Yan; Zhu, Yongqun

2015-01-23

Leucine-rich repeat G-protein-coupled receptors (LGRs) are a unique class of G-protein-coupled receptors characterized by a large extracellular domain to recognize ligands and regulate many important developmental processes. Among the three groups of LGRs, group B members (LGR4-6) recognize R-spondin family proteins (Rspo1-4) to stimulate Wnt signaling. In this study, we successfully utilized the "hybrid leucine-rich repeat technique," which fused LGR4 with the hagfish VLR protein, to obtain two recombinant human LGR4 proteins, LGR415 and LGR49. We determined the crystal structures of ligand-free LGR415 and the LGR49-Rspo1 complex. LGR4 exhibits a twisted horseshoe-like structure. Rspo1 adopts a flat and β-fold architecture and is bound in the concave surface of LGR4 in the complex through electrostatic and hydrophobic interactions. All the Rspo1-binding residues are conserved in LGR4-6, suggesting that LGR4-6 bind R-spondins through an identical surface. Structural analysis of our LGR4-Rspo1 complex with the previously determined LGR4 and LGR5 structures revealed that the concave surface of LGR4 is the sole binding site for R-spondins, suggesting a one-site binding model of LGR4-6 in ligand recognition. The molecular mechanism of LGR4-6 is distinct from the two-step mechanism of group A receptors LGR1-3 and the multiple-interface binding model of group C receptors LGR7-8, suggesting LGRs utilize the divergent mechanisms for ligand recognition. Our structures, together with previous reports, provide a comprehensive understanding of the ligand recognition by LGRs. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Bioluminescence resonance energy transfer system for measuring dynamic protein-protein interactions in bacteria.

Science.gov (United States)

Cui, Boyu; Wang, Yao; Song, Yunhong; Wang, Tietao; Li, Changfu; Wei, Yahong; Luo, Zhao-Qing; Shen, Xihui

2014-05-20

Protein-protein interactions are important for virtually every biological process, and a number of elegant approaches have been designed to detect and evaluate such interactions. However, few of these methods allow the detection of dynamic and real-time protein-protein interactions in bacteria. Here we describe a bioluminescence resonance energy transfer (BRET) system based on the bacterial luciferase LuxAB. We found that enhanced yellow fluorescent protein (eYFP) accepts the emission from LuxAB and emits yellow fluorescence. Importantly, BRET occurred when LuxAB and eYFP were fused, respectively, to the interacting protein pair FlgM and FliA. Furthermore, we observed sirolimus (i.e., rapamycin)-inducible interactions between FRB and FKBP12 and a dose-dependent abolishment of such interactions by FK506, the ligand of FKBP12. Using this system, we showed that osmotic stress or low pH efficiently induced multimerization of the regulatory protein OmpR and that the multimerization induced by low pH can be reversed by a neutralizing agent, further indicating the usefulness of this system in the measurement of dynamic interactions. This method can be adapted to analyze dynamic protein-protein interactions and the importance of such interactions in bacterial processes such as development and pathogenicity. Real-time measurement of protein-protein interactions in prokaryotes is highly desirable for determining the roles of protein complex in the development or virulence of bacteria, but methods that allow such measurement are not available. Here we describe the development of a bioluminescence resonance energy transfer (BRET) technology that meets this need. The use of endogenous excitation light in this strategy circumvents the requirement for the sophisticated instrument demanded by standard fluorescence resonance energy transfer (FRET). Furthermore, because the LuxAB substrate decanal is membrane permeable, the assay can be performed without lysing the bacterial cells

Locating binding poses in protein-ligand systems using reconnaissance metadynamics

Science.gov (United States)

Söderhjelm, Pär; Tribello, Gareth A.; Parrinello, Michele

2012-01-01

A molecular dynamics-based protocol is proposed for finding and scoring protein-ligand binding poses. This protocol uses the recently developed reconnaissance metadynamics method, which employs a self-learning algorithm to construct a bias that pushes the system away from the kinetic traps where it would otherwise remain. The exploration of phase space with this algorithm is shown to be roughly six to eight times faster than unbiased molecular dynamics and is only limited by the time taken to diffuse about the surface of the protein. We apply this method to the well-studied trypsin–benzamidine system and show that we are able to refind all the poses obtained from a reference EADock blind docking calculation. These poses can be scored based on the length of time the system remains trapped in the pose. Alternatively, one can perform dimensionality reduction on the output trajectory and obtain a map of phase space that can be used in more expensive free-energy calculations. PMID:22440749

Bystander protein protects potential vaccine-targeting ligands against intestinal proteolysis.

Science.gov (United States)

Reuter, Fabian; Bade, Steffen; Hirst, Timothy R; Frey, Andreas

2009-07-20

Endowing mucosal vaccines with ligands that target antigen to mucosal lymphoid tissues may improve immunization efficacy provided that the ligands withstand the proteolytic environment of the gastro-intestinal tract until they reach their destination. Our aim was to investigate whether and how three renowned ligands - Ulex europaeus agglutinin I and the B subunits of cholera toxin and E. coli heat-labile enterotoxin - master this challenge. We assessed the digestive power of natural murine intestinal fluid (natIF) using assays for trypsin, chymotrypsin and pancreatic elastase along with a test for nonspecific proteolysis. The natIF was compared with simulated murine intestinal fluid (simIF) that resembled the trypsin, chymotrypsin and elastase activities of its natural counterpart but lacked or contained albumins as additional protease substrates. The ligands were exposed to the digestive fluids and degradation was determined. The studies revealed that (i) the three pancreatic endoproteases constitute only one third of the total protease activity of natIF and (ii) the ligands resist proteolysis in natIF and protein-enriched simIF over 3 h but (iii) are partially destroyed in simIF that lacks additional protease substrate. We assume that the proteins of natIF are preferred substrates for the intestinal proteases and thus can protect vaccine-targeting ligands from destruction.

A control on hydrophobic and hydrophilic interactions between HEWL and metal Schiff-base complexes comprising of different metal ions and ligands

Energy Technology Data Exchange (ETDEWEB)

Koley Seth, Banabithi; Ray, Aurkie; Basu, Samita, E-mail: samita.basu@saha.ac.in

2015-05-15

The structural effects of different copper(II) and nickel(II) Schiff base complexes on hen egg white lysozyme (HEWL) have been investigated through steady state and time resolved absorption and fluorescence, and circular dichroism spectroscopy. The Schiff base ligands with N{sub 4} donor atoms show both hydrophobic and hydrophilic interactions, however hydrophilic interaction prevails with ligands having N{sub 2}O{sub 2} donor atoms. Variation of metal ions from Cu{sup 2+} to Ni{sup 2+} with each type of Schiff base ligand increases the probability of hydrophilic over hydrophobic interactions, which supports their significance in regulating the binding affinity between HEWL and metal complexes. On photo-excitation the complexes comprising of Cu{sup 2+} ion instead of Ni{sup 2+} ion and ligands with N{sub 4} donor system rather than N{sub 2}O{sub 2} donor system, increases the probability of intersystem crossing to populate the corresponding triplet state as observed from laser flash photolysis study. The better binding affinity of nickel complexes with different selectivities compared to copper complexes towards HEWL emphasizes the potentiality of less explored nickel complexes in drug–protein interactions. - Highlights: • Ni{sup II} and Cu{sup II} -Schiff base complexes bind hen egg white lysozyme spontaneously. • Both hydrophobic and hydrophilic interactions are effective for N{sub 4} ligands. • For N{sub 2}O{sub 2} ligands the hydrophilic is predominant over hydrophobic interaction. • Binding affinity and selectivity of Ni{sup II}-complexes are better than Cu{sup II}-complexes. • Replacement of Cu{sup 2+} by Ni{sup 2+} in a ligand enhances chance of hydrophilic interaction.

On the analysis and comparison of conformer-specific essential dynamics upon ligand binding to a protein

International Nuclear Information System (INIS)

Grosso, Marcos; Kalstein, Adrian; Parisi, Gustavo; Fernandez-Alberti, Sebastian; Roitberg, Adrian E.

2015-01-01

The native state of a protein consists of an equilibrium of conformational states on an energy landscape rather than existing as a single static state. The co-existence of conformers with different ligand-affinities in a dynamical equilibrium is the basis for the conformational selection model for ligand binding. In this context, the development of theoretical methods that allow us to analyze not only the structural changes but also changes in the fluctuation patterns between conformers will contribute to elucidate the differential properties acquired upon ligand binding. Molecular dynamics simulations can provide the required information to explore these features. Its use in combination with subsequent essential dynamics analysis allows separating large concerted conformational rearrangements from irrelevant fluctuations. We present a novel procedure to define the size and composition of essential dynamics subspaces associated with ligand-bound and ligand-free conformations. These definitions allow us to compare essential dynamics subspaces between different conformers. Our procedure attempts to emphasize the main similarities and differences between the different essential dynamics in an unbiased way. Essential dynamics subspaces associated to conformational transitions can also be analyzed. As a test case, we study the glutaminase interacting protein (GIP), composed of a single PDZ domain. Both GIP ligand-free state and glutaminase L peptide-bound states are analyzed. Our findings concerning the relative changes in the flexibility pattern upon binding are in good agreement with experimental Nuclear Magnetic Resonance data

On the analysis and comparison of conformer-specific essential dynamics upon ligand binding to a protein

Energy Technology Data Exchange (ETDEWEB)

Grosso, Marcos; Kalstein, Adrian; Parisi, Gustavo; Fernandez-Alberti, Sebastian, E-mail: sfalberti@gmail.com [Universidad Nacional de Quilmes, Roque Saenz Peña 352, B1876BXD Bernal (Argentina); Roitberg, Adrian E. [Departments of Physics and Chemistry, University of Florida, Gainesville, Florida 32611 (United States)

2015-06-28

The native state of a protein consists of an equilibrium of conformational states on an energy landscape rather than existing as a single static state. The co-existence of conformers with different ligand-affinities in a dynamical equilibrium is the basis for the conformational selection model for ligand binding. In this context, the development of theoretical methods that allow us to analyze not only the structural changes but also changes in the fluctuation patterns between conformers will contribute to elucidate the differential properties acquired upon ligand binding. Molecular dynamics simulations can provide the required information to explore these features. Its use in combination with subsequent essential dynamics analysis allows separating large concerted conformational rearrangements from irrelevant fluctuations. We present a novel procedure to define the size and composition of essential dynamics subspaces associated with ligand-bound and ligand-free conformations. These definitions allow us to compare essential dynamics subspaces between different conformers. Our procedure attempts to emphasize the main similarities and differences between the different essential dynamics in an unbiased way. Essential dynamics subspaces associated to conformational transitions can also be analyzed. As a test case, we study the glutaminase interacting protein (GIP), composed of a single PDZ domain. Both GIP ligand-free state and glutaminase L peptide-bound states are analyzed. Our findings concerning the relative changes in the flexibility pattern upon binding are in good agreement with experimental Nuclear Magnetic Resonance data.

istar: a web platform for large-scale protein-ligand docking.

Directory of Open Access Journals (Sweden)

Hongjian Li

Full Text Available Protein-ligand docking is a key computational method in the design of starting points for the drug discovery process. We are motivated by the desire to automate large-scale docking using our popular docking engine idock and thus have developed a publicly-accessible web platform called istar. Without tedious software installation, users can submit jobs using our website. Our istar website supports 1 filtering ligands by desired molecular properties and previewing the number of ligands to dock, 2 monitoring job progress in real time, and 3 visualizing ligand conformations and outputting free energy and ligand efficiency predicted by idock, binding affinity predicted by RF-Score, putative hydrogen bonds, and supplier information for easy purchase, three useful features commonly lacked on other online docking platforms like DOCK Blaster or iScreen. We have collected 17,224,424 ligands from the All Clean subset of the ZINC database, and revamped our docking engine idock to version 2.0, further improving docking speed and accuracy, and integrating RF-Score as an alternative rescoring function. To compare idock 2.0 with the state-of-the-art AutoDock Vina 1.1.2, we have carried out a rescoring benchmark and a redocking benchmark on the 2,897 and 343 protein-ligand complexes of PDBbind v2012 refined set and CSAR NRC HiQ Set 24Sept2010 respectively, and an execution time benchmark on 12 diverse proteins and 3,000 ligands of different molecular weight. Results show that, under various scenarios, idock achieves comparable success rates while outperforming AutoDock Vina in terms of docking speed by at least 8.69 times and at most 37.51 times. When evaluated on the PDBbind v2012 core set, our istar platform combining with RF-Score manages to reproduce Pearson's correlation coefficient and Spearman's correlation coefficient of as high as 0.855 and 0.859 respectively between the experimental binding affinity and the predicted binding affinity of the docked

istar: a web platform for large-scale protein-ligand docking.

Science.gov (United States)

Li, Hongjian; Leung, Kwong-Sak; Ballester, Pedro J; Wong, Man-Hon

2014-01-01

Protein-ligand docking is a key computational method in the design of starting points for the drug discovery process. We are motivated by the desire to automate large-scale docking using our popular docking engine idock and thus have developed a publicly-accessible web platform called istar. Without tedious software installation, users can submit jobs using our website. Our istar website supports 1) filtering ligands by desired molecular properties and previewing the number of ligands to dock, 2) monitoring job progress in real time, and 3) visualizing ligand conformations and outputting free energy and ligand efficiency predicted by idock, binding affinity predicted by RF-Score, putative hydrogen bonds, and supplier information for easy purchase, three useful features commonly lacked on other online docking platforms like DOCK Blaster or iScreen. We have collected 17,224,424 ligands from the All Clean subset of the ZINC database, and revamped our docking engine idock to version 2.0, further improving docking speed and accuracy, and integrating RF-Score as an alternative rescoring function. To compare idock 2.0 with the state-of-the-art AutoDock Vina 1.1.2, we have carried out a rescoring benchmark and a redocking benchmark on the 2,897 and 343 protein-ligand complexes of PDBbind v2012 refined set and CSAR NRC HiQ Set 24Sept2010 respectively, and an execution time benchmark on 12 diverse proteins and 3,000 ligands of different molecular weight. Results show that, under various scenarios, idock achieves comparable success rates while outperforming AutoDock Vina in terms of docking speed by at least 8.69 times and at most 37.51 times. When evaluated on the PDBbind v2012 core set, our istar platform combining with RF-Score manages to reproduce Pearson's correlation coefficient and Spearman's correlation coefficient of as high as 0.855 and 0.859 respectively between the experimental binding affinity and the predicted binding affinity of the docked conformation. istar

A multi-protein receptor-ligand complex underlies combinatorial dendrite guidance choices in C. elegans

Science.gov (United States)

Zou, Wei; Shen, Ao; Dong, Xintong; Tugizova, Madina; Xiang, Yang K; Shen, Kang

2016-01-01

Ligand receptor interactions instruct axon guidance during development. How dendrites are guided to specific targets is less understood. The C. elegans PVD sensory neuron innervates muscle-skin interface with its elaborate dendritic branches. Here, we found that LECT-2, the ortholog of leukocyte cell-derived chemotaxin-2 (LECT2), is secreted from the muscles and required for muscle innervation by PVD. Mosaic analyses showed that LECT-2 acted locally to guide the growth of terminal branches. Ectopic expression of LECT-2 from seam cells is sufficient to redirect the PVD dendrites onto seam cells. LECT-2 functions in a multi-protein receptor-ligand complex that also contains two transmembrane ligands on the skin, SAX-7/L1CAM and MNR-1, and the neuronal transmembrane receptor DMA-1. LECT-2 greatly enhances the binding between SAX-7, MNR-1 and DMA-1. The activation of DMA-1 strictly requires all three ligands, which establishes a combinatorial code to precisely target and pattern dendritic arbors. DOI: http://dx.doi.org/10.7554/eLife.18345.001 PMID:27705746

Structure-wise discrimination of adenine and guanine by proteins on the basis of their nonbonded interactions.

Science.gov (United States)

Usha, S; Selvaraj, S

2015-01-01

We have analyzed the nonbonded interactions of the structurally similar moieties, adenine and guanine forming complexes with proteins. The results comprise (a) the amino acid-ligand atom preferences, (b) solvent accessibility of ligand atoms before and after complex formation with proteins, and (c) preferred amino acid residue atoms involved in the interactions. We have observed that the amino acid preferences involved in the hydrogen bonding interactions vary for adenine and guanine. The structural variation between the purine atoms is clearly reflected by their burial tendency in the solvent environment. Correlation of the mean amino acid preference values show the variation that exists between adenine and guanine preferences of all the amino acid residues. All our observations provide evidence for the discriminating nature of the proteins in recognizing adenine and guanine.

G-LoSA for Prediction of Protein-Ligand Binding Sites and Structures.

Science.gov (United States)

Lee, Hui Sun; Im, Wonpil

2017-01-01

Recent advances in high-throughput structure determination and computational protein structure prediction have significantly enriched the universe of protein structure. However, there is still a large gap between the number of available protein structures and that of proteins with annotated function in high accuracy. Computational structure-based protein function prediction has emerged to reduce this knowledge gap. The identification of a ligand binding site and its structure is critical to the determination of a protein's molecular function. We present a computational methodology for predicting small molecule ligand binding site and ligand structure using G-LoSA, our protein local structure alignment and similarity measurement tool. All the computational procedures described here can be easily implemented using G-LoSA Toolkit, a package of standalone software programs and preprocessed PDB structure libraries. G-LoSA and G-LoSA Toolkit are freely available to academic users at http://compbio.lehigh.edu/GLoSA . We also illustrate a case study to show the potential of our template-based approach harnessing G-LoSA for protein function prediction.

LigSearch: a knowledge-based web server to identify likely ligands for a protein target

Energy Technology Data Exchange (ETDEWEB)

Beer, Tjaart A. P. de; Laskowski, Roman A. [European Bioinformatics Institute (EMBL–EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD (United Kingdom); Duban, Mark-Eugene [Northwestern University Feinberg School of Medicine, Chicago, Illinois (United States); Chan, A. W. Edith [University College London, London WC1E 6BT (United Kingdom); Anderson, Wayne F. [Northwestern University Feinberg School of Medicine, Chicago, Illinois (United States); Thornton, Janet M., E-mail: thornton@ebi.ac.uk [European Bioinformatics Institute (EMBL–EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD (United Kingdom)

2013-12-01

LigSearch is a web server for identifying ligands likely to bind to a given protein. Identifying which ligands might bind to a protein before crystallization trials could provide a significant saving in time and resources. LigSearch, a web server aimed at predicting ligands that might bind to and stabilize a given protein, has been developed. Using a protein sequence and/or structure, the system searches against a variety of databases, combining available knowledge, and provides a clustered and ranked output of possible ligands. LigSearch can be accessed at http://www.ebi.ac.uk/thornton-srv/databases/LigSearch.

Ligand Binding Domain Protein in Tetracycline-Inducible Expression ...

African Journals Online (AJOL)

binding domain proteins in E. coli using a tetracycline inducible system. To allow for ... development of molecular ligands with improved therapeutic windows. Keywords: Nuclear receptor ..... functional recombinant cannabinoid receptor CB2 in ...

Database of ligand-induced domain movements in enzymes

Directory of Open Access Journals (Sweden)

Hayward Steven

2009-03-01

Full Text Available Abstract Background Conformational change induced by the binding of a substrate or coenzyme is a poorly understood stage in the process of enzyme catalysed reactions. For enzymes that exhibit a domain movement, the conformational change can be clearly characterized and therefore the opportunity exists to gain an understanding of the mechanisms involved. The development of the non-redundant database of protein domain movements contains examples of ligand-induced domain movements in enzymes, but this valuable data has remained unexploited. Description The domain movements in the non-redundant database of protein domain movements are those found by applying the DynDom program to pairs of crystallographic structures contained in Protein Data Bank files. For each pair of structures cross-checking ligands in their Protein Data Bank files with the KEGG-LIGAND database and using methods that search for ligands that contact the enzyme in one conformation but not the other, the non-redundant database of protein domain movements was refined down to a set of 203 enzymes where a domain movement is apparently triggered by the binding of a functional ligand. For these cases, ligand binding information, including hydrogen bonds and salt-bridges between the ligand and specific residues on the enzyme is presented in the context of dynamical information such as the regions that form the dynamic domains, the hinge bending residues, and the hinge axes. Conclusion The presentation at a single website of data on interactions between a ligand and specific residues on the enzyme alongside data on the movement that these interactions induce, should lead to new insights into the mechanisms of these enzymes in particular, and help in trying to understand the general process of ligand-induced domain closure in enzymes. The website can be found at: http://www.cmp.uea.ac.uk/dyndom/enzymeList.do

Ligand-induced dynamics of heterotrimeric G protein-coupled receptor-like kinase complexes.

