Molecular dynamics simulation of intrinsically disordered proteins

Intrinsically disordered proteins are biomolecules that do not have a definite 3D structure; therefore, their dynamical simulation cannot start from a known list of atomistic positions, such as a Protein Data Bank file. We describe a method to start a computer simulation of these proteins. The first step of the procedure is the creation of a multi-rod configuration of the molecule, derived from its primary sequence. This structure is dynamically evolved in vacuo until its gyration radius reaches the experimental average value; at this point solvent molecules, in explicit or implicit implementation, are added to the protein and a regular molecular dynamics simulation follows. We have applied this procedure to the simulation of tau, one of the largest totally disordered proteins.     

A 3D model for the human hepatic asialoglycoprotein receptor (ASGP-R)

The human hepatic Asialoglycoprotein Receptor (ASGP-R) consists of two different types of liver specific membrane glycoproteins that bind to terminal galactose and N-acetylgalactosamine residues of serum glycoproteins. The two different polypeptide chains are referred to as two receptor subunits, HH1 and HH2, which are both involved in the activity of the functional receptor. This receptor has served as a model for understanding receptor-mediated endocytosis and carbohydrate mediated recognition phenomena. Here models for the C-terminal extracellular region of both HH1 and HH2 subunit are presented. The standard homology building procedure was modified in order to make it suitable for the modeling problem at hand. The models for the extracellular regions of HH1 and HH2 were initially constructed by exploiting several fragments, belonging to proteins of known 3D structure, and showing high local sequence similarity with respect to the glycoproteins of interest. Putative binding sites were first hypothesized on the basis of the comparison with other complexes of lectins, the crystal structure of which was available in the Protein Data Bank. A model for the complex involving the HH2 subunit and the typical high affinity ligand N-acetylgalactosamine (NacGal) was refined as the first by a suitable combination of MD simulations and Energy Minimization calculations, since it seemed to quickly converge to a plausible structure. An intermediate model for HH1 was then rebuilt on the basis of the refined model for HH2. It was then submitted to a sequence of molecular dynamics simulations with templates which took into account the secondary structure prediction for a final refinement. The structures of small regions of the models, located around the binding sites, were compared with more recent crystallographic data regarding a complex involving the mutant of Mannose Binding Protein QPDWGH (1BCH entry in the Protein Data Bank) and NacGal. This mutant shows high local sequence similarity with HH1 and HH2 at the binding sites. On the basis of the above comparison, different locations of the binding sites were also considered. In addition to other expected interactions, two hydrophobic interactions were observed in the models with Trp residues (positions 243 in HH1 and 181 or 267 in HH2 respectively) and His residues (positions 256 in HHI and 184 in HH2.respectively). The quality of the models was evaluated by the Procheck program and they seemed plausible. This observation together with analogies found between binding sites of the models and IBCH supported the validity of the models. A further validation element arose by comparison between experimental binding data available in the literature about the homologous rat receptor subunits and theoretical interaction energies evaluated, by means of the DOCK 3.5 program, in models for the rat subunits obtained from the corresponding human ones. The new modeling procedure used here appears to be a well-suited method for structural analysis of small regions, located around the ligands, in proteins of unknown 3D structure.     

Structure of glutathione S-transferase of the filarial parasite Wuchereria bancrofti: a target for drug development against adult worm

A three dimensional structural model of Glutathione-S-transferase (GST) of the lymphatic filarial parasite Wuchereria bancrofti (wb) was constructed by homology modeling. The three dimensional X-ray crystal structure of porcine -class GST with PDB ID: 2gsr-A chain protein with 42% sequential and functional homology was used as the template. The model of wbGST built by MODELLER6v2 was analyzed by the PROCHECK programs. Ramachandran plot analysis showed that 93.5% of the residues are in the core region followed by 5.4 and 1.1% residues in the allowed and generously allowed regions, respectively. None of the non-glycine residues is in disallowed regions. The PROSA II z-score and the energy graph for the final model further confirmed the quality of the modeled structure. The computationally modeled three-dimensional (3D) structure of wbGST has been submitted to the Protein Data Bank (PDB) (PDB ID: 1SFM and RCSB ID: RCSB021668). 1SFM was used for docking with GST inhibitors by Hex4.2 macromolecular docking using spherical polar Fourier correlations.Figure: A three-dimensional (3D) structure of Glutathione-S-transferase (GST) of the lymphatic filarial parasite Wuchereria bancrofti (wb) was constructed by homology modeling. This modeled 3D structure of wbGST has been submitted to the Protein Data Bank (PDB) (PDB ID: 1SFM and RCSB ID: RCSB021668).     

A 3D Model for the Human Hepatic Asialoglycoprotein Receptor (ASGP-R)

Abstract

The human hepatic Asialoglycoprotein Receptor (ASGP-R) consists of two different types of liver specific membrane glycoproteins that bind to terminal galactose and acetylgalactosamine residues of serum glycoproteins. The two different polypeptide chains are referred to as two receptor subunits, HH1 and HH2, which are both involved in the activity of the functional receptor. This receptor has served as a model for understanding receptor-mediated endocytosis and carbohydrate mediated recognition phenomena.

Here models for the C-terminal extracellular region of both HH1 and HH2 subunit are presented. The standard homology building procedure was modified in order to make it suitable for the modeling problem at hand. The models for the extracellular regions of HH1 and HH2 were initially constructed by exploiting several fragments, belonging to proteins of known 3D structure, and showing high local sequence similarity with respect to the glycoproteins of interest. Putative binding sites were first hypothesized on the basis of the comparison with other complexes of lectins, the crystal structure of which was available in the Protein Data Bank. A model for the complex involving the HH2 subunit and the typical high affinity ligand N-acetylgalactosamine (NacGal) was refined as the first by a suitable combination of MD simulations and Energy Minimization calculations, since it seemed to quickly converge to a plausible structure. An intermediate model for HH1 was then rebuilt on the basis of the refined model for HH2. It was then submitted to a sequence of molecular dynamics simulations with templates which took into account the secondary structure prediction for a final refinement. The structures of small regions of the models, located around the binding sites, were compared with more recent crystallographic data regarding a complex involving the mutant of Mannose Binding Protein QPDWGH (1BCH entry in the Protein Data Bank) and NacGal. This mutant shows high local sequence similarity with HH1 and HH2 at the binding sites. On the basis of the above comparison, different locations of the binding sites were also considered. In addition to other expected interactions, two hydrophobic interactions were observed in the models with Trp residues (positions 243 in HH1 and 181 or 267 in HH2 respectively) and His residues (positions 256 in HH1 and 184 in HH2 respectively). The quality of the models was evaluated by the Procheck program and they seemed plausible. This observation together with analogies found between binding sites of the models and 1BCH supported the validity of the models. A further validation element arose by comparison between experimental binding data available in the literature about the homologous rat receptor subunits and theoretical interaction energies evaluated, by means of the DOCK 3.5 program, in models for the rat subunits obtained from the corresponding human ones. The new modeling procedure used here appears to be a well-suited method for structural analysis of small regions, located around the ligands, in proteins of unknown 3D structure.     

Homology modelling and molecular dynamics simulations of a protein serine/threonine phosphatase stp1 in Staphylococcus aureus N315: a potential drug target

The prevalence of methicillin-resistant Staphylococcus aureus and vanomycin intermediate S. aureus infections is on the rise, globally. This poses a huge challenge due to limited therapeutic options and the limited number of bacterial-specific drug targets available for due conservation with the human host. A serine/threonine phosphatase/kinase stp1/stk1 phospho-signalling system in S. aureus, which is just beginning to be understood, has been shown to be of importance in virulence and susceptibility to glycopeptide antibiotics. In this study, 3D structure of stp1 (clinical strain of S. aureus N315) was predicted using a homology modelling tool MODELLER. The validation of the predicted model was done using various tools such as PROCHECK, ERRAT, VERIFY-3D and ProSA. Molecular dynamics (MD) study was carried out using GROMACS to refine the least energy model generated from MODELLER9v11 and it was compared with the template. The template used was the crystal structure of serine/threonine phosphatase stp1 in Streptoccocus agalactiae (Protein Data Bank ID: 2PK0) with 38% identity with the query. Various validation tools showed the quality of the model generated using MODELLER. PROCHECK predicted 100% residues in the allowed region, ERRAT with overall quality factor of 76.47, VERIFY-3D with average score of >0.2 in 81.78% of residues, WHATIF with packaging quality score of > ? 5 for all residues and ProSA with Z-score of ? 7.02. MD simulation of the protein showed some fluctuations in the aqueous environment and changes in the ligand binding residues after simulation. The availability of the 3D-structural information of a viable drug target in S. aureus stp1 is expected to facilitate structure–activity relationship and interactions with proteins.     