Directory of Open Access Journals (Sweden)

Meral Tunc-Ozdemir

Full Text Available Arabidopsis, 7-transmembrane Regulator of G signaling protein 1 (AtRGS1 modulates canonical G protein signaling by promoting the inactive state of heterotrimeric G protein complex on the plasma membrane. It is known that plant leucine-rich repeat receptor-like kinases (LRR RLKs phosphorylate AtRGS1 in vitro but little is known about the in vivo interaction, molecular dynamics, or the cellular consequences of this interaction.Therefore, a subset of the known RLKs that phosphorylate AtRGS1 were selected for elucidation, namely, BAK1, BIR1, FLS2. Several microscopies for both static and dynamic protein-protein interactions were used to follow in vivo interactions between the RLKs and AtRGS1 after the presentation of the Pathogen-associated Molecular Pattern, Flagellin 22 (Flg22. These microscopies included Förster Resonance Energy Transfer, Bimolecular Fluoresence Complementation, and Cross Number and Brightness Fluorescence Correlation Spectroscopy. In addition, reactive oxygen species and calcium changes in living cells were quantitated using luminometry and R-GECO1 microscopy.The LRR RLKs BAK1 and BIR1, interact with AtRGS1 at the plasma membrane. The RLK ligand flg22 sets BAK1 in motion toward AtRGS1 and BIR1 away, both returning to the baseline orientations by 10 minutes. The C-terminal tail of AtRGS1 is important for the interaction with BAK1 and for the tempo of the AtRGS1/BIR1 dynamics. This window of time corresponds to the flg22-induced transient production of reactive oxygen species and calcium release which are both attenuated in the rgs1 and the bak1 null mutants.A temporal model of these interactions is proposed. flg22 binding induces nearly instantaneous dimerization between FLS2 and BAK1. Phosphorylated BAK1 interacts with and enables AtRGS1 to move away from BIR1 and AtRGS1 becomes phosphorylated leading to its endocytosis thus leading to de-repression by permitting AtGPA1 to exchange GDP for GTP. Finally, the G protein complex

Converging ligand-binding free energies obtained with free-energy perturbations at the quantum mechanical level.

Science.gov (United States)

Olsson, Martin A; Söderhjelm, Pär; Ryde, Ulf

2016-06-30

In this article, the convergence of quantum mechanical (QM) free-energy simulations based on molecular dynamics simulations at the molecular mechanics (MM) level has been investigated. We have estimated relative free energies for the binding of nine cyclic carboxylate ligands to the octa-acid deep-cavity host, including the host, the ligand, and all water molecules within 4.5 Å of the ligand in the QM calculations (158-224 atoms). We use single-step exponential averaging (ssEA) and the non-Boltzmann Bennett acceptance ratio (NBB) methods to estimate QM/MM free energy with the semi-empirical PM6-DH2X method, both based on interaction energies. We show that ssEA with cumulant expansion gives a better convergence and uses half as many QM calculations as NBB, although the two methods give consistent results. With 720,000 QM calculations per transformation, QM/MM free-energy estimates with a precision of 1 kJ/mol can be obtained for all eight relative energies with ssEA, showing that this approach can be used to calculate converged QM/MM binding free energies for realistic systems and large QM partitions. © 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc. © 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.

Probing the role of backbone hydrogen bonds in protein-peptide interactions by amide-to-ester mutations

DEFF Research Database (Denmark)

Eildal, Jonas N N; Hultqvist, Greta; Balle, Thomas

2013-01-01

-protein interactions, those of the PDZ domain family involve formation of intermolecular hydrogen bonds: C-termini or internal linear motifs of proteins bind as β-strands to form an extended antiparallel β-sheet with the PDZ domain. Whereas extensive work has focused on the importance of the amino acid side chains...... of the protein ligand, the role of the backbone hydrogen bonds in the binding reaction is not known. Using amide-to-ester substitutions to perturb the backbone hydrogen-bonding pattern, we have systematically probed putative backbone hydrogen bonds between four different PDZ domains and peptides corresponding...... to natural protein ligands. Amide-to-ester mutations of the three C-terminal amides of the peptide ligand severely affected the affinity with the PDZ domain, demonstrating that hydrogen bonds contribute significantly to ligand binding (apparent changes in binding energy, ΔΔG = 1.3 to >3.8 kcal mol(-1...

"Precipitation on Nanoparticles": Attractive Intermolecular Interactions Stabilize Specific Ligand Ratios on the Surfaces of Nanoparticles.

Science.gov (United States)

Chu, Zonglin; Han, Yanxiao; Kral, Petr; Klajn, Rafal

2018-04-19

Confining organic molecules to the surfaces of inorganic nanoparticles can induce intermolecular interactions between them, which can affect the composition of the mixed self-assembled monolayers obtained by co-adsorption from solution of two different molecules. Here, we study co-adsorption of two thiolated ligands-a dialkylviologen and a zwitterionic sulfobetaine-that can interact with each other electrostatically, onto gold nanoparticles. Consequently, the nanoparticles favor a narrow range of ratios of these two molecules that is largely independent of the molar ratio in solution. We show that changing the solution molar ratio of two ligands by a factor of ~5,000 affects the on-nanoparticle ratio of these ligands by only 3 times. This behavior is reminiscent of the formation of insoluble inorganic salts (e.g., AgCl), which similarly compensate positive and negative charges upon crystallizing. Our results pave the way towards developing well-defined hybrid organic-inorganic nanostructures. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Simple Ligand–Receptor Interaction Descriptor (SILIRID for alignment-free binding site comparison

Directory of Open Access Journals (Sweden)

Vladimir Chupakhin

2014-06-01

Full Text Available We describe SILIRID (Simple Ligand–Receptor Interaction Descriptor, a novel fixed size descriptor characterizing protein–ligand interactions. SILIRID can be obtained from the binary interaction fingerprints (IFPs by summing up the bits corresponding to identical amino acids. This results in a vector of 168 integer numbers corresponding to the product of the number of entries (20 amino acids and one cofactor and 8 interaction types per amino acid (hydrophobic, aromatic face to face, aromatic edge to face, H-bond donated by the protein, H-bond donated by the ligand, ionic bond with protein cation and protein anion, and interaction with metal ion. Efficiency of SILIRID to distinguish different protein binding sites has been examined in similarity search in sc-PDB database, a druggable portion of the Protein Data Bank, using various protein–ligand complexes as queries. The performance of retrieval of structurally and evolutionary related classes of proteins was comparable to that of state-of-the-art approaches (ROC AUC ≈ 0.91. SILIRID can efficiently be used to visualize chemogenomic space covered by sc-PDB using Generative Topographic Mapping (GTM: sc-PDB SILIRID data form clusters corresponding to different protein types.

Molecular features related to HIV integrase inhibition obtained from structure- and ligand-based approaches.

Directory of Open Access Journals (Sweden)

Luciana L de Carvalho

Full Text Available Among several biological targets to treat AIDS, HIV integrase is a promising enzyme that can be employed to develop new anti-HIV agents. The aim of this work is to propose a mechanistic interpretation of HIV-1 integrase inhibition and to rationalize the molecular features related to the binding affinity of studied ligands. A set of 79 HIV-1 integrase inhibitors and its relationship with biological activity are investigated employing 2D and 3D QSAR models, docking analysis and DFT studies. Analyses of docking poses and frontier molecular orbitals revealed important features on the main ligand-receptor interactions. 2D and 3D models presenting good internal consistency, predictive power and stability were obtained in all cases. Significant correlation coefficients (r(2 = 0.908 and q(2= 0.643 for 2D model; r(2= 0.904 and q(2= 0.719 for 3D model were obtained, indicating the potential of these models for untested compounds. The generated holograms and contribution maps revealed important molecular requirements to HIV-1 IN inhibition and several evidences for molecular modifications. The final models along with information resulting from molecular orbitals, 2D contribution and 3D contour maps should be useful in the design of new inhibitors with increased potency and selectivity within the chemical diversity of the data.

Synthesis, characterization, DNA/protein interaction and cytotoxicity studies of Cu(II) and Co(II) complexes derived from dipyridyl triazole ligands

Science.gov (United States)

Zhang, Wei; Yao, Di; Wei, Yi; Tang, Jie; Bian, He-Dong; Huang, Fu-Ping; Liang, Hong

2016-06-01

Four different transition metal complexes containing dipyridyl triazole ligands, namely [Cu(abpt)2Cl2]·2H2O (1), [Cu(abpt)2(ClO4)2] (2), [Co2(abpt)2(H2O)2Cl2]·Cl2·4H2O (3) and [Co2(Hbpt)2(CH3OH)2(NO3)2] (4) have been designed, synthesized and further structurally characterized by X-ray crystallography, ESI-MS, elemental analysis, IR and Raman spectroscopy. In these complexes, the both ligands act as bidentate ligands with N, N donors. DNA binding interactions with calf thymus DNA (ct-DNA) of the ligand and its complexes 1 ~ 4 were investigated via electronic absorption, fluorescence quenching, circular dichroism and viscosity measurements as well as confocal Laser Raman spectroscopy. The results show these complexes are able to bind to DNA via the non-covalent mode i.e. intercalation and groove binding or electrostatic interactions. The interactions with bovine serum albumin (BSA) were also studied using UV-Vis and fluorescence spectroscopic methods which indicated that fluorescence quenching of BSA by these compounds was the presence of both static and dynamic quenching. Moreover, the in vitro cytotoxic effects of the complexes against four cell lines SK-OV-3, HL-7702, BEL7404 and NCI-H460 showed the necessity of the coordination action on the biological properties on the respective complex and that all four complexes exhibited substantial cytotoxic activity.

Rosetta Ligand docking with flexible XML protocols.

Science.gov (United States)

Lemmon, Gordon; Meiler, Jens

2012-01-01

RosettaLigand is premiere software for predicting how a protein and a small molecule interact. Benchmark studies demonstrate that 70% of the top scoring RosettaLigand predicted interfaces are within 2Å RMSD from the crystal structure [1]. The latest release of Rosetta ligand software includes many new features, such as (1) docking of multiple ligands simultaneously, (2) representing ligands as fragments for greater flexibility, (3) redesign of the interface during docking, and (4) an XML script based interface that gives the user full control of the ligand docking protocol.

ESI-MS studies of the reactions of novel platinum(II) complexes containing O,O'-chelated acetylacetonate and sulfur ligands with selected model proteins.

Science.gov (United States)

Marzo, Tiziano; De Pascali, Sandra A; Gabbiani, Chiara; Fanizzi, Francesco P; Messori, Luigi; Pratesi, Alessandro

2017-08-01

A group of mixed-ligand Pt(II) complexes bearing acetylacetonate and sulphur ligands were recently developed in the University of Lecce as a new class of prospective anticancer agents that manifested promising pharma-cological properties in preliminary in vitro and in vivo tests. Though modelled on the basis of cisplatin, these Pt(II) complexes turned out to exhibit a profoundly distinct mode of action as they were found to act mainly on non-genomic targets rather than on DNA. Accordingly, we have explored here their reactions with two representative model proteins through an established ESI-MS procedure with the aim to describe their general interaction mechanism with protein targets. A pronounced reactivity with the tested proteins was indeed documented; the nature of the resulting metallodrug-protein interactions could be characterised in depth in the various cases. Preferential binding to protein targets compared to DNA is supported by independent ICP-OES measurements. The implications of these findings are discussed.

Structure and ligand-binding properties of the biogenic amine-binding protein from the saliva of a blood-feeding insect vector of Trypanosoma cruzi

Energy Technology Data Exchange (ETDEWEB)

Xu, Xueqing; Chang, Bianca W. [NIH/NIAID, 12735 Twinbrook Parkway, Rockville, MD 20852 (United States); Mans, Ben J. [NIH/NIAID, 12735 Twinbrook Parkway, Rockville, MD 20852 (United States); Agricultural Research Council, Onderstepoort 0110 (South Africa); Ribeiro, Jose M. C.; Andersen, John F., E-mail: jandersen@niaid.nih.gov [NIH/NIAID, 12735 Twinbrook Parkway, Rockville, MD 20852 (United States)

2013-01-01

Biogenic amine-binding proteins mediate the anti-inflammatory and antihemostatic activities of blood-feeding insect saliva. The structure of the amine-binding protein from R. prolixus reveals the interaction of biogenic amine ligands with the protein. Proteins that bind small-molecule mediators of inflammation and hemostasis are essential for blood-feeding by arthropod vectors of infectious disease. In ticks and triatomine insects, the lipocalin protein family is greatly expanded and members have been shown to bind biogenic amines, eicosanoids and ADP. These compounds are potent mediators of platelet activation, inflammation and vascular tone. In this paper, the structure of the amine-binding protein (ABP) from Rhodnius prolixus, a vector of the trypanosome that causes Chagas disease, is described. ABP binds the biogenic amines serotonin and norepinephrine with high affinity. A complex with tryptamine shows the presence of a binding site for a single ligand molecule in the central cavity of the β-barrel structure. The cavity contains significant additional volume, suggesting that this protein may have evolved from the related nitrophorin proteins, which bind a much larger heme ligand in the central cavity.

Structure and ligand-binding properties of the biogenic amine-binding protein from the saliva of a blood-feeding insect vector of Trypanosoma cruzi

International Nuclear Information System (INIS)

Xu, Xueqing; Chang, Bianca W.; Mans, Ben J.; Ribeiro, Jose M. C.; Andersen, John F.

2013-01-01

Biogenic amine-binding proteins mediate the anti-inflammatory and antihemostatic activities of blood-feeding insect saliva. The structure of the amine-binding protein from R. prolixus reveals the interaction of biogenic amine ligands with the protein. Proteins that bind small-molecule mediators of inflammation and hemostasis are essential for blood-feeding by arthropod vectors of infectious disease. In ticks and triatomine insects, the lipocalin protein family is greatly expanded and members have been shown to bind biogenic amines, eicosanoids and ADP. These compounds are potent mediators of platelet activation, inflammation and vascular tone. In this paper, the structure of the amine-binding protein (ABP) from Rhodnius prolixus, a vector of the trypanosome that causes Chagas disease, is described. ABP binds the biogenic amines serotonin and norepinephrine with high affinity. A complex with tryptamine shows the presence of a binding site for a single ligand molecule in the central cavity of the β-barrel structure. The cavity contains significant additional volume, suggesting that this protein may have evolved from the related nitrophorin proteins, which bind a much larger heme ligand in the central cavity

Acetylation of pregnane X receptor protein determines selective function independent of ligand activation

International Nuclear Information System (INIS)

Biswas, Arunima; Pasquel, Danielle; Tyagi, Rakesh Kumar; Mani, Sridhar

2011-01-01

Research highlights: → Pregnane X receptor (PXR), a major regulatory protein, is modified by acetylation. → PXR undergoes dynamic deacetylation upon ligand-mediated activation. → SIRT1 partially mediates PXR deacetylation. → PXR deacetylation per se induces lipogenesis mimicking ligand-mediated activation. -- Abstract: Pregnane X receptor (PXR), like other members of its class of nuclear receptors, undergoes post-translational modification [PTM] (e.g., phosphorylation). However, it is unknown if acetylation (a major and common form of protein PTM) is observed on PXR and, if it is, whether it is of functional consequence. PXR has recently emerged as an important regulatory protein with multiple ligand-dependent functions. In the present work we show that PXR is indeed acetylated in vivo. SIRT1 (Sirtuin 1), a NAD-dependent class III histone deacetylase and a member of the sirtuin family of proteins, partially mediates deacetylation of PXR. Most importantly, the acetylation status of PXR regulates its selective function independent of ligand activation.

Information assessment on predicting protein-protein interactions

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Gerstein Mark

2004-10-01

Full Text Available Abstract Background Identifying protein-protein interactions is fundamental for understanding the molecular machinery of the cell. Proteome-wide studies of protein-protein interactions are of significant value, but the high-throughput experimental technologies suffer from high rates of both false positive and false negative predictions. In addition to high-throughput experimental data, many diverse types of genomic data can help predict protein-protein interactions, such as mRNA expression, localization, essentiality, and functional annotation. Evaluations of the information contributions from different evidences help to establish more parsimonious models with comparable or better prediction accuracy, and to obtain biological insights of the relationships between protein-protein interactions and other genomic information. Results Our assessment is based on the genomic features used in a Bayesian network approach to predict protein-protein interactions genome-wide in yeast. In the special case, when one does not have any missing information about any of the features, our analysis shows that there is a larger information contribution from the functional-classification than from expression correlations or essentiality. We also show that in this case alternative models, such as logistic regression and random forest, may be more effective than Bayesian networks for predicting interactions. Conclusions In the restricted problem posed by the complete-information subset, we identified that the MIPS and Gene Ontology (GO functional similarity datasets as the dominating information contributors for predicting the protein-protein interactions under the framework proposed by Jansen et al. Random forests based on the MIPS and GO information alone can give highly accurate classifications. In this particular subset of complete information, adding other genomic data does little for improving predictions. We also found that the data discretizations used in the

Identifying Interactions that Determine Fragment Binding at Protein Hotspots.

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Radoux, Chris J; Olsson, Tjelvar S G; Pitt, Will R; Groom, Colin R; Blundell, Tom L

2016-05-12

Locating a ligand-binding site is an important first step in structure-guided drug discovery, but current methods do little to suggest which interactions within a pocket are the most important for binding. Here we illustrate a method that samples atomic hotspots with simple molecular probes to produce fragment hotspot maps. These maps specifically highlight fragment-binding sites and their corresponding pharmacophores. For ligand-bound structures, they provide an intuitive visual guide within the binding site, directing medicinal chemists where to grow the molecule and alerting them to suboptimal interactions within the original hit. The fragment hotspot map calculation is validated using experimental binding positions of 21 fragments and subsequent lead molecules. The ligands are found in high scoring areas of the fragment hotspot maps, with fragment atoms having a median percentage rank of 97%. Protein kinase B and pantothenate synthetase are examined in detail. In each case, the fragment hotspot maps are able to rationalize a Free-Wilson analysis of SAR data from a fragment-based drug design project.

Metal-ligand interactions

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Ervin, Kent M.

Experimental studies of the interactions of small transition-metal cluster anions with carbonyl ligands are reviewed and compared with neutral and cationic clusters. Under thermal conditions, the reaction rates of transition-metal clusters with carbon monoxide are measured as a function of cluster size. Saturation limits for carbon monoxide addition can be related to the geometric structures of the clusters. Both energy-resolved threshold collision-induced dissociation experiments and time-resolved photodissociation experiments are used to measure metal-carbonyl binding energies. For platinum and palladium trimer anions, the carbonyl binding energies are assigned to different geometric binding sites. Platinum and palladium cluster anions catalyse the oxidation of carbon monoxide to carbon dioxide in a full catalytic cycle at thermal energies.

@TOME-2: a new pipeline for comparative modeling of protein-ligand complexes.

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Pons, Jean-Luc; Labesse, Gilles

2009-07-01

@TOME 2.0 is new web pipeline dedicated to protein structure modeling and small ligand docking based on comparative analyses. @TOME 2.0 allows fold recognition, template selection, structural alignment editing, structure comparisons, 3D-model building and evaluation. These tasks are routinely used in sequence analyses for structure prediction. In our pipeline the necessary software is efficiently interconnected in an original manner to accelerate all the processes. Furthermore, we have also connected comparative docking of small ligands that is performed using protein-protein superposition. The input is a simple protein sequence in one-letter code with no comment. The resulting 3D model, protein-ligand complexes and structural alignments can be visualized through dedicated Web interfaces or can be downloaded for further studies. These original features will aid in the functional annotation of proteins and the selection of templates for molecular modeling and virtual screening. Several examples are described to highlight some of the new functionalities provided by this pipeline. The server and its documentation are freely available at http://abcis.cbs.cnrs.fr/AT2/

Towards accurate free energy calculations in ligand protein-binding studies.

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Steinbrecher, Thomas; Labahn, Andreas

2010-01-01

Cells contain a multitude of different chemical reaction paths running simultaneously and quite independently next to each other. This amazing feat is enabled by molecular recognition, the ability of biomolecules to form stable and specific complexes with each other and with their substrates. A better understanding of this process, i.e. of the kinetics, structures and thermodynamic properties of biomolecule binding, would be invaluable in the study of biological systems. In addition, as the mode of action of many pharmaceuticals is based upon their inhibition or activation of biomolecule targets, predictive models of small molecule receptor binding are very helpful tools in rational drug design. Since the goal here is normally to design a new compound with a high inhibition strength, one of the most important thermodynamic properties is the binding free energy DeltaG(0). The prediction of binding constants has always been one of the major goals in the field of computational chemistry, because the ability to reliably assess a hypothetical compound's binding properties without having to synthesize it first would save a tremendous amount of work. The different approaches to this question range from fast and simple empirical descriptor methods to elaborate simulation protocols aimed at putting the computation of free energies onto a solid foundation of statistical thermodynamics. While the later methods are still not suited for the screenings of thousands of compounds that are routinely performed in computational drug design studies, they are increasingly put to use for the detailed study of protein ligand interactions. This review will focus on molecular mechanics force field based free energy calculations and their application to the study of protein ligand interactions. After a brief overview of other popular methods for the calculation of free energies, we will describe recent advances in methodology and a variety of exemplary studies of molecular dynamics

Accuracy comparison of several common implicit solvent models and their implementations in the context of protein-ligand binding.