An empirical approach for structure-based prediction of carbohydrate-binding sites on proteins

A computer program system was developed to predict carbohydrate-binding sites on three-dimensional (3D) protein structures. The programs search for binding sites by referring to the empirical rules derived from the known 3D structures of carbohydrate-protein complexes. A total of 80 non-redundant carbohydrate-protein complex structures were selected from the Protein Data Bank for the empirical rule construction. The performance of the prediction system was tested on 50 known complex structures to determine whether the system could detect the known binding sites. The known monosaccharide-binding sites were detected among the best three predictions in 59% of the cases, which covered 69% of the polysaccharide-binding sites in the target proteins, when the performance was evaluated by the overlap between residue patches of predicted and known binding sites.     

Modelling a binuclear metal binding site in the photosynthetic reaction centre II

Based on the experimental data and homologous sites in Protein Data Bank (PDB) a model for metal binding sites in D1/D2 heterodimer has been proposed. On searching for tetranuclear and binuclear Mn binding sites in the PDB, a suitable sequence homology in thermolysin and D1 could be observed. From the homology and site-directed mutagenesis data, a model for binuclear Mn-Ca or Mn-Mn has been built and it is extended to a tetranuclear Mn centre.     

Prediction of transition metal-binding sites from apo protein structures

Metal ions are crucial for protein function. They participate in enzyme catalysis, play regulatory roles, and help maintain protein structure. Current tools for predicting metal-protein interactions are based on proteins crystallized with their metal ions present (holo forms). However, a majority of resolved structures are free of metal ions (apo forms). Moreover, metal binding is a dynamic process, often involving conformational rearrangement of the binding pocket. Thus, effective predictions need to be based on the structure of the apo state. Here, we report an approach that identifies transition metal-binding sites in apo forms with a resulting selectivity >95%. Applying the approach to apo forms in the Protein Data Bank and structural genomics initiative identifies a large number of previously unknown, putative metal-binding sites, and their amino acid residues, in some cases providing a first clue to the function of the protein.     

Brownian dynamics simulations of the recognition of the scorpion toxin P05 with the small-conductance calcium-activated potassium channels

The recognition of the scorpion toxin P05 and the small-conductance, calcium-activated potassium (SK) channels, rsk1, rsk2, and rsk3, has been studied by means of the Brownian dynamics (BD) method. All of the 25 available structures of P05 in the RCSB Protein Data Bank determined by NMR were considered during the simulation, which indicated that the conformation of P05 affects both the recognition and binding between the two proteins significantly. Comparing the top four high-frequency structures of P05 binding to the SK channels, we found that the rsk2 channel, with high frequencies and lowest electrostatic interaction energies (E (int)(ES)), is the most favorable for P05 binding, while rsk3 is intermediate, and rsk1 is the least favorable. Among the 25 structures of P05, the 13th structure docks into the binding site of the rsk2 channel with the highest probability and most favorable electrostatic interactions. From the P05-rsk2 channel binding model, we identified the residues critical for the recognition of these two proteins through triplet contact analyses. P05 locates around the extracellular mouth of the SK channels and contacts the SK channels using its alpha-helix rather than beta-sheets. The critical triplet contacts for recognition between P05 and the rsk2 channel are Arg6 (P05)-Asp364 (SK), Arg7 (P05)-Asn368 (SK), and Arg13 (P05)-Asp341 (SK). The structure of the P05-rsk2 complex with the most favorable electrostatic interaction energy was further refined by molecular mechanics, showing that six hydrogen bonding interactions exist between P05 and the rsk2 channel: one hydrogen bond is formed between Arg6 (P05) and Asp364(D) (rsk2); Arg7 (P05) forms three hydrogen bonds with Asp341(B) (rsk2)) and Asp364(C) (rsk2); two hydrogen bonds are formed by Arg13 (P05) with Asp341(A) (rsk2) and Asp364(B) (rsk2). The simulation results are in good agreement with the previous molecular biological experiments and can explain the binding phenomena between P05 and SK channels at the level of molecular structure. The consistency between the results of the BD simulations and the experimental data indicated that our 3D model of the P05-rsk2 channel complex is reasonable and can be employed in further biological studies, such as rational design of the novel therapeutic agents blocking the small-conductance, calcium-activated and apamin-sensitive potassium channels, and for mutagenesis studies in both toxins and SK channels. In particular, both the BD simulations and the molecular mechanics refinements indicate that residue Asp364 of the rsk2 channel is critical for its recognition and binding functionality towards P05. This phenomenon has not been appreciated in the previous mutagenesis experiments, indicating that this might be a new clue for further functional study of SK channels.     