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Katkova, E V; Onufriev, A V; Aguilar, B; Sulimov, V B

2017-03-01

more by different parameterization and less by methods and indicates the need for further improvement of implicit solvent models parameterization. Within the same parameterization, various implicit methods give practically the same correlation with results obtained in explicit solvent model for ligands and proteins: e.g. correlation values of polar ligand solvation energies and the corresponding energies in the frame of explicit solvent were 0.953-0.966 for the APBS program, the GBNSR6 program and all models used in the DISOLV program. The DISOLV program proved to be on a par with the other used programs in the case of proteins and ligands solvation energy calculation. However, the solution of the Poisson-Boltzmann equation (APBS program) and Generalized Born method (implemented in the GBNSR6 program) proved to be the most accurate in calculating the desolvation energies of complexes. Copyright © 2016 Elsevier Inc. All rights reserved.

Alignment of non-covalent interactions at protein-protein interfaces.

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Hongbo Zhu

Full Text Available BACKGROUND: The study and comparison of protein-protein interfaces is essential for the understanding of the mechanisms of interaction between proteins. While there are many methods for comparing protein structures and protein binding sites, so far no methods have been reported for comparing the geometry of non-covalent interactions occurring at protein-protein interfaces. METHODOLOGY/PRINCIPAL FINDINGS: Here we present a method for aligning non-covalent interactions between different protein-protein interfaces. The method aligns the vector representations of van der Waals interactions and hydrogen bonds based on their geometry. The method has been applied to a dataset which comprises a variety of protein-protein interfaces. The alignments are consistent to a large extent with the results obtained using two other complementary approaches. In addition, we apply the method to three examples of protein mimicry. The method successfully aligns respective interfaces and allows for recognizing conserved interface regions. CONCLUSIONS/SIGNIFICANCE: The Galinter method has been validated in the comparison of interfaces in which homologous subunits are involved, including cases of mimicry. The method is also applicable to comparing interfaces involving non-peptidic compounds. Galinter assists users in identifying local interface regions with similar patterns of non-covalent interactions. This is particularly relevant to the investigation of the molecular basis of interaction mimicry.

GalaxyDock BP2 score: a hybrid scoring function for accurate protein-ligand docking

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Baek, Minkyung; Shin, Woong-Hee; Chung, Hwan Won; Seok, Chaok

2017-07-01

Protein-ligand docking is a useful tool for providing atomic-level understanding of protein functions in nature and design principles for artificial ligands or proteins with desired properties. The ability to identify the true binding pose of a ligand to a target protein among numerous possible candidate poses is an essential requirement for successful protein-ligand docking. Many previously developed docking scoring functions were trained to reproduce experimental binding affinities and were also used for scoring binding poses. However, in this study, we developed a new docking scoring function, called GalaxyDock BP2 Score, by directly training the scoring power of binding poses. This function is a hybrid of physics-based, empirical, and knowledge-based score terms that are balanced to strengthen the advantages of each component. The performance of the new scoring function exhibits significant improvement over existing scoring functions in decoy pose discrimination tests. In addition, when the score is used with the GalaxyDock2 protein-ligand docking program, it outperformed other state-of-the-art docking programs in docking tests on the Astex diverse set, the Cross2009 benchmark set, and the Astex non-native set. GalaxyDock BP2 Score and GalaxyDock2 with this score are freely available at http://galaxy.seoklab.org/softwares/galaxydock.html.

Quality control methodology for high-throughput protein-protein interaction screening.

Science.gov (United States)

Vazquez, Alexei; Rual, Jean-François; Venkatesan, Kavitha

2011-01-01

Protein-protein interactions are key to many aspects of the cell, including its cytoskeletal structure, the signaling processes in which it is involved, or its metabolism. Failure to form protein complexes or signaling cascades may sometimes translate into pathologic conditions such as cancer or neurodegenerative diseases. The set of all protein interactions between the proteins encoded by an organism constitutes its protein interaction network, representing a scaffold for biological function. Knowing the protein interaction network of an organism, combined with other sources of biological information, can unravel fundamental biological circuits and may help better understand the molecular basics of human diseases. The protein interaction network of an organism can be mapped by combining data obtained from both low-throughput screens, i.e., "one gene at a time" experiments and high-throughput screens, i.e., screens designed to interrogate large sets of proteins at once. In either case, quality controls are required to deal with the inherent imperfect nature of experimental assays. In this chapter, we discuss experimental and statistical methodologies to quantify error rates in high-throughput protein-protein interactions screens.

Categorizing Biases in High-Confidence High-Throughput Protein-Protein Interaction Data Sets*

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Yu, Xueping; Ivanic, Joseph; Memišević, Vesna; Wallqvist, Anders; Reifman, Jaques

2011-01-01

We characterized and evaluated the functional attributes of three yeast high-confidence protein-protein interaction data sets derived from affinity purification/mass spectrometry, protein-fragment complementation assay, and yeast two-hybrid experiments. The interacting proteins retrieved from these data sets formed distinct, partially overlapping sets with different protein-protein interaction characteristics. These differences were primarily a function of the deployed experimental technologies used to recover these interactions. This affected the total coverage of interactions and was especially evident in the recovery of interactions among different functional classes of proteins. We found that the interaction data obtained by the yeast two-hybrid method was the least biased toward any particular functional characterization. In contrast, interacting proteins in the affinity purification/mass spectrometry and protein-fragment complementation assay data sets were over- and under-represented among distinct and different functional categories. We delineated how these differences affected protein complex organization in the network of interactions, in particular for strongly interacting complexes (e.g. RNA and protein synthesis) versus weak and transient interacting complexes (e.g. protein transport). We quantified methodological differences in detecting protein interactions from larger protein complexes, in the correlation of protein abundance among interacting proteins, and in their connectivity of essential proteins. In the latter case, we showed that minimizing inherent methodology biases removed many of the ambiguous conclusions about protein essentiality and protein connectivity. We used these findings to rationalize how biological insights obtained by analyzing data sets originating from different sources sometimes do not agree or may even contradict each other. An important corollary of this work was that discrepancies in biological insights did not

A domain-based approach to predict protein-protein interactions

Directory of Open Access Journals (Sweden)

Resat Haluk

2007-06-01

Full Text Available Abstract Background Knowing which proteins exist in a certain organism or cell type and how these proteins interact with each other are necessary for the understanding of biological processes at the whole cell level. The determination of the protein-protein interaction (PPI networks has been the subject of extensive research. Despite the development of reasonably successful methods, serious technical difficulties still exist. In this paper we present DomainGA, a quantitative computational approach that uses the information about the domain-domain interactions to predict the interactions between proteins. Results DomainGA is a multi-parameter optimization method in which the available PPI information is used to derive a quantitative scoring scheme for the domain-domain pairs. Obtained domain interaction scores are then used to predict whether a pair of proteins interacts. Using the yeast PPI data and a series of tests, we show the robustness and insensitivity of the DomainGA method to the selection of the parameter sets, score ranges, and detection rules. Our DomainGA method achieves very high explanation ratios for the positive and negative PPIs in yeast. Based on our cross-verification tests on human PPIs, comparison of the optimized scores with the structurally observed domain interactions obtained from the iPFAM database, and sensitivity and specificity analysis; we conclude that our DomainGA method shows great promise to be applicable across multiple organisms. Conclusion We envision the DomainGA as a first step of a multiple tier approach to constructing organism specific PPIs. As it is based on fundamental structural information, the DomainGA approach can be used to create potential PPIs and the accuracy of the constructed interaction template can be further improved using complementary methods. Explanation ratios obtained in the reported test case studies clearly show that the false prediction rates of the template networks constructed

High Throughput, Label-free Screening Small Molecule Compound Libraries for Protein-Ligands using Combination of Small Molecule Microarrays and a Special Ellipsometry-based Optical Scanner.

Science.gov (United States)

Landry, James P; Fei, Yiyan; Zhu, X D

2011-12-01

Small-molecule compounds remain the major source of therapeutic and preventative drugs. Developing new drugs against a protein target often requires screening large collections of compounds with diverse structures for ligands or ligand fragments that exhibit sufficiently affinity and desirable inhibition effect on the target before further optimization and development. Since the number of small molecule compounds is large, high-throughput screening (HTS) methods are needed. Small-molecule microarrays (SMM) on a solid support in combination with a suitable binding assay form a viable HTS platform. We demonstrate that by combining an oblique-incidence reflectivity difference optical scanner with SMM we can screen 10,000 small-molecule compounds on a single glass slide for protein ligands without fluorescence labeling. Furthermore using such a label-free assay platform we can simultaneously acquire binding curves of a solution-phase protein to over 10,000 immobilized compounds, thus enabling full characterization of protein-ligand interactions over a wide range of affinity constants.

A general approach for developing system-specific functions to score protein-ligand docked complexes using support vector inductive logic programming.

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Amini, Ata; Shrimpton, Paul J; Muggleton, Stephen H; Sternberg, Michael J E

2007-12-01

Despite the increased recent use of protein-ligand and protein-protein docking in the drug discovery process due to the increases in computational power, the difficulty of accurately ranking the binding affinities of a series of ligands or a series of proteins docked to a protein receptor remains largely unsolved. This problem is of major concern in lead optimization procedures and has lead to the development of scoring functions tailored to rank the binding affinities of a series of ligands to a specific system. However, such methods can take a long time to develop and their transferability to other systems remains open to question. Here we demonstrate that given a suitable amount of background information a new approach using support vector inductive logic programming (SVILP) can be used to produce system-specific scoring functions. Inductive logic programming (ILP) learns logic-based rules for a given dataset that can be used to describe properties of each member of the set in a qualitative manner. By combining ILP with support vector machine regression, a quantitative set of rules can be obtained. SVILP has previously been used in a biological context to examine datasets containing a series of singular molecular structures and properties. Here we describe the use of SVILP to produce binding affinity predictions of a series of ligands to a particular protein. We also for the first time examine the applicability of SVILP techniques to datasets consisting of protein-ligand complexes. Our results show that SVILP performs comparably with other state-of-the-art methods on five protein-ligand systems as judged by similar cross-validated squares of their correlation coefficients. A McNemar test comparing SVILP to CoMFA and CoMSIA across the five systems indicates our method to be significantly better on one occasion. The ability to graphically display and understand the SVILP-produced rules is demonstrated and this feature of ILP can be used to derive hypothesis for

A specific interdomain interaction preserves the structural and binding properties of the ModA protein from the phytopathogen Xanthomonas citri domain interaction and transport in ModA.

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Santacruz-Perez, Carolina; Pegos, Vanessa Rodrigues; Honorato, Rodrigo V; Verli, Hugo; Lindahl, Erik; Barbosa, João Alexandre Ribeiro Gonçalves; Balan, Andrea

2013-11-01

The periplasmic-binding proteins in ATP-binding cassette systems (ABC Transporters) are responsible for the capture and delivery of ligands to their specific transporters, triggering a series of ATP-driven conformational changes that leads to the transport of the ligand. Structurally consisting of two lobes, the proteins change conformation after interaction with the ligand. The structure of the molybdate-binding protein (ModA) from Xanthomonas citri, bound to molybdate, was previously solved by our group and an interdomain interaction, mediated by a salt bridge between K127 and D59, apparently supports the binding properties and keeps the domains closed. To determinate the importance of this interaction, we built two ModA mutants, K127S and D59A, and analysed their functional and structural properties. Based on a set of spectroscopic experiments, crystallisation trials, structure determination and molecular dynamics (MD) simulations, we showed that the salt bridge is essential to maintain the structure and binding properties. Additionally, the MD simulations revealed that this mutant adopted a more compact structure that packed down the ligand-binding pocket. From the closed bound to open structure, the positioning of the helices forming the dipole and the salt bridge are essential to induce an intermediate state. Copyright © 2013 Elsevier Inc. All rights reserved.

Ligand Binding Induces Conformational Changes in Human Cellular Retinol-binding Protein 1 (CRBP1) Revealed by Atomic Resolution Crystal Structures.

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Silvaroli, Josie A; Arne, Jason M; Chelstowska, Sylwia; Kiser, Philip D; Banerjee, Surajit; Golczak, Marcin

2016-04-15

Important in regulating the uptake, storage, and metabolism of retinoids, cellular retinol-binding protein 1 (CRBP1) is essential for trafficking vitamin A through the cytoplasm. However, the molecular details of ligand uptake and targeted release by CRBP1 remain unclear. Here we report the first structure of CRBP1 in a ligand-free form as well as ultra-high resolution structures of this protein bound to either all-trans-retinol or retinylamine, the latter a therapeutic retinoid that prevents light-induced retinal degeneration. Superpositioning of human apo- and holo-CRBP1 revealed major differences within segments surrounding the entrance to the retinoid-binding site. These included α-helix II and hairpin turns between β-strands βC-βD and βE-βF as well as several side chains, such as Phe-57, Tyr-60, and Ile-77, that change their orientations to accommodate the ligand. Additionally, we mapped hydrogen bond networks inside the retinoid-binding cavity and demonstrated their significance for the ligand affinity. Analyses of the crystallographic B-factors indicated several regions with higher backbone mobility in the apoprotein that became more rigid upon retinoid binding. This conformational flexibility of human apo-CRBP1 facilitates interaction with the ligands, whereas the more rigid holoprotein structure protects the labile retinoid moiety during vitamin A transport. These findings suggest a mechanism of induced fit upon ligand binding by mammalian cellular retinol-binding proteins. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Entropy in molecular recognition by proteins.

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Caro, José A; Harpole, Kyle W; Kasinath, Vignesh; Lim, Jackwee; Granja, Jeffrey; Valentine, Kathleen G; Sharp, Kim A; Wand, A Joshua

2017-06-20

Molecular recognition by proteins is fundamental to molecular biology. Dissection of the thermodynamic energy terms governing protein-ligand interactions has proven difficult, with determination of entropic contributions being particularly elusive. NMR relaxation measurements have suggested that changes in protein conformational entropy can be quantitatively obtained through a dynamical proxy, but the generality of this relationship has not been shown. Twenty-eight protein-ligand complexes are used to show a quantitative relationship between measures of fast side-chain motion and the underlying conformational entropy. We find that the contribution of conformational entropy can range from favorable to unfavorable, which demonstrates the potential of this thermodynamic variable to modulate protein-ligand interactions. For about one-quarter of these complexes, the absence of conformational entropy would render the resulting affinity biologically meaningless. The dynamical proxy for conformational entropy or "entropy meter" also allows for refinement of the contributions of solvent entropy and the loss in rotational-translational entropy accompanying formation of high-affinity complexes. Furthermore, structure-based application of the approach can also provide insight into long-lived specific water-protein interactions that escape the generic treatments of solvent entropy based simply on changes in accessible surface area. These results provide a comprehensive and unified view of the general role of entropy in high-affinity molecular recognition by proteins.

Virtual Ligand Screening Using PL-PatchSurfer2, a Molecular Surface-Based Protein-Ligand Docking Method.

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Shin, Woong-Hee; Kihara, Daisuke

2018-01-01

Virtual screening is a computational technique for predicting a potent binding compound for a receptor protein from a ligand library. It has been a widely used in the drug discovery field to reduce the efforts of medicinal chemists to find hit compounds by experiments.Here, we introduce our novel structure-based virtual screening program, PL-PatchSurfer, which uses molecular surface representation with the three-dimensional Zernike descriptors, which is an effective mathematical representation for identifying physicochemical complementarities between local surfaces of a target protein and a ligand. The advantage of the surface-patch description is its tolerance on a receptor and compound structure variation. PL-PatchSurfer2 achieves higher accuracy on apo form and computationally modeled receptor structures than conventional structure-based virtual screening programs. Thus, PL-PatchSurfer2 opens up an opportunity for targets that do not have their crystal structures. The program is provided as a stand-alone program at http://kiharalab.org/plps2 . We also provide files for two ligand libraries, ChEMBL and ZINC Drug-like.

Application of encoded library technology (ELT) to a protein-protein interaction target: discovery of a potent class of integrin lymphocyte function-associated antigen 1 (LFA-1) antagonists.

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Kollmann, Christopher S; Bai, Xiaopeng; Tsai, Ching-Hsuan; Yang, Hongfang; Lind, Kenneth E; Skinner, Steven R; Zhu, Zhengrong; Israel, David I; Cuozzo, John W; Morgan, Barry A; Yuki, Koichi; Xie, Can; Springer, Timothy A; Shimaoka, Motomu; Evindar, Ghotas

2014-04-01

The inhibition of protein-protein interactions remains a challenge for traditional small molecule drug discovery. Here we describe the use of DNA-encoded library technology for the discovery of small molecules that are potent inhibitors of the interaction between lymphocyte function-associated antigen 1 and its ligand intercellular adhesion molecule 1. A DNA-encoded library with a potential complexity of 4.1 billion compounds was exposed to the I-domain of the target protein and the bound ligands were affinity selected, yielding an enriched small-molecule hit family. Compounds representing this family were synthesized without their DNA encoding moiety and found to inhibit the lymphocyte function-associated antigen 1/intercellular adhesion molecule-1 interaction with submicromolar potency in both ELISA and cell adhesion assays. Re-synthesized compounds conjugated to DNA or a fluorophore were demonstrated to bind to cells expressing the target protein. Copyright © 2014 Elsevier Ltd. All rights reserved.

Interactions between alkaline earth cations and oxo ligands. DFT study of the affinity of the Mg²+ cation for phosphoryl ligands.

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da Costa, Leonardo Moreira; de Mesquita Carneiro, José Walkimar; Paes, Lilian Weitzel Coelho

2011-08-01

DFT (B3LYP/6-31+G(d)) calculations of Mg(2+) affinities for a set of phosphoryl ligands were performed. Two types of ligands were studied: a set of trivalent [O = P(R)] and a set of pentavalent phosphoryl ligands [O = P(R)(3)] (R = H, F, Cl, Br, OH, OCH(3), CH(3), CN, NH(2) and NO(2)), with R either bound directly to the phosphorus atom or to the para position of a phenyl ring. The affinity of the Mg(2+) cation for the ligands was quantified by means of the enthalpy for the substitution of one water molecule in the [Mg(H(2)O)(6)](2+) complex for a ligand. The enthalpy of substitution was correlated with electronic and geometric parameters. Electron-donor groups increase the interaction between the cation and the ligand, while electron-acceptor groups decrease the interaction enthalpy.

The role of ligands on the equilibria between functional states of a G protein-coupled receptor.

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Kim, Tae Hun; Chung, Ka Young; Manglik, Aashish; Hansen, Alexandar L; Dror, Ron O; Mildorf, Thomas J; Shaw, David E; Kobilka, Brian K; Prosser, R Scott

2013-06-26

G protein-coupled receptors exhibit a wide variety of signaling behaviors in response to different ligands. When a small label was incorporated on the cytosolic interface of transmembrane helix 6 (Cys-265), (19)F NMR spectra of the β2 adrenergic receptor (β2AR) reconstituted in maltose/neopentyl glycol detergent micelles revealed two distinct inactive states, an activation intermediate state en route to activation, and, in the presence of a G protein mimic, a predominant active state. Analysis of the spectra as a function of temperature revealed that for all ligands, the activation intermediate is entropically favored and enthalpically disfavored. β2AR enthalpy changes toward activation are notably lower than those observed with rhodopsin, a likely consequence of basal activity and the fact that the ionic lock and other interactions stabilizing the inactive state of β2AR are weaker. Positive entropy changes toward activation likely reflect greater mobility (configurational entropy) in the cytoplasmic domain, as confirmed through an order parameter analysis. Ligands greatly influence the overall changes in enthalpy and entropy of the system and the corresponding changes in population and amplitude of motion of given states, suggesting a complex landscape of states and substates.

Detection of site-specific binding and co-binding of ligands to macromolecules using 19F NMR

International Nuclear Information System (INIS)

Jenkins, B.G.

1991-01-01

Study of ligand-macromolecular interactions by 19 F nuclear magnetic resonance (NMR) spectroscopy affords many opportunities for obtaining molecular biochemical and pharmaceutical information. This is due to the absence of a background fluorine signal, as well as the relatively high sensitivity of 19 F NMR. Use of fluorine-labeled ligands enables one to probe not only binding and co-binding phenomena to macromolecules, but also can provide data on binding constants, stoichiometries, kinetics, and conformational properties of these complexes. Under conditions of slow exchange and macromolecule-induced chemical shifts, multiple 19 F NMR resonances can be observed for free and bound ligands. These shifted resonances are a direct correlate of the concentration of ligand bound in a specific state rather than the global concentrations of bound or free ligand which are usually determined using other techniques such as absorption spectroscopy or equilibrium dialysis. Examples of these interactions are demonstrated both from the literature and from interactions of 5-fluorotryptophan, 5-fluorosalicylic acid, flurbiprofen, and sulindac sulfide with human serum albumin. Other applications of 19 F NMR to study of these interactions in vivo, as well for receptor binding and metabolic tracing of fluorinated drugs and proteins are discussed

Systematic Characterisation of Cellular Localisation and Expression Profiles of Proteins Containing MHC Ligands

DEFF Research Database (Denmark)

Juncker, Agnieszka; Larsen, Mette Voldby; Weinhold, Nils

2009-01-01

Background: Presentation of peptides on Major Histocompatibility Complex (MHC) molecules is the cornerstone in immune system activation and increased knowledge of the characteristics of MHC ligands and their source proteins is highly desirable. Methodology/Principal Finding: In the present large......-scale study, we used a large data set of proteins containing experimentally identified MHC class I or II ligands and examined the proteins according to their expression profiles at the mRNA level and their Gene Ontology (GO) classification within the cellular component ontology. Proteins encoded by highly...

Complement factor H-related proteins CFHR2 and CFHR5 represent novel ligands for the infection-associated CRASP proteins of Borrelia burgdorferi.

Directory of Open Access Journals (Sweden)

Corinna Siegel

2010-10-01

Full Text Available One virulence property of Borrelia burgdorferi is its resistance to innate immunity, in particular to complement-mediated killing. Serum-resistant B. burgdorferi express up to five distinct complement regulator-acquiring surface proteins (CRASP which interact with complement regulator factor H (CFH and factor H-like protein 1 (FHL1 or factor H-related protein 1 (CFHR1. In the present study we elucidate the role of the infection-associated CRASP-3 and CRASP-5 protein to serve as ligands for additional complement regulatory proteins as well as for complement resistance of B. burgdorferi.To elucidate whether CRASP-5 and CRASP-3 interact with various human proteins, both borrelial proteins were immobilized on magnetic beads. Following incubation with human serum, bound proteins were eluted and separated by Glycine-SDS-PAGE. In addition to CFH and CFHR1, complement regulators CFHR2 and CFHR5 were identified as novel ligands for both borrelial proteins by employing MALDI-TOF. To further assess the contributions of CRASP-3 and CRASP-5 to complement resistance, a serum-sensitive B. garinii strain G1 which lacks all CFH-binding proteins was used as a valuable model for functional analyses. Both CRASPs expressed on the B. garinii outer surface bound CFH as well as CFHR1 and CFHR2 in ELISA. In contrast, live B. garinii bound CFHR1, CFHR2, and CFHR5 and only miniscute amounts of CFH as demonstrated by serum adsorption assays and FACS analyses. Further functional analysis revealed that upon NHS incubation, CRASP-3 or CRASP-5 expressing borreliae were killed by complement.In the absence of CFH and the presence of CFHR1, CFHR2 and CFHR5, assembly and integration of the membrane attack complex was not efficiently inhibited indicating that CFH in co-operation with CFHR1, CFHR2 and CFHR5 supports complement evasion of B. burgdorferi.

Topology and weights in a protein domain interaction network--a novel way to predict protein interactions.

Science.gov (United States)

Wuchty, Stefan

2006-05-23

While the analysis of unweighted biological webs as diverse as genetic, protein and metabolic networks allowed spectacular insights in the inner workings of a cell, biological networks are not only determined by their static grid of links. In fact, we expect that the heterogeneity in the utilization of connections has a major impact on the organization of cellular activities as well. We consider a web of interactions between protein domains of the Protein Family database (PFAM), which are weighted by a probability score. We apply metrics that combine the static layout and the weights of the underlying interactions. We observe that unweighted measures as well as their weighted counterparts largely share the same trends in the underlying domain interaction network. However, we only find weak signals that weights and the static grid of interactions are connected entities. Therefore assuming that a protein interaction is governed by a single domain interaction, we observe strong and significant correlations of the highest scoring domain interaction and the confidence of protein interactions in the underlying interactions of yeast and fly. Modeling an interaction between proteins if we find a high scoring protein domain interaction we obtain 1, 428 protein interactions among 361 proteins in the human malaria parasite Plasmodium falciparum. Assessing their quality by a logistic regression method we observe that increasing confidence of predicted interactions is accompanied by high scoring domain interactions and elevated levels of functional similarity and evolutionary conservation. Our results indicate that probability scores are randomly distributed, allowing to treat static grid and weights of domain interactions as separate entities. In particular, these finding confirms earlier observations that a protein interaction is a matter of a single interaction event on domain level. As an immediate application, we show a simple way to predict potential protein interactions

In silico modeling and experimental evidence of coagulant protein interaction with precursors for nanoparticle functionalization.

Science.gov (United States)

Okoli, Chuka; Sengottaiyan, Selvaraj; Arul Murugan, N; Pavankumar, Asalapuram R; Agren, Hans; Kuttuva Rajarao, Gunaratna

2013-10-01

The design of novel protein-nanoparticle hybrid systems has applications in many fields of science ranging from biomedicine, catalysis, water treatment, etc. The main barrier in devising such tool is lack of adequate information or poor understanding of protein-ligand chemistry. Here, we establish a new strategy based on computational modeling for protein and precursor linkers that can decorate the nanoparticles. Moringa oleifera (MO2.1) seed protein that has coagulation and antimicrobial properties was used. Superparamagnetic nanoparticles (SPION) with precursor ligands were used for the protein-ligand interaction studies. The molecular docking studies reveal that there are two binding sites, one is located at the core binding site; tetraethoxysilane (TEOS) or 3-aminopropyl trimethoxysilane (APTES) binds to this site while the other one is located at the side chain residues where trisodium citrate (TSC) or Si60 binds to this site. The protein-ligand distance profile analysis explains the differences in functional activity of the decorated SPION. Experimentally, TSC-coated nanoparticles showed higher coagulation activity as compared to TEOS- and APTES-coated SPION. To our knowledge, this is the first report on in vitro experimental data, which endorses the computational modeling studies as a powerful tool to design novel precursors for functionalization of nanomaterials; and develop interface hybrid systems for various applications.

A scalable and accurate method for classifying protein-ligand binding geometries using a MapReduce approach.

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Estrada, T; Zhang, B; Cicotti, P; Armen, R S; Taufer, M

2012-07-01

We present a scalable and accurate method for classifying protein-ligand binding geometries in molecular docking. Our method is a three-step process: the first step encodes the geometry of a three-dimensional (3D) ligand conformation into a single 3D point in the space; the second step builds an octree by assigning an octant identifier to every single point in the space under consideration; and the third step performs an octree-based clustering on the reduced conformation space and identifies the most dense octant. We adapt our method for MapReduce and implement it in Hadoop. The load-balancing, fault-tolerance, and scalability in MapReduce allow screening of very large conformation spaces not approachable with traditional clustering methods. We analyze results for docking trials for 23 protein-ligand complexes for HIV protease, 21 protein-ligand complexes for Trypsin, and 12 protein-ligand complexes for P38alpha kinase. We also analyze cross docking trials for 24 ligands, each docking into 24 protein conformations of the HIV protease, and receptor ensemble docking trials for 24 ligands, each docking in a pool of HIV protease receptors. Our method demonstrates significant improvement over energy-only scoring for the accurate identification of native ligand geometries in all these docking assessments. The advantages of our clustering approach make it attractive for complex applications in real-world drug design efforts. We demonstrate that our method is particularly useful for clustering docking results using a minimal ensemble of representative protein conformational states (receptor ensemble docking), which is now a common strategy to address protein flexibility in molecular docking. Copyright © 2012 Elsevier Ltd. All rights reserved.

Detecting local ligand-binding site similarity in nonhomologous proteins by surface patch comparison.

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Sael, Lee; Kihara, Daisuke

2012-04-01

Functional elucidation of proteins is one of the essential tasks in biology. Function of a protein, specifically, small ligand molecules that bind to a protein, can be predicted by finding similar local surface regions in binding sites of known proteins. Here, we developed an alignment free local surface comparison method for predicting a ligand molecule which binds to a query protein. The algorithm, named Patch-Surfer, represents a binding pocket as a combination of segmented surface patches, each of which is characterized by its geometrical shape, the electrostatic potential, the hydrophobicity, and the concaveness. Representing a pocket by a set of patches is effective to absorb difference of global pocket shape while capturing local similarity of pockets. The shape and the physicochemical properties of surface patches are represented using the 3D Zernike descriptor, which is a series expansion of mathematical 3D function. Two pockets are compared using a modified weighted bipartite matching algorithm, which matches similar patches from the two pockets. Patch-Surfer was benchmarked on three datasets, which consist in total of 390 proteins that bind to one of 21 ligands. Patch-Surfer showed superior performance to existing methods including a global pocket comparison method, Pocket-Surfer, which we have previously introduced. Particularly, as intended, the accuracy showed large improvement for flexible ligand molecules, which bind to pockets in different conformations. Copyright © 2011 Wiley Periodicals, Inc.

ReFlexIn: a flexible receptor protein-ligand docking scheme evaluated on HIV-1 protease.

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Simon Leis

Full Text Available For many targets of pharmaceutical importance conformational changes of the receptor protein are relevant during the ligand binding process. A new docking approach, ReFlexIn (Receptor Flexibility by Interpolation, that combines receptor flexibility with the computationally efficient potential grid representation of receptor molecules has been evaluated on the retroviral HIV-1 (Human Immunodeficiency Virus 1 protease system. An approximate inclusion of receptor flexibility is achieved by using interpolation between grid representations of individual receptor conformations. For the retroviral protease the method was tested on an ensemble of protease structures crystallized in the presence of different ligands and on a set of structures obtained from morphing between the unbound and a ligand-bound protease structure. Docking was performed on ligands known to bind to the protease and several non-binders. For the binders the ReFlexIn method yielded in almost all cases ligand placements in similar or closer agreement with experiment than docking to any of the ensemble members without degrading the discrimination with respect to non-binders. The improved docking performance compared to docking to rigid receptors allows for systematic virtual screening applications at very small additional computational cost.

On the analysis of protein-protein interactions via knowledge-based potentials for the prediction of protein-protein docking

DEFF Research Database (Denmark)

Feliu, Elisenda; Aloy, Patrick; Oliva, Baldo

2011-01-01

Development of effective methods to screen binary interactions obtained by rigid-body protein-protein docking is key for structure prediction of complexes and for elucidating physicochemical principles of protein-protein binding. We have derived empirical knowledge-based potential functions for s...... and with independence of the partner. This information is encoded at the residue level and could be easily incorporated in the initial grid scoring for Fast Fourier Transform rigid-body docking methods.......Development of effective methods to screen binary interactions obtained by rigid-body protein-protein docking is key for structure prediction of complexes and for elucidating physicochemical principles of protein-protein binding. We have derived empirical knowledge-based potential functions...... for selecting rigid-body docking poses. These potentials include the energetic component that provides the residues with a particular secondary structure and surface accessibility. These scoring functions have been tested on a state-of-art benchmark dataset and on a decoy dataset of permanent interactions. Our...

Challenges predicting ligand-receptor interactions of promiscuous proteins: the nuclear receptor PXR.

Directory of Open Access Journals (Sweden)

Sean Ekins

2009-12-01

Full Text Available Transcriptional regulation of some genes involved in xenobiotic detoxification and apoptosis is performed via the human pregnane X receptor (PXR which in turn is activated by structurally diverse agonists including steroid hormones. Activation of PXR has the potential to initiate adverse effects, altering drug pharmacokinetics or perturbing physiological processes. Reliable computational prediction of PXR agonists would be valuable for pharmaceutical and toxicological research. There has been limited success with structure-based modeling approaches to predict human PXR activators. Slightly better success has been achieved with ligand-based modeling methods including quantitative structure-activity relationship (QSAR analysis, pharmacophore modeling and machine learning. In this study, we present a comprehensive analysis focused on prediction of 115 steroids for ligand binding activity towards human PXR. Six crystal structures were used as templates for docking and ligand-based modeling approaches (two-, three-, four- and five-dimensional analyses. The best success at external prediction was achieved with 5D-QSAR. Bayesian models with FCFP_6 descriptors were validated after leaving a large percentage of the dataset out and using an external test set. Docking of ligands to the PXR structure co-crystallized with hyperforin had the best statistics for this method. Sulfated steroids (which are activators were consistently predicted as non-activators while, poorly predicted steroids were docked in a reverse mode compared to 5alpha-androstan-3beta-ol. Modeling of human PXR represents a complex challenge by virtue of the large, flexible ligand-binding cavity. This study emphasizes this aspect, illustrating modest success using the largest quantitative data set to date and multiple modeling approaches.

Detecting protein-protein interactions in living cells

DEFF Research Database (Denmark)

Gottschalk, Marie; Bach, Anders; Hansen, Jakob Lerche

2009-01-01

to the endogenous C-terminal peptide of the NMDA receptor, as evaluated by a cell-free protein-protein interaction assay. However, it is important to address both membrane permeability and effect in living cells. Therefore a bioluminescence resonance energy transfer (BRET) assay was established, where the C......-terminal of the NMDA receptor and PDZ2 of PSD-95 were fused to green fluorescent protein (GFP) and Renilla luciferase (Rluc) and expressed in COS7 cells. A robust and specific BRET signal was obtained by expression of the appropriate partner proteins and subsequently, the assay was used to evaluate a Tat......The PDZ domain mediated interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treatment of ischemic brain diseases. We have recently developed a number of peptide analogues with improved affinity for the PDZ domains of PSD-95 compared...

Human cancer protein-protein interaction network: a structural perspective.

Directory of Open Access Journals (Sweden)

Gozde Kar

2009-12-01

proteins: Erbb3, a multi interface, and Raf1, a single interface hub. The results reveal that affinity of interactions of the multi-interface hub tends to be higher than that of the single-interface hub. These findings might be important in obtaining new targets in cancer as well as finding the details of specific binding regions of putative cancer drug candidates.

A credit-card library approach for disrupting protein-protein interactions.

Science.gov (United States)

Xu, Yang; Shi, Jin; Yamamoto, Noboru; Moss, Jason A; Vogt, Peter K; Janda, Kim D

2006-04-15

Protein-protein interfaces are prominent in many therapeutically important targets. Using small organic molecules to disrupt protein-protein interactions is a current challenge in chemical biology. An important example of protein-protein interactions is provided by the Myc protein, which is frequently deregulated in human cancers. Myc belongs to the family of basic helix-loop-helix leucine zipper (bHLH-ZIP) transcription factors. It is biologically active only as heterodimer with the bHLH-ZIP protein Max. Herein, we report a new strategy for the disruption of protein-protein interactions that has been corroborated through the design and synthesis of a small parallel library composed of 'credit-card' compounds. These compounds are derived from a planar, aromatic scaffold and functionalized with four points of diversity. From a 285 membered library, several hits were obtained that disrupted the c-Myc-Max interaction and cellular functions of c-Myc. The IC50 values determined for this small focused library for the disruption of Myc-Max dimerization are quite potent, especially since small molecule antagonists of protein-protein interactions are notoriously difficult to find. Furthermore, several of the compounds were active at the cellular level as shown by their biological effects on Myc action in chicken embryo fibroblast assays. In light of our findings, this approach is considered a valuable addition to the armamentarium of new molecules being developed to interact with protein-protein interfaces. Finally, this strategy for disrupting protein-protein interactions should prove applicable to other families of proteins.

Topology and weights in a protein domain interaction network – a novel way to predict protein interactions

Directory of Open Access Journals (Sweden)

Wuchty Stefan

2006-05-01

Full Text Available Abstract Background While the analysis of unweighted biological webs as diverse as genetic, protein and metabolic networks allowed spectacular insights in the inner workings of a cell, biological networks are not only determined by their static grid of links. In fact, we expect that the heterogeneity in the utilization of connections has a major impact on the organization of cellular activities as well. Results We consider a web of interactions between protein domains of the Protein Family database (PFAM, which are weighted by a probability score. We apply metrics that combine the static layout and the weights of the underlying interactions. We observe that unweighted measures as well as their weighted counterparts largely share the same trends in the underlying domain interaction network. However, we only find weak signals that weights and the static grid of interactions are connected entities. Therefore assuming that a protein interaction is governed by a single domain interaction, we observe strong and significant correlations of the highest scoring domain interaction and the confidence of protein interactions in the underlying interactions of yeast and fly. Modeling an interaction between proteins if we find a high scoring protein domain interaction we obtain 1, 428 protein interactions among 361 proteins in the human malaria parasite Plasmodium falciparum. Assessing their quality by a logistic regression method we observe that increasing confidence of predicted interactions is accompanied by high scoring domain interactions and elevated levels of functional similarity and evolutionary conservation. Conclusion Our results indicate that probability scores are randomly distributed, allowing to treat static grid and weights of domain interactions as separate entities. In particular, these finding confirms earlier observations that a protein interaction is a matter of a single interaction event on domain level. As an immediate application, we

Cancer exosomes and NKG2D receptor-ligand interactions: impairing NKG2D-mediated cytotoxicity and anti-tumour immune surveillance.

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Mincheva-Nilsson, Lucia; Baranov, Vladimir

2014-10-01

Human cancers constitutively produce and release endosome-derived nanometer-sized vesicles called exosomes that carry biologically active proteins, messenger and micro RNAs and serve as vehicles of intercellular communication. The tumour exosomes are present in the blood, urine and various malignant effusions such as peritoneal and pleural fluid of cancer patients and can modulate immune cells and responses thus deranging the immune system of cancer patients and giving advantage to the cancer to establish and spread itself. Here, the role of exosomes in the NKG2D receptor-ligand system's interactions is discussed. The activating NK cell receptor NKG2D and its multiple ligands, the MHC class I-related chain (MIC) A/B and the retinoic acid transcript-1/UL-16 binding proteins (RAET1/ULBP) 1-6 comprise a powerful stress-inducible danger detector system that targets infected, inflamed and malignantly transformed cells and plays a decisive role in anti-tumour immune surveillance. Mounting evidence reveals that the MIC- and RAET1/ULBP ligand family members are enriched in the endosomal compartment of various tumour cells and expressed and released into the intercellular space and bodily fluids on exosomes thus preserving their entire molecule, three-dimensional protein structure and biologic activity. The NKG2D ligand-expressing exosomes serve as decoys with a powerful ability to down regulate the cognate receptor and impair the cytotoxic function of NK-, NKT-, gamma/delta- and cytotoxic T cells. This review summarizes recent findings concerning the role of NKG2D receptor-ligand system in cancer with emphasis on regulation of NKG2D ligand expression and the immunosuppressive role of exosomally expressed NKG2D ligands. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparison of the ligand binding properties of two homologous rat apocellular retinol-binding proteins expressed in Escherichia coli.

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Levin, M S; Locke, B; Yang, N C; Li, E; Gordon, J I

1988-11-25

Cellular retinol-binding protein (CRBP) and cellular retinol-binding protein II (CRBP II) are 132-residue cytosolic proteins which have 56% amino acid sequence identity and bind all-trans-retinol as their endogenous ligand. They belong to a family of cytoplasmic proteins which have evolved to bind distinct hydrophobic ligands. Their patterns of tissue-specific and developmental regulation are distinct. We have compared the ligand binding properties of rat apo-CRBP and apo-CRBP II that have been expressed in Escherichia coli. Several observations indicate that the E. coli-derived apoproteins are structurally similar to the native rat proteins: they co-migrate on isoelectric focusing gels; and when complexed with all-trans-retinol, their absorption and excitation/emission spectra are nearly identical to those of the authentic rat holoproteins. Comparative lifetime and acrylamide quenching studies suggest that there are differences in the conformations of apo-CRBP and apo-CRBP II. The interaction of E. coli-derived apo-CRBP and apo-CRBP II with a variety of retinoids was analyzed using spectroscopic techniques. Both apoproteins formed high affinity complexes with all-trans-retinol (K'd approximately 10 nM). In direct binding assays, all-trans-retinal bound to both apoproteins (K'd approximately 50 nM for CRBP; K'd approximately 90 nM for CRBP II). However, all-trans-retinal could displace all-trans-retinol bound to CRBP II but not to CRBP. These observations suggests that there is a specific yet distinct interaction between these two proteins and all-trans-retinal. Apo-CRBP and apo-CRBP II did not demonstrate significant binding to either retinoic acid or methyl retinoate, an uncharged derivative of all-trans-retinoic acid. This indicates that the carboxymethyl group of methyl retinoate cannot be sterically accommodated in their binding pockets and that failure to bind retinoic acid probably is not simply due to the negative charge of its C-15 carboxylate group

Incorporation of protein flexibility and conformational energy penalties in docking screens to improve ligand discovery

Science.gov (United States)

Fischer, Marcus; Coleman, Ryan G.; Fraser, James S.; Shoichet, Brian K.

2014-07-01

Proteins fluctuate between alternative conformations, which presents a challenge for ligand discovery because such flexibility is difficult to treat computationally owing to problems with conformational sampling and energy weighting. Here we describe a flexible docking method that samples and weights protein conformations using experimentally derived conformations as a guide. The crystallographically refined occupancies of these conformations, which are observable in an apo receptor structure, define energy penalties for docking. In a large prospective library screen, we identified new ligands that target specific receptor conformations of a cavity in cytochrome c peroxidase, and we confirm both ligand pose and associated receptor conformation predictions by crystallography. The inclusion of receptor flexibility led to ligands with new chemotypes and physical properties. By exploiting experimental measures of loop and side-chain flexibility, this method can be extended to the discovery of new ligands for hundreds of targets in the Protein Data Bank for which similar experimental information is available.