基于分子模拟的离子交换层析中静电相互作用研究

将分子模拟方法引入到蛋白质离子交换层析中的静电相互作用研究。选用蛋清溶菌酶和牛胰凝乳蛋白酶为模型蛋白质,阳离子交换吸附剂SP Sepharose FF等为模型层析介质。从蛋白质数据库(PDB)中获得蛋白质三维结构数据,分析了介质孔径和配基分布,以点电荷模拟离子交换层析介质的功能配基,构筑了蛋白质-介质配基模拟表面体系。采用MCCE、Delphi和GRASP等程序包进行了分子模拟计算,考察了作用方向、作用距离、盐浓度、pH等对蛋白质和模拟配基平面间静电相互作用的影响。结果表明,宏观的层析平衡常数与微观分子模拟计算的相互作用能量参数间存在良好的线性关系。     

The pearl necklace model in protein A chromatography: Molecular mechanisms at the resin interface

Staphylococcal protein A chromatography is an established core technology for monoclonal antibody purification and capture in the downstream processing. MabSelect SuRe involves a tetrameric chain of a recombinant form of the B domain of staphylococcal protein A, called the Z-domain. Little is known about the stoichiometry, binding orientation, or preferred binding. We analyzed small-angle X-ray scattering data of the antibody–protein A complex immobilized in an industrial highly relevant chromatographic resin at different antibody concentrations. From scattering data, we computed the normalized radial density distributions. We designed three-dimensional (3D) models with protein data bank crystallographic structures of an IgG1 (the isoform of trastuzumab, used here; Protein Data Bank: 1HZH) and the staphylococcal protein A B domain (the native form of the recombinant structure contained in MabSelect SuRe resin; Protein Data Bank: 1BDD). We computed different binding conformations for different antibody to protein A stoichiometries (1:1, 2:1, and 3:1) and compared the normalized radial density distributions computed from 3D models with those obtained from the experimental data. In the linear range of the isotherm we favor a 1:1 ratio, with the antibody binding to the outer domains in the protein A chain at very low and high concentrations. In the saturation region, a 2:1 ratio is more likely to occur. A 3:1 stoichiometry is excluded because of steric effects.     

The SuMo server: 3D search for protein functional sites

SUMMARY: We provide the scientific community with a web server which gives access to SuMo, a bioinformatic system for finding similarities in arbitrary 3D structures or substructures of proteins. SuMo is based on a unique representation of macromolecules using selected triplets of chemical groups having their own geometry and symmetry, regardless of the restrictive notions of main chain and lateral chains of amino acids. The heuristic for extracting similar sites was used to drive two major large-scale approaches. First, searching for ligand binding sites onto a query structure has been made possible by comparing the structure against each of the ligand binding sites found in the Protein Data Bank (PDB). Second, the reciprocal process, i.e. searching for a given 3D site of interest among the structures of the PDB is also possible and helps detect cross-reacting targets in drug design projects. AVAILABILITY: The web server is freely accessible to academia through http://sumo-pbil.ibcp.fr and full support is available from MEDIT (http://www.medit.fr). CONTACT: mjambon@burnham.org.     

Integration of molecular dynamics simulation and hotspot residues grafting for de novo scFv design against Salmonella Typhi TolC protein

With the development of de novo binders for protein targets from non‐related scaffolds, many possibilities for therapeutics and diagnostics have been created. In this study, we described the use of de novo design approach to create single‐chain fragment variable (scFv) for Salmonella enterica subspecies enterica serovar Typhi TolC protein. Typhoid fever is a global health concern in developing and underdeveloped countries. Rapid typhoid diagnostics will improve disease management and therapy. In this work, molecular dynamics simulation was first performed on a homology model of TolC protein in POPE membrane bilayer to obtain the central structure that was subsequently used as the target for scFv design. Potential hotspot residues capable of anchoring the binders to the target were identified by docking “disembodied” amino acid residues against TolC surface. Next, scFv scaffolds were selected from Protein Data Bank to harbor the computed hotspot residues. The hotspot residues were then incorporated into the scFv scaffold complementarity determining regions. The designs recapitulated binding energy, shape complementarity, and interface surface area of natural protein‐antibody interfaces. This approach has yielded 5 designs with high binding affinity against TolC that may be beneficial for the future development of antigen‐based detection agents for typhoid diagnostics.     