AFD: an application for bi-molecular interaction using axial frequency distribution.

Science.gov (United States)

Raza, Saad; Azam, Syed Sikander

2018-03-06

Conformational flexibility and generalized structural features are responsible for specific phenomena existing in biological pathways. With advancements in computational chemistry, novel approaches and new methods are required to compare the dynamic nature of biomolecules, which are crucial not only to address dynamic functional relationships but also to gain detailed insights into the disturbance and positional fluctuation responsible for functional shifts. Keeping this in mind, axial frequency distribution (AFD) has been developed, designed, and implemented. AFD can profoundly represent distribution and density of ligand atom around a particular atom or set of atoms. It enabled us to obtain an explanation of local movements and rotations, which are not significantly highlighted by any other structural and dynamical parameters. AFD can be implemented on biological models representing ligand and protein interactions. It shows a comprehensive view of the binding pattern of ligand by exploring the distribution of atoms relative to the x-y plane of the system. By taking a relative centroid on protein or ligand, molecular interactions like hydrogen bonds, van der Waals, polar or ionic interaction can be analyzed to cater the ligand movement, stabilization or flexibility with respect to the protein. The AFD graph resulted in the residual depiction of bi-molecular interaction in gradient form which can yield specific information depending upon the system of interest.

Conformational transitions and interactions underlying the function of membrane embedded receptor protein kinases.

Science.gov (United States)

Bocharov, Eduard V; Sharonov, Georgy V; Bocharova, Olga V; Pavlov, Konstantin V

2017-09-01

Among membrane receptors, the single-span receptor protein kinases occupy a broad but specific functional niche determined by distinctive features of the underlying transmembrane signaling mechanisms that are briefly overviewed on the basis of some of the most representative examples, followed by a more detailed discussion of several hierarchical levels of organization and interactions involved. All these levels, including single-molecule interactions (e.g., dimerization, liganding, chemical modifications), local processes (e.g. lipid membrane perturbations, cytoskeletal interactions), and larger scale phenomena (e.g., effects of membrane surface shape or electrochemical potential gradients) appear to be closely integrated to achieve the observed diversity of the receptor functioning. Different species of receptor protein kinases meet their specific functional demands through different structural features defining their responses to stimulation, but certain common patterns exist. Signaling by receptor protein kinases is typically associated with the receptor dimerization and clustering, ligand-induced rearrangements of receptor domains through allosteric conformational transitions with involvement of lipids, release of the sequestered lipids, restriction of receptor diffusion, cytoskeleton and membrane shape remodeling. Understanding of complexity and continuity of the signaling processes can help identifying currently neglected opportunities for influencing the receptor signaling with potential therapeutic implications. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova. Copyright © 2017 Elsevier B.V. All rights reserved.

Specificity of molecular interactions in transient protein-protein interaction interfaces.

Science.gov (United States)

Cho, Kyu-il; Lee, KiYoung; Lee, Kwang H; Kim, Dongsup; Lee, Doheon

2006-11-15

In this study, we investigate what types of interactions are specific to their biological function, and what types of interactions are persistent regardless of their functional category in transient protein-protein heterocomplexes. This is the first approach to analyze protein-protein interfaces systematically at the molecular interaction level in the context of protein functions. We perform systematic analysis at the molecular interaction level using classification and feature subset selection technique prevalent in the field of pattern recognition. To represent the physicochemical properties of protein-protein interfaces, we design 18 molecular interaction types using canonical and noncanonical interactions. Then, we construct input vector using the frequency of each interaction type in protein-protein interface. We analyze the 131 interfaces of transient protein-protein heterocomplexes in PDB: 33 protease-inhibitors, 52 antibody-antigens, 46 signaling proteins including 4 cyclin dependent kinase and 26 G-protein. Using kNN classification and feature subset selection technique, we show that there are specific interaction types based on their functional category, and such interaction types are conserved through the common binding mechanism, rather than through the sequence or structure conservation. The extracted interaction types are C(alpha)-- H...O==C interaction, cation...anion interaction, amine...amine interaction, and amine...cation interaction. With these four interaction types, we achieve the classification success rate up to 83.2% with leave-one-out cross-validation at k = 15. Of these four interaction types, C(alpha)--H...O==C shows binding specificity for protease-inhibitor complexes, while cation-anion interaction is predominant in signaling complexes. The amine ... amine and amine...cation interaction give a minor contribution to the classification accuracy. When combined with these two interactions, they increase the accuracy by 3.8%. In the case of

Macromolecular Competition Titration Method: Accessing Thermodynamics of the Unmodified Macromolecule–Ligand Interactions Through Spectroscopic Titrations of Fluorescent Analogs

Science.gov (United States)

Bujalowski, Wlodzimierz; Jezewska, Maria J.

2011-01-01

Analysis of thermodynamically rigorous binding isotherms provides fundamental information about the energetics of the ligand–macromolecule interactions and often an invaluable insight about the structure of the formed complexes. The Macromolecular Competition Titration (MCT) method enables one to quantitatively obtain interaction parameters of protein–nucleic acid interactions, which may not be available by other methods, particularly for the unmodified long polymer lattices and specific nucleic acid substrates, if the binding is not accompanied by adequate spectroscopic signal changes. The method can be applied using different fluorescent nucleic acids or fluorophores, although the etheno-derivatives of nucleic acid are especially suitable as they are relatively easy to prepare, have significant blue fluorescence, their excitation band lies far from the protein absorption spectrum, and the modification eliminates the possibility of base pairing with other nucleic acids. The MCT method is not limited to the specific size of the reference nucleic acid. Particularly, a simple analysis of the competition titration experiments is described in which the fluorescent, short fragment of nucleic acid, spanning the exact site-size of the protein–nucleic acid complex, and binding with only a 1:1 stoichiometry to the protein, is used as a reference macromolecule. Although the MCT method is predominantly discussed as applied to studying protein–nucleic acid interactions, it can generally be applied to any ligand–macromolecule system by monitoring the association reaction using the spectroscopic signal originating from the reference macromolecule in the presence of the competing macromolecule, whose interaction parameters with the ligand are to be determined. PMID:21195223

Adaptor proteins intersectin 1 and 2 bind similar proline-rich ligands but are differentially recognized by SH2 domain-containing proteins.

Directory of Open Access Journals (Sweden)

Olga Novokhatska

Full Text Available BACKGROUND: Scaffolding proteins of the intersectin (ITSN family, ITSN1 and ITSN2, are crucial for the initiation stage of clathrin-mediated endocytosis. These proteins are closely related but have implications in distinct pathologies. To determine how these proteins could be separated in certain cell pathways we performed a comparative study of ITSNs. METHODOLOGY/PRINCIPAL FINDINGS: We have shown that endogenous ITSN1 and ITSN2 colocalize and form a complex in cells. A structural comparison of five SH3 domains, which mediated most ITSNs protein-protein interactions, demonstrated a similarity of their ligand-binding sites. We showed that the SH3 domains of ITSN2 bound well-established interactors of ITSN1 as well as newly identified ITSNs protein partners. A search for a novel interacting interface revealed multiple tyrosines that could be phosphorylated in ITSN2. Phosphorylation of ITSN2 isoforms but not ITSN1 short isoform was observed in various cell lines. EGF stimulation of HeLa cells enhanced tyrosine phosphorylation of ITSN2 isoforms and enabled their recognition by the SH2 domains of the Fyn, Fgr and Abl1 kinases, the regulatory subunit of PI3K, the adaptor proteins Grb2 and Crk, and phospholipase C gamma. The SH2 domains mentioned were unable to bind ITSN1 short isoform. CONCLUSIONS/SIGNIFICANCE: Our results indicate that during evolution of vertebrates ITSN2 acquired a novel protein-interaction interface that allows its specific recognition by the SH2 domains of signaling proteins. We propose that these data could be important to understand the functional diversity of paralogous ITSN proteins.

Measuring binding of protein to gel-bound ligands using magnetic levitation.

Science.gov (United States)

Shapiro, Nathan D; Mirica, Katherine A; Soh, Siowling; Phillips, Scott T; Taran, Olga; Mace, Charles R; Shevkoplyas, Sergey S; Whitesides, George M

2012-03-28

This paper describes the use of magnetic levitation (MagLev) to measure the association of proteins and ligands. The method starts with diamagnetic gel beads that are functionalized covalently with small molecules (putative ligands). Binding of protein to the ligands within the bead causes a change in the density of the bead. When these beads are suspended in a paramagnetic aqueous buffer and placed between the poles of two NbFeB magnets with like poles facing, the changes in the density of the bead on binding of protein result in changes in the levitation height of the bead that can be used to quantify the amount of protein bound. This paper uses a reaction-diffusion model to examine the physical principles that determine the values of rate and equilibrium constants measured by this system, using the well-defined model system of carbonic anhydrase and aryl sulfonamides. By tuning the experimental protocol, the method is capable of quantifying either the concentration of protein in a solution, or the binding affinities of a protein to several resin-bound small molecules simultaneously. Since this method requires no electricity and only a single piece of inexpensive equipment, it may find use in situations where portability and low cost are important, such as in bioanalysis in resource-limited settings, point-of-care diagnosis, veterinary medicine, and plant pathology. It still has several practical disadvantages. Most notably, the method requires relatively long assay times and cannot be applied to large proteins (>70 kDa), including antibodies. The design and synthesis of beads with improved characteristics (e.g., larger pore size) has the potential to resolve these problems.

Detecting Local Ligand-Binding Site Similarity in Non-Homologous Proteins by Surface Patch Comparison

Science.gov (United States)

Sael, Lee; Kihara, Daisuke

2012-01-01

Functional elucidation of proteins is one of the essential tasks in biology. Function of a protein, specifically, small ligand molecules that bind to a protein, can be predicted by finding similar local surface regions in binding sites of known proteins. Here, we developed an alignment free local surface comparison method for predicting a ligand molecule which binds to a query protein. The algorithm, named Patch-Surfer, represents a binding pocket as a combination of segmented surface patches, each of which is characterized by its geometrical shape, the electrostatic potential, the hydrophobicity, and the concaveness. Representing a pocket by a set of patches is effective to absorb difference of global pocket shape while capturing local similarity of pockets. The shape and the physicochemical properties of surface patches are represented using the 3D Zernike descriptor, which is a series expansion of mathematical 3D function. Two pockets are compared using a modified weighted bipartite matching algorithm, which matches similar patches from the two pockets. Patch-Surfer was benchmarked on three datasets, which consist in total of 390 proteins that bind to one of 21 ligands. Patch-Surfer showed superior performance to existing methods including a global pocket comparison method, Pocket-Surfer, which we have previously introduced. Particularly, as intended, the accuracy showed large improvement for flexible ligand molecules, which bind to pockets in different conformations. PMID:22275074

A PM7 dynamic residue-ligand interactions energy landscape of the BACE1 inhibitory pathway by hydroxyethylamine compounds. Part I: The flap closure process.

Science.gov (United States)

Gueto-Tettay, Carlos; Martinez-Consuegra, Alejandro; Zuchniarz, Joshua; Gueto-Tettay, Luis Roberto; Drosos-Ramírez, Juan Carlos

2017-09-01

BACE1 is an enzyme of scientific interest because it participates in the progression of Alzheimer's disease. Hydroxyethylamines (HEAs) are a family of compounds which exhibit inhibitory activity toward BACE1 at a nanomolar level, favorable pharmacokinetic properties and oral bioavailability. The first step in the inhibition of BACE1 by HEAs consists of their entrance into the protease active site and the resultant conformational change in the protein, from Apo to closed form. These two conformations differ in the position of an antiparallel loop (called the flap) which covers the entrance to the catalytic site. For BACE1, closure of this flap is vital to its catalytic activity and to inhibition of the enzyme due to the new interactions thereby formed with the ligand. In the present study a dynamic energy landscape of residue-ligand interaction energies (ReLIE) measured for 112 amino acids in the BACE1 active site and its immediate vicinity during the closure of the flap induced by 8 HEAs of different inhibitory power is presented. A total of 6.272 million ReLIE calculations, based on the PM7 semiempirical method, provided a deep and quantitative view of the first step in the inhibition of the aspartyl protease. The information suggests that residues Asp93, Asp289, Thr292, Thr293, Asn294 and Arg296 are anchor points for the ligand, accounting for approximately 45% of the total protein-ligand interaction. Additionally, flap closure improved the BACE1-HEA interaction by around 25%. Furthermore, the inhibitory activity of HEAs could be related to the capacity of these ligands to form said anchor point interactions and maintain them over time: the lack of some of these anchor interactions delayed flap closure or impeded it completely, or even caused the flap to reopen. The methodology employed here could be used as a tool to evaluate future structural modifications which lead to improvements in the favorability and stability of BACE1-HEA ReLIEs, aiding in the design of

Exploring the Ligand-Protein Networks in Traditional Chinese Medicine: Current Databases, Methods, and Applications

Directory of Open Access Journals (Sweden)

Mingzhu Zhao

2013-01-01

Full Text Available The traditional Chinese medicine (TCM, which has thousands of years of clinical application among China and other Asian countries, is the pioneer of the “multicomponent-multitarget” and network pharmacology. Although there is no doubt of the efficacy, it is difficult to elucidate convincing underlying mechanism of TCM due to its complex composition and unclear pharmacology. The use of ligand-protein networks has been gaining significant value in the history of drug discovery while its application in TCM is still in its early stage. This paper firstly surveys TCM databases for virtual screening that have been greatly expanded in size and data diversity in recent years. On that basis, different screening methods and strategies for identifying active ingredients and targets of TCM are outlined based on the amount of network information available, both on sides of ligand bioactivity and the protein structures. Furthermore, applications of successful in silico target identification attempts are discussed in detail along with experiments in exploring the ligand-protein networks of TCM. Finally, it will be concluded that the prospective application of ligand-protein networks can be used not only to predict protein targets of a small molecule, but also to explore the mode of action of TCM.

Computational Approaches to the Chemical Equilibrium Constant in Protein-ligand Binding.

Science.gov (United States)

Montalvo-Acosta, Joel José; Cecchini, Marco

2016-12-01

The physiological role played by protein-ligand recognition has motivated the development of several computational approaches to the ligand binding affinity. Some of them, termed rigorous, have a strong theoretical foundation but involve too much computation to be generally useful. Some others alleviate the computational burden by introducing strong approximations and/or empirical calibrations, which also limit their general use. Most importantly, there is no straightforward correlation between the predictive power and the level of approximation introduced. Here, we present a general framework for the quantitative interpretation of protein-ligand binding based on statistical mechanics. Within this framework, we re-derive self-consistently the fundamental equations of some popular approaches to the binding constant and pinpoint the inherent approximations. Our analysis represents a first step towards the development of variants with optimum accuracy/efficiency ratio for each stage of the drug discovery pipeline. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Extreme sequence divergence but conserved ligand-binding specificity in Streptococcus pyogenes M protein.

Directory of Open Access Journals (Sweden)

2006-05-01

Full Text Available Many pathogenic microorganisms evade host immunity through extensive sequence variability in a protein region targeted by protective antibodies. In spite of the sequence variability, a variable region commonly retains an important ligand-binding function, reflected in the presence of a highly conserved sequence motif. Here, we analyze the limits of sequence divergence in a ligand-binding region by characterizing the hypervariable region (HVR of Streptococcus pyogenes M protein. Our studies were focused on HVRs that bind the human complement regulator C4b-binding protein (C4BP, a ligand that confers phagocytosis resistance. A previous comparison of C4BP-binding HVRs identified residue identities that could be part of a binding motif, but the extended analysis reported here shows that no residue identities remain when additional C4BP-binding HVRs are included. Characterization of the HVR in the M22 protein indicated that two relatively conserved Leu residues are essential for C4BP binding, but these residues are probably core residues in a coiled-coil, implying that they do not directly contribute to binding. In contrast, substitution of either of two relatively conserved Glu residues, predicted to be solvent-exposed, had no effect on C4BP binding, although each of these changes had a major effect on the antigenic properties of the HVR. Together, these findings show that HVRs of M proteins have an extraordinary capacity for sequence divergence and antigenic variability while retaining a specific ligand-binding function.

Analytical developments for screening of lanthanides/ligands interactions

International Nuclear Information System (INIS)

Varenne, F.

2012-01-01

This work investigates the potential of hyphenated capillary electrophoresis and inductively coupled mass spectrometry to classify different ligands according to their europium binding affinity in a hydro-organic medium. On the one hand, this method enables to evaluate the affinity of phosphorus-containing ligands in less than two hours and using less than 15 ng of ligand. On the other hand, complexation constants could be determined. The results are in excellent agreement with the values obtained by spectrophotometric titrations.Moreover, a library of copolymers for solid/liquid extraction of europium is investigated. The extraction protocol enables to classify copolymers according to their europium affinity in a hydro-organic medium. This screening requires 60 mg of copolymers. For the most promising recognition properties and selectivity La 3+ /Eu 3+ /Lu 3+ are evaluated. (author)

Quantifying high-affinity binding of hydrophobic ligands by isothermal titration calorimetry.

Science.gov (United States)

Krainer, Georg; Broecker, Jana; Vargas, Carolyn; Fanghänel, Jörg; Keller, Sandro

2012-12-18

A fast and reliable quantification of the binding thermodynamics of hydrophobic high-affinity ligands employing a new calorimetric competition experiment is described. Although isothermal titration calorimetry is the method of choice for a quantitative characterization of intermolecular interactions in solution, a reliable determination of a dissociation constant (K(D)) is typically limited to the range 100 μM > K(D) > 1 nM. Interactions displaying higher or lower K(D) values can be assessed indirectly, provided that a suitable competing ligand is available whose K(D) falls within the directly accessible affinity window. This established displacement assay, however, requires the high-affinity ligand to be soluble at high concentrations in aqueous buffer and, consequently, poses serious problems in the study of protein binding involving small-molecule ligands dissolved in organic solvents--a familiar case in many drug-discovery projects relying on compound libraries. The calorimetric competition assay introduced here overcomes this limitation, thus allowing for a detailed thermodynamic description of high-affinity receptor-ligand interactions involving poorly water-soluble compounds. Based on a single titration of receptor into a dilute mixture of the two competing ligands, this competition assay provides accurate and precise values for the dissociation constants and binding enthalpies of both high- and moderate-affinity ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation and high-affinity protein-inhibitor interactions, and explore its potential and limitations with the aid of simulations and statistical analyses.

Computational multiscale modeling in protein--ligand docking.

Science.gov (United States)

Taufer, Michela; Armen, Roger; Chen, Jianhan; Teller, Patricia; Brooks, Charles

2009-01-01

In biological systems, the binding of small molecule ligands to proteins is a crucial process for almost every aspect of biochemistry and molecular biology. Enzymes are proteins that function by catalyzing specific biochemical reactions that convert reactants into products. Complex organisms are typically composed of cells in which thousands of enzymes participate in complex and interconnected biochemical pathways. Some enzymes serve as sequential steps in specific pathways (such as energy metabolism), while others function to regulate entire pathways and cellular functions [1]. Small molecule ligands can be designed to bind to a specific enzyme and inhibit the biochemical reaction. Inhibiting the activity of key enzymes may result in the entire biochemical pathways being turned on or off [2], [3]. Many small molecule drugs marketed today function in this generic way as enzyme inhibitors. If research identifies a specific enzyme as being crucial to the progress of disease, then this enzyme may be targeted with an inhibitor, which may slow down or reverse the progress of disease. In this way, enzymes are targeted from specific pathogens (e.g., virus, bacteria, fungi) for infectious diseases [4], [5], and human enzymes are targeted for noninfectious diseases such as cardiovascular disease, cancer, diabetes, and neurodegenerative diseases [6].

Real-Time Ligand Binding Pocket Database Search Using Local Surface Descriptors

Science.gov (United States)

Chikhi, Rayan; Sael, Lee; Kihara, Daisuke

2010-01-01

Due to the increasing number of structures of unknown function accumulated by ongoing structural genomics projects, there is an urgent need for computational methods for characterizing protein tertiary structures. As functions of many of these proteins are not easily predicted by conventional sequence database searches, a legitimate strategy is to utilize structure information in function characterization. Of a particular interest is prediction of ligand binding to a protein, as ligand molecule recognition is a major part of molecular function of proteins. Predicting whether a ligand molecule binds a protein is a complex problem due to the physical nature of protein-ligand interactions and the flexibility of both binding sites and ligand molecules. However, geometric and physicochemical complementarity is observed between the ligand and its binding site in many cases. Therefore, ligand molecules which bind to a local surface site in a protein can be predicted by finding similar local pockets of known binding ligands in the structure database. Here, we present two representations of ligand binding pockets and utilize them for ligand binding prediction by pocket shape comparison. These representations are based on mapping of surface properties of binding pockets, which are compactly described either by the two dimensional pseudo-Zernike moments or the 3D Zernike descriptors. These compact representations allow a fast real-time pocket searching against a database. Thorough benchmark study employing two different datasets show that our representations are competitive with the other existing methods. Limitations and potentials of the shape-based methods as well as possible improvements are discussed. PMID:20455259

A Fragment-Based Ligand Screen Against Part of a Large Protein Machine: The ND1 Domains of the AAA+ ATPase p97/VCP.

Science.gov (United States)

Chimenti, Michael S; Bulfer, Stacie L; Neitz, R Jeffrey; Renslo, Adam R; Jacobson, Matthew P; James, Thomas L; Arkin, Michelle R; Kelly, Mark J S

2015-07-01

The ubiquitous AAA+ ATPase p97 functions as a dynamic molecular machine driving several cellular processes. It is essential in regulating protein homeostasis, and it represents a potential drug target for cancer, particularly when there is a greater reliance on the endoplasmic reticulum-associated protein degradation pathway and ubiquitin-proteasome pathway to degrade an overabundance of secreted proteins. Here, we report a case study for using fragment-based ligand design approaches against this large and dynamic hexamer, which has multiple potential binding sites for small molecules. A screen of a fragment library was conducted by surface plasmon resonance (SPR) and followed up by nuclear magnetic resonance (NMR), two complementary biophysical techniques. Virtual screening was also carried out to examine possible binding sites for the experimental hits and evaluate the potential utility of fragment docking for this target. Out of this effort, 13 fragments were discovered that showed reversible binding with affinities between 140 µM and 1 mM, binding stoichiometries of 1:1 or 2:1, and good ligand efficiencies. Structural data for fragment-protein interactions were obtained with residue-specific [U-(2)H] (13)CH3-methyl-labeling NMR strategies, and these data were compared to poses from docking. The combination of virtual screening, SPR, and NMR enabled us to find and validate a number of interesting fragment hits and allowed us to gain an understanding of the structural nature of fragment binding. © 2015 Society for Laboratory Automation and Screening.

Using chemical shift perturbation to characterise ligand binding.

Science.gov (United States)

Williamson, Mike P

2013-08-01

Chemical shift perturbation (CSP, chemical shift mapping or complexation-induced changes in chemical shift, CIS) follows changes in the chemical shifts of a protein when a ligand is added, and uses these to determine the location of the binding site, the affinity of the ligand, and/or possibly the structure of the complex. A key factor in determining the appearance of spectra during a titration is the exchange rate between free and bound, or more specifically the off-rate koff. When koff is greater than the chemical shift difference between free and bound, which typically equates to an affinity Kd weaker than about 3μM, then exchange is fast on the chemical shift timescale. Under these circumstances, the observed shift is the population-weighted average of free and bound, which allows Kd to be determined from measurement of peak positions, provided the measurements are made appropriately. (1)H shifts are influenced to a large extent by through-space interactions, whereas (13)Cα and (13)Cβ shifts are influenced more by through-bond effects. (15)N and (13)C' shifts are influenced both by through-bond and by through-space (hydrogen bonding) interactions. For determining the location of a bound ligand on the basis of shift change, the most appropriate method is therefore usually to measure (15)N HSQC spectra, calculate the geometrical distance moved by the peak, weighting (15)N shifts by a factor of about 0.14 compared to (1)H shifts, and select those residues for which the weighted shift change is larger than the standard deviation of the shift for all residues. Other methods are discussed, in particular the measurement of (13)CH3 signals. Slow to intermediate exchange rates lead to line broadening, and make Kd values very difficult to obtain. There is no good way to distinguish changes in chemical shift due to direct binding of the ligand from changes in chemical shift due to allosteric change. Ligand binding at multiple sites can often be characterised, by

LigandRFs: random forest ensemble to identify ligand-binding residues from sequence information alone

KAUST Repository

Chen, Peng; Huang, Jianhua Z; Gao, Xin

2014-01-01

Protein-ligand binding is important for some proteins to perform their functions. Protein-ligand binding sites are the residues of proteins that physically bind to ligands. Despite of the recent advances in computational prediction

Quantification of protein interaction kinetics in a micro droplet

Energy Technology Data Exchange (ETDEWEB)

Yin, L. L. [Center for Bioelectronics and Biosensors, Biodesign Institute, Arizona State University, Tempe, Arizona 85287 (United States); College of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044 (China); Wang, S. P., E-mail: shaopeng.wang@asu.edu, E-mail: njtao@asu.edu; Shan, X. N.; Tao, N. J., E-mail: shaopeng.wang@asu.edu, E-mail: njtao@asu.edu [Center for Bioelectronics and Biosensors, Biodesign Institute, Arizona State University, Tempe, Arizona 85287 (United States); Zhang, S. T. [College of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044 (China)

2015-11-15

Characterization of protein interactions is essential to the discovery of disease biomarkers, the development of diagnostic assays, and the screening for therapeutic drugs. Conventional flow-through kinetic measurements need relative large amount of sample that is not feasible for precious protein samples. We report a novel method to measure protein interaction kinetics in a single droplet with sub microliter or less volume. A droplet in a humidity-controlled environmental chamber is replacing the microfluidic channels as the reactor for the protein interaction. The binding process is monitored by a surface plasmon resonance imaging (SPRi) system. Association curves are obtained from the average SPR image intensity in the center area of the droplet. The washing step required by conventional flow-through SPR method is eliminated in the droplet method. The association and dissociation rate constants and binding affinity of an antigen-antibody interaction are obtained by global fitting of association curves at different concentrations. The result obtained by this method is accurate as validated by conventional flow-through SPR system. This droplet-based method not only allows kinetic studies for proteins with limited supply but also opens the door for high-throughput protein interaction study in a droplet-based microarray format that enables measurement of many to many interactions on a single chip.

LigandRFs: random forest ensemble to identify ligand-binding residues from sequence information alone

KAUST Repository

Chen, Peng

2014-12-03

Background Protein-ligand binding is important for some proteins to perform their functions. Protein-ligand binding sites are the residues of proteins that physically bind to ligands. Despite of the recent advances in computational prediction for protein-ligand binding sites, the state-of-the-art methods search for similar, known structures of the query and predict the binding sites based on the solved structures. However, such structural information is not commonly available. Results In this paper, we propose a sequence-based approach to identify protein-ligand binding residues. We propose a combination technique to reduce the effects of different sliding residue windows in the process of encoding input feature vectors. Moreover, due to the highly imbalanced samples between the ligand-binding sites and non ligand-binding sites, we construct several balanced data sets, for each of which a random forest (RF)-based classifier is trained. The ensemble of these RF classifiers forms a sequence-based protein-ligand binding site predictor. Conclusions Experimental results on CASP9 and CASP8 data sets demonstrate that our method compares favorably with the state-of-the-art protein-ligand binding site prediction methods.

Biotechnological Fluorescent Ligands of the Bradykinin B1 Receptor: Protein Ligands for a Peptide Receptor.

Directory of Open Access Journals (Sweden)

Xavier Charest-Morin

Full Text Available The bradykinin (BK B1 receptor (B1R is a peculiar G protein coupled receptor that is strongly regulated to the point of being inducible in immunopathology. Limited clinical evidence suggests that its expression in peripheral blood mononuclear cells is a biomarker of active inflammatory states. In an effort to develop a novel imaging/diagnostic tool, we report the rational design and testing of a fusion protein that is a ligand of the human B1R but not likely to label peptidases. This ligand is composed of a fluorescent protein (FP (enhanced green FP [EGFP] or mCherry prolonged at its N-terminus by a spacer peptide and a classical peptide agonist or antagonist (des-Arg9-BK, [Leu8]des-Arg9-BK, respectively. The design of the spacer-ligand joint peptide was validated by a competition assay for [3H]Lys-des-Arg9-BK binding to the human B1R applied to 4 synthetic peptides of 18 or 19 residues. The labeling of B1R-expressing cells with EGFP or mCherry fused with 7 of such peptides was performed in parallel (microscopy. Both assays indicated that the best design was FP-(Asn-Glyn-Lys-des-Arg9-BK; n = 15 was superior to n = 5, suggesting benefits from minimizing steric hindrance between the FP and the receptor. Cell labeling concerned mostly plasma membranes and was inhibited by a B1R antagonist. EGFP-(Asn-Gly15-Lys-des-Arg9-BK competed for the binding of [3H]Lys-des-Arg9-BK to human recombinant B1R, being only 10-fold less potent than the unlabeled form of Lys-des-Arg9-BK to do so. The fusion protein did not label HEK 293a cells expressing recombinant human BK B2 receptors or angiotensin converting enzyme. This study identifies a modular C-terminal sequence that can be adapted to protein cargoes, conferring high affinity for the BK B1R, with possible applications in diagnostic cytofluorometry, histology and drug delivery (e.g., in oncology.

Importance of a Conserved Lys/Arg Residue for Ligand/PDZ Domain Interactions as Examined by Protein Semisynthesis

DEFF Research Database (Denmark)

Pedersen, Søren W; Moran, Griffin E; Sereikaité, Vita

2016-01-01

PDZ domains are ubiquitous small protein domains that are mediators of numerous protein-protein interactions, and play a pivotal role in protein trafficking, synaptic transmission, and the assembly of signaling-transduction complexes. In recent years, PDZ domains have emerged as novel and exciting...... drug targets for diseases (in the brain in particular), so understanding the molecular details of PDZ domain interactions is of fundamental importance. PDZ domains bind to a protein partner at either a C-terminal peptide or internal peptide motifs. Here, we examined the importance of a conserved Lys...

Dr. PIAS: an integrative system for assessing the druggability of protein-protein interactions

Directory of Open Access Journals (Sweden)

Furuya Toshio

2011-02-01

Full Text Available Abstract Background The amount of data on protein-protein interactions (PPIs available in public databases and in the literature has rapidly expanded in recent years. PPI data can provide useful information for researchers in pharmacology and medicine as well as those in interactome studies. There is urgent need for a novel methodology or software allowing the efficient utilization of PPI data in pharmacology and medicine. Results To address this need, we have developed the 'Druggable Protein-protein Interaction Assessment System' (Dr. PIAS. Dr. PIAS has a meta-database that stores various types of information (tertiary structures, drugs/chemicals, and biological functions associated with PPIs retrieved from public sources. By integrating this information, Dr. PIAS assesses whether a PPI is druggable as a target for small chemical ligands by using a supervised machine-learning method, support vector machine (SVM. Dr. PIAS holds not only known druggable PPIs but also all PPIs of human, mouse, rat, and human immunodeficiency virus (HIV proteins identified to date. Conclusions The design concept of Dr. PIAS is distinct from other published PPI databases in that it focuses on selecting the PPIs most likely to make good drug targets, rather than merely collecting PPI data.

Solution characterization of the extracellular region of CD147 and its interaction with its enzyme ligand cyclophilin-A

Energy Technology Data Exchange (ETDEWEB)

Schlegel, Jennifer; Redzic, Jasmina S.; Porter, Christopher; Yurchenko, Vyacheslav; Bukrinsky, Michael; Labeikovsky, Wladimir; Armstrong, Geoffrey S.; Zhang, Fengli; Isern, Nancy G.; Degregori, James; Hodges, Robert; Eisenmesser, Elan Z.

2009-08-21

The CD147 receptor plays an integral role in numerous diseases by stimulating the expression of several protein families and serving as the receptor for extracellular cyclophilins, however, neither CD147 nor its interactions with its cyclophilin ligands have been well characterized in solution. CD147 is a unique protein in that it can function both at the cell membrane and after being released from cells where it continues to retain activity. Thus, the CD147 receptor functions through at least two mechanisms that include both cyclophilin-independent and cyclophilin-dependent modes of action. In regard to CD147 cyclophilin-independent activity, CD147 homophilic interactions are thought to underlie its activity. In regard to CD147 cyclophilin-dependent activity, cyclophilin/CD147 interactions may represent a novel means of signaling since cyclophilins are also peptidyl-prolyl isomerases.

Estimation of kinetic and thermodynamic ligand-binding parameters using computational strategies.

Science.gov (United States)

Deganutti, Giuseppe; Moro, Stefano

2017-04-01

Kinetic and thermodynamic ligand-protein binding parameters are gaining growing importance as key information to consider in drug discovery. The determination of the molecular structures, using particularly x-ray and NMR techniques, is crucial for understanding how a ligand recognizes its target in the final binding complex. However, for a better understanding of the recognition processes, experimental studies of ligand-protein interactions are needed. Even though several techniques can be used to investigate both thermodynamic and kinetic profiles for a ligand-protein complex, these procedures are very often laborious, time consuming and expensive. In the last 10 years, computational approaches have enormous potential in providing insights into each of the above effects and in parsing their contributions to the changes in both kinetic and thermodynamic binding parameters. The main purpose of this review is to summarize the state of the art of computational strategies for estimating the kinetic and thermodynamic parameters of a ligand-protein binding.

Online size-exclusion high-performance liquid chromatography light scattering and differential refractometry methods to determine degree of polymer conjugation to proteins and protein-protein or protein-ligand association states.

Science.gov (United States)

Kendrick, B S; Kerwin, B A; Chang, B S; Philo, J S

2001-12-15

Characterizing the solution structure of protein-polymer conjugates and protein-ligand interactions is important in fields such as biotechnology and biochemistry. Size-exclusion high-performance liquid chromatography with online classical light scattering (LS), refractive index (RI), and UV detection offers a powerful tool in such characterization. Novel methods are presented utilizing LS, RI, and UV signals to rapidly determine the degree of conjugation and the molecular mass of the protein conjugate. Baseline resolution of the chromatographic peaks is not required; peaks need only be sufficiently separated to represent relatively pure fractions. An improved technique for determining the polypeptide-only mass of protein conjugates is also described. These techniques are applied to determining the degree of erythropoietin glycosylation, the degree of polyethylene glycol conjugation to RNase A and brain-derived neurotrophic factor, and the solution association states of these molecules. Calibration methods for the RI, UV, and LS detectors will also be addressed, as well as online methods to determine protein extinction coefficients and dn/dc values both unconjugated and conjugated protein molecules. (c)2001 Elsevier Science.

Preclinical Testing of a Translocator Protein Ligand for the Treatment of Amyotrophic Lateral Sclerosis

Science.gov (United States)

2017-03-01

investigated the neuroprotective effect and therapeutic value of a drug : the translocator protein (TSPO) ligand PK11195 (PK), which we found to be...Translocator protein (TSPO) ligand Neuroprotective drugs In vitro models of neurodegeneration Axon preservation Neuromuscular junctions 5 3...pharmacokinetic data of the JNK study were showing that daily gavage (30 mg/kg/day) allowed an efficient delivery of the drugs in the brain and the spinal

Nanoparticle-based receptors mimic protein-ligand recognition

OpenAIRE

Riccardi, Laura; Gabrielli, Luca; Sun, Xiaohuan; Biasi, Federico De; Rastrelli, Federico; Mancin, Fabrizio; De Vivo, Marco

2017-01-01

Summary The self-assembly of a monolayer of ligands on the surface of noble-metal nanoparticles dictates the fundamental nanoparticle's behavior and its functionality. In this combined computational-experimental study, we analyze the structure, organization, and dynamics of functionalized coating thiols in monolayer-protected gold nanoparticles (AuNPs). We explain how functionalized coating thiols self-organize through a delicate and somehow counterintuitive balance of interactions within the...

Affinity-based, biophysical methods to detect and analyze ligand binding to recombinant proteins: matching high information content with high throughput.

Science.gov (United States)

Holdgate, Geoff A; Anderson, Malcolm; Edfeldt, Fredrik; Geschwindner, Stefan

2010-10-01

Affinity-based technologies have become impactful tools to detect, monitor and characterize molecular interactions using recombinant target proteins. This can aid the understanding of biological function by revealing mechanistic details, and even more importantly, enables the identification of new improved ligands that can modulate the biological activity of those targets in a desired fashion. The selection of the appropriate technology is a key step in that process, as each one of the currently available technologies offers a characteristic type of biophysical information about the ligand-binding event. Alongside the indisputable advantages of each of those technologies they naturally display diverse restrictions that are quite frequently related to the target system to be studied but also to the affinity, solubility and molecular size of the ligands. This paper discusses some of the theoretical and experimental aspects of the most common affinity-based methods, what type of information can be gained from each one of those approaches, and what requirements as well as limitations are expected from working with recombinant proteins on those platforms and how those can be optimally addressed.

An analysis pipeline for the inference of protein-protein interaction networks

Energy Technology Data Exchange (ETDEWEB)

Taylor, Ronald C.; Singhal, Mudita; Daly, Don S.; Gilmore, Jason M.; Cannon, William R.; Domico, Kelly O.; White, Amanda M.; Auberry, Deanna L.; Auberry, Kenneth J.; Hooker, Brian S.; Hurst, G. B.; McDermott, Jason E.; McDonald, W. H.; Pelletier, Dale A.; Schmoyer, Denise A.; Wiley, H. S.

2009-12-01

An analysis pipeline has been created for deployment of a novel algorithm, the Bayesian Estimator of Protein-Protein Association Probabilities (BEPro), for use in the reconstruction of protein-protein interaction networks. We have combined the Software Environment for BIological Network Inference (SEBINI), an interactive environment for the deployment and testing of network inference algorithms that use high-throughput data, and the Collective Analysis of Biological Interaction Networks (CABIN), software that allows integration and analysis of protein-protein interaction and gene-to-gene regulatory evidence obtained from multiple sources, to allow interactions computed by BEPro to be stored, visualized, and further analyzed. Incorporating BEPro into SEBINI and automatically feeding the resulting inferred network into CABIN, we have created a structured workflow for protein-protein network inference and supplemental analysis from sets of mass spectrometry bait-prey experiment data. SEBINI demo site: https://www.emsl.pnl.gov /SEBINI/ Contact: ronald.taylor@pnl.gov. BEPro is available at http://www.pnl.gov/statistics/BEPro3/index.htm. Contact: ds.daly@pnl.gov. CABIN is available at http://www.sysbio.org/dataresources/cabin.stm. Contact: mudita.singhal@pnl.gov.

Kinase Substrate Sensor (KISS), a mammalian in situ protein interaction sensor.

Science.gov (United States)

Lievens, Sam; Gerlo, Sarah; Lemmens, Irma; De Clercq, Dries J H; Risseeuw, Martijn D P; Vanderroost, Nele; De Smet, Anne-Sophie; Ruyssinck, Elien; Chevet, Eric; Van Calenbergh, Serge; Tavernier, Jan

2014-12-01

Probably every cellular process is governed by protein-protein interaction (PPIs), which are often highly dynamic in nature being modulated by in- or external stimuli. Here we present KISS, for KInase Substrate Sensor, a mammalian two-hybrid approach designed to map intracellular PPIs and some of the dynamic features they exhibit. Benchmarking experiments indicate that in terms of sensitivity and specificity KISS is on par with other binary protein interaction technologies while being complementary with regard to the subset of PPIs it is able to detect. We used KISS to evaluate interactions between different types of proteins, including transmembrane proteins, expressed at their native subcellular location. In situ analysis of endoplasmic reticulum stress-induced clustering of the endoplasmic reticulum stress sensor ERN1 and ligand-dependent β-arrestin recruitment to GPCRs illustrated the method's potential to study functional PPI modulation in complex cellular processes. Exploring its use as a tool for in cell evaluation of pharmacological interference with PPIs, we showed that reported effects of known GPCR antagonists and PPI inhibitors are properly recapitulated. In a three-hybrid setup, KISS was able to map interactions between small molecules and proteins. Taken together, we established KISS as a sensitive approach for in situ analysis of protein interactions and their modulation in a changing cellular context or in response to pharmacological challenges. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

Energy Technology Data Exchange (ETDEWEB)

Matsushita, Y., E-mail: kurita@cs.tut.ac.jp; Murakawa, T., E-mail: kurita@cs.tut.ac.jp; Shimamura, K., E-mail: kurita@cs.tut.ac.jp; Oishi, M., E-mail: kurita@cs.tut.ac.jp; Ohyama, T., E-mail: kurita@cs.tut.ac.jp; Kurita, N., E-mail: kurita@cs.tut.ac.jp [Department of Computer Science and Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi, Aichi, 441-8580 (Japan)

2015-02-27

The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA.

Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

International Nuclear Information System (INIS)

Matsushita, Y.; Murakawa, T.; Shimamura, K.; Oishi, M.; Ohyama, T.; Kurita, N.

2015-01-01

The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA

Dispatched and Scube Mediate the Efficient Secretion of the Cholesterol-Modified Hedgehog Ligand

Directory of Open Access Journals (Sweden)

Hanna Tukachinsky

2012-08-01

Full Text Available The Hedgehog (Hh signaling pathway plays critical roles in metazoan development and in cancer. How the Hh ligand is secreted and spreads to distant cells is unclear, given its covalent modification with a hydrophobic cholesterol molecule, which makes it stick to membranes. We demonstrate that Hh ligand secretion from vertebrate cells is accomplished via two distinct and synergistic cholesterol-dependent binding events, mediated by two proteins that are essential for vertebrate Hh signaling: the membrane protein Dispatched (Disp and a member of the Scube family of secreted proteins. Cholesterol modification is sufficient for a heterologous protein to interact with Scube and to be secreted in a Scube-dependent manner. Disp and Scube recognize different structural aspects of cholesterol similarly to how Niemann-Pick disease proteins 1 and 2 interact with cholesterol, suggesting a hand-off mechanism for transferring Hh from Disp to Scube. Thus, Disp and Scube cooperate to dramatically enhance the secretion and solubility of the cholesterol-modified Hh ligand.

Identification of ligand-selective peptidic ActRIIB-antagonists using phage display technology

Directory of Open Access Journals (Sweden)

Kotaro Sakamoto

2017-09-01

Full Text Available ActRIIB (activin receptor type-2B is an activin receptor subtype constitutively expressed in the whole body, playing a role in cellular proliferation, differentiation, and metabolism. For its various physiological activities, ActRIIB interacts with activin and multiple other ligands including myostatin (MSTN, growth differentiation factor 11 (GDF11, and bone morphogenetic protein 9 (BMP9. Notably, the protein-protein interaction (PPI between ActRIIB and MSTN negatively controls muscular development. Therefore, this PPI has been targeted for effective treatment of muscle degenerative diseases such as muscular dystrophy and sarcopenia. Here, we report the identification of ligand-selective peptidic ActRIIB-antagonists by phage display technology. Our peptides bound to the extracellular domain of ActRIIB, inhibited PPIs between ActRIIB expressed on the cell surface and its ligands, and subsequently suppressed activation of Smad that serves as the downstream signal of the ActRIIB pathway. Interestingly, these peptidic antagonists displayed different ligand selectivities; the AR2mini peptide inhibited multiple ligands (activin A, MSTN, GDF11, and BMP9, AR9 inhibited MSTN and GDF11, while AR8 selectively inhibited MSTN. This is the first report of artificial peptidic ActRIIB-antagonists possessing ligand-selectivity.

Coupling ligand recognition to protein folding in an engineered variant of rabbit ileal lipid binding protein.

Science.gov (United States)

Kouvatsos, Nikolaos; Meldrum, Jill K; Searle, Mark S; Thomas, Neil R

2006-11-28

We have engineered a variant of the beta-clam shell protein ILBP which lacks the alpha-helical motif that caps the central binding cavity; the mutant protein is sufficiently destabilised that it is unfolded under physiological conditions, however, it unexpectedly binds its natural bile acid substrates with high affinity forming a native-like beta-sheet rich structure and demonstrating strong thermodynamic coupling between ligand binding and protein folding.

Small molecule inhibitors of bromodomain-acetyl-lysine interactions.

Science.gov (United States)

Brand, Michael; Measures, Angelina R; Measures, Angelina M; Wilson, Brian G; Cortopassi, Wilian A; Alexander, Rikki; Höss, Matthias; Hewings, David S; Rooney, Timothy P C; Paton, Robert S; Conway, Stuart J

2015-01-16

Bromodomains are protein modules that bind to acetylated lysine residues. Their interaction with histone proteins suggests that they function as "readers" of histone lysine acetylation, a component of the proposed "histone code". Bromodomain-containing proteins are often found as components of larger protein complexes with roles in fundamental cellular process including transcription. The publication of two potent ligands for the BET bromodomains in 2010 demonstrated that small molecules can inhibit the bromodomain-acetyl-lysine protein-protein interaction. These molecules display strong phenotypic effects in a number of cell lines and affect a range of cancers in vivo. This work stimulated intense interest in developing further ligands for the BET bromodomains and the design of ligands for non-BET bromodomains. Here we review the recent progress in the field with particular attention paid to ligand design, the assays employed in early ligand discovery, and the use of computational approaches to inform ligand design.

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

Science.gov (United States)

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

2016-04-06

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

Dynamic strength of the interaction between lung surfactant protein D (SP-D) and saccharide ligands

DEFF Research Database (Denmark)

Thormann, Esben; Dreyer, Jakob K; Simonsen, Adam C

2007-01-01

In order to investigate the dynamic strength of the interaction between lung surfactant protein D (SP-D) and different sugars, maltose, mannose, glucose, and galactose, we have used an atomic force microscope to monitor the interaction on a single molecule scale. The experiment is performed...

DomPep--a general method for predicting modular domain-mediated protein-protein interactions.

Directory of Open Access Journals (Sweden)

Lei Li

Full Text Available Protein-protein interactions (PPIs are frequently mediated by the binding of a modular domain in one protein to a short, linear peptide motif in its partner. The advent of proteomic methods such as peptide and protein arrays has led to the accumulation of a wealth of interaction data for modular interaction domains. Although several computational programs have been developed to predict modular domain-mediated PPI events, they are often restricted to a given domain type. We describe DomPep, a method that can potentially be used to predict PPIs mediated by any modular domains. DomPep combines proteomic data with sequence information to achieve high accuracy and high coverage in PPI prediction. Proteomic binding data were employed to determine a simple yet novel parameter Ligand-Binding Similarity which, in turn, is used to calibrate Domain Sequence Identity and Position-Weighted-Matrix distance, two parameters that are used in constructing prediction models. Moreover, DomPep can be used to predict PPIs for both domains with experimental binding data and those without. Using the PDZ and SH2 domain families as test cases, we show that DomPep can predict PPIs with accuracies superior to existing methods. To evaluate DomPep as a discovery tool, we deployed DomPep to identify interactions mediated by three human PDZ domains. Subsequent in-solution binding assays validated the high accuracy of DomPep in predicting authentic PPIs at the proteome scale. Because DomPep makes use of only interaction data and the primary sequence of a domain, it can be readily expanded to include other types of modular domains.

Functional glass slides for in vitro evaluation of interactions between osteosarcoma TE85 cells and mineral-binding ligands

Energy Technology Data Exchange (ETDEWEB)

Song, Jie; Chen, Julia; Klapperich, Catherine M.; Eng, Vincent; Bertozzi, Carolyn R.

2004-07-20

Primary amine-functionalized glass slides obtained through a multi-step plasma treatment were conjugated with anionic amino acids that are frequently found as mineral binding elements in acidic extracellular matrix components of natural bone. The modified glass surfaces were characterized by X-ray photoelectron spectroscopy (XPS) and contact angle measurements. Human osteosarcoma TE85 cells were cultured on these functionalized slides and analyses on both protein and gene expression levels were performed to probe the ''biocompatibility'' of the surface ligands. Cell attachment and proliferation on anionic surfaces were either better than or comparable to those of cells cultured on tissue culture polystyrene (TCPS). The modified glass surfaces promoted the expression of osteocalcin, alkaline phosphatase activity and ECM proteins such as fibronectin and vitronectin under differentiation culture conditions. Transcript analysis using gene chip microarrays confirmed that culturing TE85 cells on anionic surfaces did not activate apoptotic pathways. Collectively, these results suggest that the potential mineral-binding anionic ligands examined here do not exert significant adverse effects on the expression of important osteogenic markers of TE85 cells. This work paves the way for the incorporation of these ligands into 3-dimensional artificial bone-like scaffolds.

The role of electrostatics in protein-protein interactions of a monoclonal antibody.

Science.gov (United States)

Roberts, D; Keeling, R; Tracka, M; van der Walle, C F; Uddin, S; Warwicker, J; Curtis, R

2014-07-07

Understanding how protein-protein interactions depend on the choice of buffer, salt, ionic strength, and pH is needed to have better control over protein solution behavior. Here, we have characterized the pH and ionic strength dependence of protein-protein interactions in terms of an interaction parameter kD obtained from dynamic light scattering and the osmotic second virial coefficient B22 measured by static light scattering. A simplified protein-protein interaction model based on a Baxter adhesive potential and an electric double layer force is used to separate out the contributions of longer-ranged electrostatic interactions from short-ranged attractive forces. The ionic strength dependence of protein-protein interactions for solutions at pH 6.5 and below can be accurately captured using a Deryaguin-Landau-Verwey-Overbeek (DLVO) potential to describe the double layer forces. In solutions at pH 9, attractive electrostatics occur over the ionic strength range of 5-275 mM. At intermediate pH values (7.25 to 8.5), there is a crossover effect characterized by a nonmonotonic ionic strength dependence of protein-protein interactions, which can be rationalized by the competing effects of long-ranged repulsive double layer forces at low ionic strength and a shorter ranged electrostatic attraction, which dominates above a critical ionic strength. The change of interactions from repulsive to attractive indicates a concomitant change in the angular dependence of protein-protein interaction from isotropic to anisotropic. In the second part of the paper, we show how the Baxter adhesive potential can be used to predict values of kD from fitting to B22 measurements, thus providing a molecular basis for the linear correlation between the two protein-protein interaction parameters.

Direct detection of ligand binding to Sepharose-immobilised protein using saturation transfer double difference (STDD) NMR spectroscopy

International Nuclear Information System (INIS)

Haselhorst, Thomas; Muenster-Kuehnel, Anja K.; Oschlies, Melanie; Tiralongo, Joe; Gerardy-Schahn, Rita; Itzstein, Mark von

2007-01-01

We report an easy and direct application of 'Saturation Transfer Double Difference' (STDD) NMR spectroscopy to identify ligands that bind to a Sepharose-immobilised target protein. The model protein, cytidine 5'-monophosphate sialic acid (CMP-Sia) synthetase, was expressed as a Strep-Tag II fusion protein and immobilised on Strep-Tactin Sepharose. STD NMR experiments of the protein-enriched Sepharose matrix in the presence of a binding ligand (cytidine 5'-triphosphate, CTP) and a non-binding ligand (α/β-glucose) clearly show that CTP binds to the immobilised enzyme, whereas glucose has no affinity. This approach has three major advantages: (a) only low quantities of protein are required, (b) no specialised NMR technology or the application of additional data analysis by non-routine methods is required, and (c) easy multiple use of the immobilised protein is available

Optimizing scoring function of protein-nucleic acid interactions with both affinity and specificity.

Directory of Open Access Journals (Sweden)

Zhiqiang Yan

Full Text Available Protein-nucleic acid (protein-DNA and protein-RNA recognition is fundamental to the regulation of gene expression. Determination of the structures of the protein-nucleic acid recognition and insight into their interactions at molecular level are vital to understanding the regulation function. Recently, quantitative computational approach has been becoming an alternative of experimental technique for predicting the structures and interactions of biomolecular recognition. However, the progress of protein-nucleic acid structure prediction, especially protein-RNA, is far behind that of the protein-ligand and protein-protein structure predictions due to the lack of reliable and accurate scoring function for quantifying the protein-nucleic acid interactions. In this work, we developed an accurate scoring function (named as SPA-PN, SPecificity and Affinity of the Protein-Nucleic acid interactions for protein-nucleic acid interactions by incorporating both the specificity and affinity into the optimization strategy. Specificity and affinity are two requirements of highly efficient and specific biomolecular recognition. Previous quantitative descriptions of the biomolecular interactions considered the affinity, but often ignored the specificity owing to the challenge of specificity quantification. We applied our concept of intrinsic specificity to connect the conventional specificity, which circumvents the challenge of specificity quantification. In addition to the affinity optimization, we incorporated the quantified intrinsic specificity into the optimization strategy of SPA-PN. The testing results and comparisons with other scoring functions validated that SPA-PN performs well on both the prediction of binding affinity and identification of native conformation. In terms of its performance, SPA-PN can be widely used to predict the protein-nucleic acid structures and quantify their interactions.

Spatio-temporal Characterization of Ligand-Receptor Interactions in Blood Stem-Cell Rolling

KAUST Repository

Al Alwan, Bader

2017-08-16

One of the most important issues in the research on hematopoietic stem/progenitor cells (HSPCs) is to understand the mechanism of the homing process of these cells to the bone marrow after being transplanted into patients and establish the production of various blood cell types. The HSPCs first come in contact with the endothelial cells. This contact is known as adhesion and occurs through a multi-step paradigm ending with transmigration to the bone marrow niche. The initial step of the homing, tethering and rolling of HSPCs is mediated by P- and E-Selectins expressed on the endothelial cell surface through their interactions with the ligands expressed by HSPCs. Here we developed a novel experimental method to unravel the molecular mechanisms of the selectin-ligands interactions in vitro at the single molecule level by combining microfluidics and single-molecule fluorescence imaging. Our method enables direct visualization of the nanoscale spatiotemporal dynamics of the E-selectin-ligand (PSGL-1) interactions under conditions of shear stress acting on the cells at the molecular level in real time.

Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides

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Weonu Choe

2016-12-01

Full Text Available The rapidly increasing application of antibodies has inspired the development of several novel methods to isolate and target antibodies using smart biomaterials that mimic the binding of Fc-receptors to antibodies. The Fc-binding domain of antibodies is the primary binding site for e.g., effector proteins and secondary antibodies, whereas antigens bind to the Fab region. Protein A, G, and L, surface proteins expressed by pathogenic bacteria, are well known to bind immunoglobulin and have been widely exploited in antibody purification strategies. Several difficulties are encountered when bacterial proteins are used in antibody research and application. One of the major obstacles hampering the use of bacterial proteins is sample contamination with trace amounts of these proteins, which can invoke an immune response in the host. Many research groups actively develop synthetic ligands that are able to selectively and strongly bind to antibodies. Among the reported ligands, peptides that bind to the Fc-domain of antibodies are attractive tools in antibody research. Besides their use as high affinity ligands in antibody purification chromatography, Fc-binding peptides are applied e.g., to localize antibodies on nanomaterials and to increase the half-life of proteins in serum. In this review, recent developments of Fc-binding peptides are presented and their binding characteristics and diverse applications are discussed.

Evaluating the binding efficiency of pheromone binding protein with its natural ligand using molecular docking and fluorescence analysis

Science.gov (United States)

Ilayaraja, Renganathan; Rajkumar, Ramalingam; Rajesh, Durairaj; Muralidharan, Arumugam Ramachandran; Padmanabhan, Parasuraman; Archunan, Govindaraju

2014-06-01

Chemosignals play a crucial role in social and sexual communication among inter- and intra-species. Chemical cues are bound with protein that is present in the pheromones irrespective of sex are commonly called as pheromone binding protein (PBP). In rats, the pheromone compounds are bound with low molecular lipocalin protein α2u-globulin (α2u). We reported farnesol is a natural endogenous ligand (compound) present in rat preputial gland as a bound volatile compound. In the present study, an attempt has been made through computational method to evaluating the binding efficiency of α2u with the natural ligand (farnesol) and standard fluorescent molecule (2-naphthol). The docking analysis revealed that the binding energy of farnesol and 2-naphthol was almost equal and likely to share some binding pocket of protein. Further, to extrapolate the results generated through computational approach, the α2u protein was purified and subjected to fluorescence titration and binding assay. The results showed that the farnesol is replaced by 2-naphthol with high hydrophobicity of TYR120 in binding sites of α2u providing an acceptable dissociation constant indicating the binding efficiency of α2u. The obtained results are in corroboration with the data made through computational approach.

A review of the thermodynamics of protein association to ligands, protein adsorption, and adsorption isotherms

DEFF Research Database (Denmark)

Mollerup, Jørgen

2008-01-01

The application of thermodynamic models in the development of chromatographic separation processes is discussed. The paper analyses the thermodynamic principles of protein adsorption. It can be modeled either as a reversible association between the adsorbate and the ligands or as a steady...

E-selectin ligand-1 (ESL-1) is a novel adiponectin binding protein on cell adhesion

Energy Technology Data Exchange (ETDEWEB)

Yamamoto, Hiroyasu; Kuroda, Nana; Uekita, Hiromi; Kochi, Ikoi; Matsumoto, Akane; Niinaga, Ryu [Department of Biomedical Informatics, Division of Health Sciences, Osaka University Graduate School of Medicine, Osaka (Japan); Funahashi, Tohru; Shimomura, Iichiro [Department of Metabolic Medicine, Osaka University Graduate School of Medicine, Osaka (Japan); Kihara, Shinji, E-mail: skihara@sahs.med.osaka-u.ac.jp [Department of Biomedical Informatics, Division of Health Sciences, Osaka University Graduate School of Medicine, Osaka (Japan)

2016-02-05

Background: Adiponectin (APN) is an adipocyte-derived bioactive molecule with anti-diabetic and anti-atherogenic properties. Although anti-diabetic effects are mostly mediated by the adiponectin receptors AdipoR1 and AdipoR2, the anti-atherogenic mechanisms have not been fully elucidated. Methods and Results: In this study, we identified E-selectin ligand (ESL)-1 as a novel APN-binding protein by mass spectrometry analysis of HepG2 cell-derived immunoprecipitant with an anti-APN antibody. Cell adhesion assays using fluorescence-labelled monocyte cell line THP-1 cells and human umbilical vein endothelial cells (HUVECs) revealed that APN-pre-treated THP-1 cells had reduced binding ability to HUVECs. This APN-mediated suppressive effect on monocyte binding to endothelial cells was partially abrogated by targeting ESL-1 with shRNA in THP-1 cells. In addition, serial mutagenesis analysis disclosed that five extracellular amino acids close to the N-terminus of ESL-1 were essential for binding with APN. Conclusion: Our results highlight the fact that interaction between APN and ESL-1 could provide a fundamental mechanism underlying the anti-atherogenic properties of APN. - Highlights: • E-selectin ligand (ESL)-1 was identified as an adiponectin (APN)-binding protein. • ESL-1 bound to APN at its N-terminal 6th-10th amino acids. • shESL-1 reduced the suppressive effect of APN on adhesion of THP-1 cells to HUVECs. • Interaction with ESL may be involved in the anti-atherogenic effects of APN.

E-selectin ligand-1 (ESL-1) is a novel adiponectin binding protein on cell adhesion

International Nuclear Information System (INIS)

Yamamoto, Hiroyasu; Kuroda, Nana; Uekita, Hiromi; Kochi, Ikoi; Matsumoto, Akane; Niinaga, Ryu; Funahashi, Tohru; Shimomura, Iichiro; Kihara, Shinji

2016-01-01

Background: Adiponectin (APN) is an adipocyte-derived bioactive molecule with anti-diabetic and anti-atherogenic properties. Although anti-diabetic effects are mostly mediated by the adiponectin receptors AdipoR1 and AdipoR2, the anti-atherogenic mechanisms have not been fully elucidated. Methods and Results: In this study, we identified E-selectin ligand (ESL)-1 as a novel APN-binding protein by mass spectrometry analysis of HepG2 cell-derived immunoprecipitant with an anti-APN antibody. Cell adhesion assays using fluorescence-labelled monocyte cell line THP-1 cells and human umbilical vein endothelial cells (HUVECs) revealed that APN-pre-treated THP-1 cells had reduced binding ability to HUVECs. This APN-mediated suppressive effect on monocyte binding to endothelial cells was partially abrogated by targeting ESL-1 with shRNA in THP-1 cells. In addition, serial mutagenesis analysis disclosed that five extracellular amino acids close to the N-terminus of ESL-1 were essential for binding with APN. Conclusion: Our results highlight the fact that interaction between APN and ESL-1 could provide a fundamental mechanism underlying the anti-atherogenic properties of APN. - Highlights: • E-selectin ligand (ESL)-1 was identified as an adiponectin (APN)-binding protein. • ESL-1 bound to APN at its N-terminal 6th-10th amino acids. • shESL-1 reduced the suppressive effect of APN on adhesion of THP-1 cells to HUVECs. • Interaction with ESL may be involved in the anti-atherogenic effects of APN.

Functional aspects of protein flexibility

DEFF Research Database (Denmark)

Teilum, Kaare; Olsen, Johan G; Kragelund, Birthe B

2009-01-01

this into an intuitive perception of protein function is challenging. Flexibility is of overwhelming importance for protein function, and the changes in protein structure during interactions with binding partners can be dramatic. The present review addresses protein flexibility, focusing on protein-ligand interactions...

Protein-protein interaction network-based detection of functionally similar proteins within species.

Science.gov (United States)

Song, Baoxing; Wang, Fen; Guo, Yang; Sang, Qing; Liu, Min; Li, Dengyun; Fang, Wei; Zhang, Deli

2012-07-01

Although functionally similar proteins across species have been widely studied, functionally similar proteins within species showing low sequence similarity have not been examined in detail. Identification of these proteins is of significant importance for understanding biological functions, evolution of protein families, progression of co-evolution, and convergent evolution and others which cannot be obtained by detection of functionally similar proteins across species. Here, we explored a method of detecting functionally similar proteins within species based on graph theory. After denoting protein-protein interaction networks using graphs, we split the graphs into subgraphs using the 1-hop method. Proteins with functional similarities in a species were detected using a method of modified shortest path to compare these subgraphs and to find the eligible optimal results. Using seven protein-protein interaction networks and this method, some functionally similar proteins with low sequence similarity that cannot detected by sequence alignment were identified. By analyzing the results, we found that, sometimes, it is difficult to separate homologous from convergent evolution. Evaluation of the performance of our method by gene ontology term overlap showed that the precision of our method was excellent. Copyright © 2012 Wiley Periodicals, Inc.

The interactions between lipase and pyridinium ligands investigated by electrochemical and spectrophotometric methods

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Simona Patriche

2016-04-01

Full Text Available The interaction between pyridinium ligands derived from 4,4’-bipyridine (N,N’-bis(p-bromophenacyl-4,4’-bipyridinium dibromide – Lr and (N,N’-bis(p-bromophenacyl-1,2-bis (4-pyridyl ethane dibromide – Lm with lipase enzyme was evaluated. The stability of the pyridinium ligands, having an essential role in biological systems, in 0.1 M KNO3 as supporting electrolyte is influenced by the lipase concentration added. The pH and conductometry measurements in aqueous solution suggest a rapid ionic exchange process. The behavior of pyridinium ligands in the presence of lipase is investigated by cyclic voltammetry and UV/Vis spectroscopy, which indicated bindings and changes from the interaction between them. The voltammograms recorded on the glassy carbon electrode showed a more intense electronic transfer for the Lr interaction with lipase compared to Lm, which is due to the absence of mobile ethylene groups from Lr structure.

Analysis of protein interactions at native chloroplast membranes by ellipsometry.

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Verena Kriechbaumer

Full Text Available Membrane bound receptors play vital roles in cell signaling, and are the target for many drugs, yet their interactions with ligands are difficult to study by conventional techniques due to the technical difficulty of monitoring these interactions in lipid environments. In particular, the ability to analyse the behaviour of membrane proteins in their native membrane environment is limited. Here, we have developed a quantitative approach to detect specific interactions between low-abundance chaperone receptors within native chloroplast membranes and their soluble chaperone partners. Langmuir-Schaefer film deposition was used to deposit native chloroplasts onto gold-coated glass slides, and interactions between the molecular chaperones Hsp70 and Hsp90 and their receptors in the chloroplast membranes were detected and quantified by total internal reflection ellipsometry (TIRE. We show that native chloroplast membranes deposited on gold-coated glass slides using Langmuir-Schaefer films retain functional receptors capable of binding chaperones with high specificity and affinity. Taking into account the low chaperone receptor abundance in native membranes, these binding properties are consistent with data generated using soluble forms of the chloroplast chaperone receptors, OEP61 and Toc64. Therefore, we conclude that chloroplasts have the capacity to selectively bind chaperones, consistent with the notion that chaperones play an important role in protein targeting to chloroplasts. Importantly, this method of monitoring by TIRE does not require any protein labelling. This novel combination of techniques should be applicable to a wide variety of membranes and membrane protein receptors, thus presenting the opportunity to quantify protein interactions involved in fundamental cellular processes, and to screen for drugs that target membrane proteins.

Elucidating Ligand-Modulated Conformational Landscape of GPCRs Using Cloud-Computing Approaches.

Science.gov (United States)

Shukla, Diwakar; Lawrenz, Morgan; Pande, Vijay S

2015-01-01

G-protein-coupled receptors (GPCRs) are a versatile family of membrane-bound signaling proteins. Despite the recent successes in obtaining crystal structures of GPCRs, much needs to be learned about the conformational changes associated with their activation. Furthermore, the mechanism by which ligands modulate the activation of GPCRs has remained elusive. Molecular simulations provide a way of obtaining detailed an atomistic description of GPCR activation dynamics. However, simulating GPCR activation is challenging due to the long timescales involved and the associated challenge of gaining insights from the "Big" simulation datasets. Here, we demonstrate how cloud-computing approaches have been used to tackle these challenges and obtain insights into the activation mechanism of GPCRs. In particular, we review the use of Markov state model (MSM)-based sampling algorithms for sampling milliseconds of dynamics of a major drug target, the G-protein-coupled receptor β2-AR. MSMs of agonist and inverse agonist-bound β2-AR reveal multiple activation pathways and how ligands function via modulation of the ensemble of activation pathways. We target this ensemble of conformations with computer-aided drug design approaches, with the goal of designing drugs that interact more closely with diverse receptor states, for overall increased efficacy and specificity. We conclude by discussing how cloud-based approaches present a powerful and broadly available tool for studying the complex biological systems routinely. © 2015 Elsevier Inc. All rights reserved.

Rates and equilibrium constants of the ligand-induced conformational transition of an HCN ion channel protein domain determined by DEER spectroscopy.

Science.gov (United States)

Collauto, Alberto; DeBerg, Hannah A; Kaufmann, Royi; Zagotta, William N; Stoll, Stefan; Goldfarb, Daniella

2017-06-14

Ligand binding can induce significant conformational changes in proteins. The mechanism of this process couples equilibria associated with the ligand binding event and the conformational change. Here we show that by combining the application of W-band double electron-electron resonance (DEER) spectroscopy with microfluidic rapid freeze quench (μRFQ) it is possible to resolve these processes and obtain both equilibrium constants and reaction rates. We studied the conformational transition of the nitroxide labeled, isolated carboxy-terminal cyclic-nucleotide binding domain (CNBD) of the HCN2 ion channel upon binding of the ligand 3',5'-cyclic adenosine monophosphate (cAMP). Using model-based global analysis, the time-resolved data of the μRFQ DEER experiments directly provide fractional populations of the open and closed conformations as a function of time. We modeled the ligand-induced conformational change in the protein using a four-state model: apo/open (AO), apo/closed (AC), bound/open (BO), bound/closed (BC). These species interconvert according to AC + L ⇌ AO + L ⇌ BO ⇌ BC. By analyzing the concentration dependence of the relative contributions of the closed and open conformations at equilibrium, we estimated the equilibrium constants for the two conformational equilibria and the open-state ligand dissociation constant. Analysis of the time-resolved μRFQ DEER data gave estimates for the intrinsic rates of ligand binding and unbinding as well as the rates of the conformational change. This demonstrates that DEER can quantitatively resolve both the thermodynamics and the kinetics of ligand binding and the associated conformational change.

Detecting mutually exclusive interactions in protein-protein interaction maps.

KAUST Repository

Sánchez Claros, Carmen

2012-06-08

Comprehensive protein interaction maps can complement genetic and biochemical experiments and allow the formulation of new hypotheses to be tested in the system of interest. The computational analysis of the maps may help to focus on interesting cases and thereby to appropriately prioritize the validation experiments. We show here that, by automatically comparing and analyzing structurally similar regions of proteins of known structure interacting with a common partner, it is possible to identify mutually exclusive interactions present in the maps with a sensitivity of 70% and a specificity higher than 85% and that, in about three fourth of the correctly identified complexes, we also correctly recognize at least one residue (five on average) belonging to the interaction interface. Given the present and continuously increasing number of proteins of known structure, the requirement of the knowledge of the structure of the interacting proteins does not substantially impact on the coverage of our strategy that can be estimated to be around 25%. We also introduce here the Estrella server that embodies this strategy, is designed for users interested in validating specific hypotheses about the functional role of a protein-protein interaction and it also allows access to pre-computed data for seven organisms.

Detecting mutually exclusive interactions in protein-protein interaction maps.

KAUST Repository

Sá nchez Claros, Carmen; Tramontano, Anna

2012-01-01

Comprehensive protein interaction maps can complement genetic and biochemical experiments and allow the formulation of new hypotheses to be tested in the system of interest. The computational analysis of the maps may help to focus on interesting cases and thereby to appropriately prioritize the validation experiments. We show here that, by automatically comparing and analyzing structurally similar regions of proteins of known structure interacting with a common partner, it is possible to identify mutually exclusive interactions present in the maps with a sensitivity of 70% and a specificity higher than 85% and that, in about three fourth of the correctly identified complexes, we also correctly recognize at least one residue (five on average) belonging to the interaction interface. Given the present and continuously increasing number of proteins of known structure, the requirement of the knowledge of the structure of the interacting proteins does not substantially impact on the coverage of our strategy that can be estimated to be around 25%. We also introduce here the Estrella server that embodies this strategy, is designed for users interested in validating specific hypotheses about the functional role of a protein-protein interaction and it also allows access to pre-computed data for seven organisms.

Investigation of the Copper Binding Site And the Role of Histidine As a Ligand in Riboflavin Binding Protein

Energy Technology Data Exchange (ETDEWEB)

Smith, S.R.; Bencze, K.Z.; Russ, K.A.; Wasiukanis, K.; Benore-Parsons, M.; Stemmler, T.L.

2009-05-26

Riboflavin Binding Protein (RBP) binds copper in a 1:1 molar ratio, forming a distinct well-ordered type II site. The nature of this site has been examined using X-ray absorption and pulsed electron paramagnetic resonance (EPR) spectroscopies, revealing a four coordinate oxygen/nitrogen rich environment. On the basis of analysis of the Cambridge Structural Database, the average protein bound copper-ligand bond length of 1.96 {angstrom}, obtained by extended x-ray absorption fine structure (EXAFS), is consistent with four coordinate Cu(I) and Cu(II) models that utilize mixed oxygen and nitrogen ligand distributions. These data suggest a Cu-O{sub 3}N coordination state for copper bound to RBP. While pulsed EPR studies including hyperfine sublevel correlation spectroscopy and electron nuclear double resonance show clear spectroscopic evidence for a histidine bound to the copper, inclusion of a histidine in the EXAFS simulation did not lead to any significant improvement in the fit.

Dispatched and scube mediate the efficient secretion of the cholesterol-modified hedgehog ligand.

Science.gov (United States)

Tukachinsky, Hanna; Kuzmickas, Ryan P; Jao, Cindy Y; Liu, Jing; Salic, Adrian

2012-08-30

The Hedgehog (Hh) signaling pathway plays critical roles in metazoan development and in cancer. How the Hh ligand is secreted and spreads to distant cells is unclear, given its covalent modification with a hydrophobic cholesterol molecule, which makes it stick to membranes. We demonstrate that Hh ligand secretion from vertebrate cells is accomplished via two distinct and synergistic cholesterol-dependent binding events, mediated by two proteins that are essential for vertebrate Hh signaling: the membrane protein Dispatched (Disp) and a member of the Scube family of secreted proteins. Cholesterol modification is sufficient for a heterologous protein to interact with Scube and to be secreted in a Scube-dependent manner. Disp and Scube recognize different structural aspects of cholesterol similarly to how Niemann-Pick disease proteins 1 and 2 interact with cholesterol, suggesting a hand-off mechanism for transferring Hh from Disp to Scube. Thus, Disp and Scube cooperate to dramatically enhance the secretion and solubility of the cholesterol-modified Hh ligand. Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.

Ligand deconstruction: Why some fragment binding positions are conserved and others are not

Science.gov (United States)

Kozakov, Dima; Hall, David R.; Jehle, Stefan; Luo, Lingqi; Ochiana, Stefan O.; Jones, Elizabeth V.; Pollastri, Michael; Allen, Karen N.; Whitty, Adrian; Vajda, Sandor

2015-01-01

Fragment-based drug discovery (FBDD) relies on the premise that the fragment binding mode will be conserved on subsequent expansion to a larger ligand. However, no general condition has been established to explain when fragment binding modes will be conserved. We show that a remarkably simple condition can be developed in terms of how fragments coincide with binding energy hot spots—regions of the protein where interactions with a ligand contribute substantial binding free energy—the locations of which can easily be determined computationally. Because a substantial fraction of the free energy of ligand binding comes from interacting with the residues in the energetically most important hot spot, a ligand moiety that sufficiently overlaps with this region will retain its location even when other parts of the ligand are removed. This hypothesis is supported by eight case studies. The condition helps identify whether a protein is suitable for FBDD, predicts the size of fragments required for screening, and determines whether a fragment hit can be extended into a higher affinity ligand. Our results show that ligand binding sites can usefully be thought of in terms of an anchor site, which is the top-ranked hot spot and dominates the free energy of binding, surrounded by a number of weaker satellite sites that confer improved affinity and selectivity for a particular ligand and that it is the intrinsic binding potential of the protein surface that determines whether it can serve as a robust binding site for a suitably optimized ligand. PMID:25918377

Ligand deconstruction: Why some fragment binding positions are conserved and others are not.

Science.gov (United States)

Kozakov, Dima; Hall, David R; Jehle, Stefan; Jehle, Sefan; Luo, Lingqi; Ochiana, Stefan O; Jones, Elizabeth V; Pollastri, Michael; Allen, Karen N; Whitty, Adrian; Vajda, Sandor

2015-05-19

Fragment-based drug discovery (FBDD) relies on the premise that the fragment binding mode will be conserved on subsequent expansion to a larger ligand. However, no general condition has been established to explain when fragment binding modes will be conserved. We show that a remarkably simple condition can be developed in terms of how fragments coincide with binding energy hot spots--regions of the protein where interactions with a ligand contribute substantial binding free energy--the locations of which can easily be determined computationally. Because a substantial fraction of the free energy of ligand binding comes from interacting with the residues in the energetically most important hot spot, a ligand moiety that sufficiently overlaps with this region will retain its location even when other parts of the ligand are removed. This hypothesis is supported by eight case studies. The condition helps identify whether a protein is suitable for FBDD, predicts the size of fragments required for screening, and determines whether a fragment hit can be extended into a higher affinity ligand. Our results show that ligand binding sites can usefully be thought of in terms of an anchor site, which is the top-ranked hot spot and dominates the free energy of binding, surrounded by a number of weaker satellite sites that confer improved affinity and selectivity for a particular ligand and that it is the intrinsic binding potential of the protein surface that determines whether it can serve as a robust binding site for a suitably optimized ligand.

Aquaporin Protein-Protein Interactions

Directory of Open Access Journals (Sweden)

Jennifer Virginia Roche

2017-10-01

Full Text Available Aquaporins are tetrameric membrane-bound channels that facilitate transport of water and other small solutes across cell membranes. In eukaryotes, they are frequently regulated by gating or trafficking, allowing for the cell to control membrane permeability in a specific manner. Protein–protein interactions play crucial roles in both regulatory processes and also mediate alternative functions such as cell adhesion. In this review, we summarize recent knowledge about aquaporin protein–protein interactions; dividing the interactions into three types: (1 interactions between aquaporin tetramers; (2 interactions between aquaporin monomers within a tetramer (hetero-tetramerization; and (3 transient interactions with regulatory proteins. We particularly focus on the structural aspects of the interactions, discussing the small differences within a conserved overall fold that allow for aquaporins to be differentially regulated in an organism-, tissue- and trigger-specific manner. A deep knowledge about these differences is needed to fully understand aquaporin function and regulation in many physiological processes, and may enable design of compounds targeting specific aquaporins for treatment of human disease.

Interaction of a non-histone chromatin protein (high-mobility group protein 2) with DNA

International Nuclear Information System (INIS)

Goodwin, G.H.; Shooter, K.V.; Johns, E.W.

1975-01-01

The interaction with DNA of the calf thymus chromatin non-histone protein termed the high-mobility group protein 2 has been studied by sedimentation analysis in the ultracentrifuge and by measuring the binding of the 125 I-labelled protein to DNA. The results have been compared with those obtained previously by us [Eur. J. Biochem. (1974) 47, 263-270] for the interaction of high-mobility group protein 1 with DNA. Although the binding parameters are similar for these two proteins, high-mobility group protein 2 differs from high-mobility group protein 1 in that the former appears to change the shape of the DNA to a more compact form. The molecular weight of high-mobility group protein 2 has been determined by equilibrium sedimentation and a mean value of 26,000 was obtained. A low level of nuclease activity detected in one preparation of high-mobility group protein 2 has been investigated. (orig.) [de

Ligand-induced association of surface immunoglobulin with the detergent insoluble cytoskeleton may involve an 89K protein

International Nuclear Information System (INIS)

Gupta, S.K.; Woda, B.

1986-01-01

Membrane immunoglobulin of B-lymphocytes is thought to play an important role in antigen recognition and cellular activation. Binding of cross-linking ligands to surface immunoglobulin (SIg) on intact cells converts it to a detergent insoluble state, and this conversion is associated with the transmission of a mitogenic signal. Insolubilized membrane proteins may be solubilized by incubating the detergent insoluble cytoskeletons in buffers which convert F-actin to G-actin [(Buffer 1), 0.34M sucrose, 0.5mM ATP, 0.5mM Dithiothrietol and lmM EDTA]. Immunoprecipitation of SIg from the detergent soluble fraction of 35 S-methionine labeled non ligand treated rat B-cells results in the co-isolation of an 89K protein and a 44K protein, presumably actin. The 89K protein is not associated with the fraction of endogenous detergent insoluble SIg. On treatment of rat B cells with cross-linking ligand (anti-Ig) the 89K protein becomes detergent insoluble along with most of the SIg and co-isolates with SIg on immunoprecipitation of the detergent insoluble, buffer l solubilized fraction. The migration of the SIg-associated 89K protein from the detergent soluble fraction to the detergent insoluble fraction after ligand treatment, suggests that this protein might be involved in linking SIg to the underlying cytoskeleton and could be involved in the transmission of a mitogenic signal

Platinum(II Complexes with Tetradentate Schiff Bases as Ligands: Synthesis, Characterization and Detection of DNA Interaction by Differential Pulse Voltammetry

Directory of Open Access Journals (Sweden)

Lijun Li

2012-01-01

Full Text Available Five sterically hindered platinum(II complexes with tetradentate schiff bases as ligands, [Pt(L] (L= N,N′-bisalicylidene-1,2-ethylenediamine (L1, N,N′-bisalicylidene-1,2-cyclohexanediamine (L2, N,N′-bis(5-hydroxyl-salicylidene-1,2-cyclohexanediamine (L3, N,N′-bisalicylidene-1,2-diphenyl-ethylenediamine (L4 and N,N′-bis(3-tert-butyl-5-methyl-salicylidene-1,2-diphenylethylenediamine (L5 have been synthesized and characterized by IR spectroscopy and elemental analysis. The sterical hindrance of antitumor drug candidates potentially makes them less susceptible to deactivation by sulphur containing proteins and helping to overcome resistance mechanisms. The interaction of these metal complexes with fish sperm single-stranded DNA (ssDNA was studied electrochemically based on the oxidation signals of guanine and adenine. Differential pulse voltammetry was employed to monitor the DNA interaction in solution by using renewable pencil graphite electrode. The results indicate that ligands with different groups can strongly affect the interaction between [Pt(L] complexes and ssDNA due to sterical hindrances and complex [Pt(L1] has the best interaction with DNA among the five complexes.

Our interests in protein-protein interactions

Indian Academy of Sciences (India)

protein interactions. Evolution of P-P partnerships. Evolution of P-P structures. Evolutionary dynamics of P-P interactions. Dynamics of P-P interaction network. Host-pathogen interactions. CryoEM mapping of gigantic protein assemblies.

Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

Energy Technology Data Exchange (ETDEWEB)

Bokoch, Michael P.; Zou, Yaozhong; Rasmussen, Søren G.F.; Liu, Corey W.; Nygaard, Rie; Rosenbaum, Daniel M.; Fung, Juan José; Choi, Hee-Jung; Thian, Foon Sun; Kobilka, Tong Sun; Puglisi, Joseph D.; Weis, William I.; Pardo, Leonardo; Prosser, R. Scott; Mueller, Luciano; Kobilka, Brian K. (Stanford-MED); (Toronto); (BMS); (UAB, Spain)

2010-01-14

G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the {beta}{sub 2} adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.

From nonspecific DNA-protein encounter complexes to the prediction of DNA-protein interactions.

Directory of Open Access Journals (Sweden)

Mu Gao

2009-03-01

Full Text Available DNA-protein interactions are involved in many essential biological activities. Because there is no simple mapping code between DNA base pairs and protein amino acids, the prediction of DNA-protein interactions is a challenging problem. Here, we present a novel computational approach for predicting DNA-binding protein residues and DNA-protein interaction modes without knowing its specific DNA target sequence. Given the structure of a DNA-binding protein, the method first generates an ensemble of complex structures obtained by rigid-body docking with a nonspecific canonical B-DNA. Representative models are subsequently selected through clustering and ranking by their DNA-protein interfacial energy. Analysis of these encounter complex models suggests that the recognition sites for specific DNA binding are usually favorable interaction sites for the nonspecific DNA probe and that nonspecific DNA-protein interaction modes exhibit some similarity to specific DNA-protein binding modes. Although the method requires as input the knowledge that the protein binds DNA, in benchmark tests, it achieves better performance in identifying DNA-binding sites than three previously established methods, which are based on sophisticated machine-learning techniques. We further apply our method to protein structures predicted through modeling and demonstrate that our method performs satisfactorily on protein models whose root-mean-square Calpha deviation from native is up to 5 A from their native structures. This study provides valuable structural insights into how a specific DNA-binding protein interacts with a nonspecific DNA sequence. The similarity between the specific DNA-protein interaction mode and nonspecific interaction modes may reflect an important sampling step in search of its specific DNA targets by a DNA-binding protein.

Evolution of protein-protein interactions

Indian Academy of Sciences (India)

Evolution of protein-protein interactions · Our interests in protein-protein interactions · Slide 3 · Slide 4 · Slide 5 · Slide 6 · Slide 7 · Slide 8 · Slide 9 · Slide 10 · Slide 11 · Slide 12 · Slide 13 · Slide 14 · Slide 15 · Slide 16 · Slide 17 · Slide 18 · Slide 19 · Slide 20.

Proteins interacting with cloning scars: a source of false positive protein-protein interactions.