Novel fold and carbohydrate specificity of the potent anti-HIV cyanobacterial lectin from Oscillatoria agardhii

Oscillatoria agardhii agglutinin (OAA) is a recently discovered cyanobacterial lectin that exhibits potent anti-HIV activity. Up to now, only its primary structure and carbohydrate binding data have been available. To elucidate the structural basis for the antiviral mechanism of OAA, we determined the structure of this lectin by x-ray crystallography at 1.2 Å resolution and mapped the specific carbohydrate recognition sites of OAA by NMR spectroscopy. The overall architecture of OAA comprises 10 β-strands that fold into a single, compact, β-barrel-like domain, creating a unique topology compared with all known protein structures in the Protein Data Bank. OAA sugar binding was tested against Man-9 and various disaccharide components of Man-9. Two symmetric carbohydrate-binding sites were located on the protein, and a preference for Manα(1–6)Man-linked sugars was found. Altogether, our structural results explain the antiviral activity OAA and add to the growing body of knowledge about antiviral lectins.     

Three-dimensional model of a substrate-bound SARS chymotrypsin-like cysteine proteinase predicted by multiple molecular dynamics simulations: catalytic efficiency regulated by substrate binding

Severe acute respiratory syndrome (SARS) is a contagious and deadly disease caused by a new coronavirus. The protein sequence of the chymotrypsin-like cysteine proteinase (CCP) responsible for SARS viral replication has been identified as a target for developing anti-SARS drugs. Here, I report the ATVRLQ(p1)A(p1')-bound CCP 3D model predicted by 420 different molecular dynamics simulations (2.0 ns for each simulation with a 1.0-fs time step). This theoretical model was released at the Protein Data Bank (PDB; code: 1P76) before the release of the first X-ray structure of CCP (PDB code: 1Q2W). In contrast to the catalytic dyad observed in X-ray structures of CCP and other coronavirus cysteine proteinases, a catalytic triad comprising Asp187, His41, and Cys145 is found in the theoretical model of the substrate-bound CCP. The simulations of the CCP complex suggest that substrate binding leads to the displacement of a water molecule entrapped by Asp187 and His41, thus converting the dyad to a more efficient catalytic triad. The CCP complex structure has an expanded active-site pocket that is useful for anti-SARS drug design. In addition, this work demonstrates that multiple molecular dynamics simulations are effective in correcting errors that result from low-sequence-identity homology modeling.     

Protein structure databases with new web services for structural biology and biomedical research

The Protein Data Bank Japan (PDBj) curates, edits and distributes protein structural data as a member of the worldwide Protein Data Bank (wwPDB) and currently processes approximately 25-30% of all deposited data in the world. Structural information is enhanced by the addition of biological and biochemical functional data as well as experimental details extracted from the literature and other databases. Several applications have been developed at PDBj for structural biology and biomedical studies: (i) a Java-based molecular graphics viewer, jV; (ii) display of electron density maps for the evaluation of structure quality; (iii) an extensive database of molecular surfaces for functional sites, eF-site, as well as a search service for similar molecular surfaces, eF-seek; (iv) identification of sequence and structural neighbors; (v) a graphical user interface to all known protein folds with links to the above applications, Protein Globe. Recent examples are shown that highlight the utility of these tools in recognizing remote homologies between pairs of protein structures and in assigning putative biochemical functions to newly determined targets from structural genomics projects.     

WebFEATURE: An interactive web tool for identifying and visualizing functional sites on macromolecular structures

WebFEATURE (http://feature.stanford.edu/webfeature/) is a web-accessible structural analysis tool that allows users to scan query structures for functional sites in both proteins and nucleic acids. WebFEATURE is the public interface to the scanning algorithm of the FEATURE package, a supervised learning algorithm for creating and identifying 3D, physicochemical motifs in molecular structures. Given an input structure or Protein Data Bank identifier (PDB ID), and a statistical model of a functional site, WebFEATURE will return rank-scored 'hits' in 3D space that identify regions in the structure where similar distributions of physicochemical properties occur relative to the site model. Users can visualize and interactively manipulate scored hits and the query structure in web browsers that support the Chime plug-in. Alternatively, results can be downloaded and visualized through other freely available molecular modeling tools, like RasMol, PyMOL and Chimera. A major application of WebFEATURE is in rapid annotation of function to structures in the context of structural genomics.     

Identification of the ligand binding sites on the molecular surface of proteins

Identification of protein biochemical functions based on their three-dimensional structures is now required in the post-genome-sequencing era. Ligand binding is one of the major biochemical functions of proteins, and thus the identification of ligands and their binding sites is the starting point for the function identification. Previously we reported our first trial on structure-based function prediction, based on the similarity searches of molecular surfaces against the functional site database. Here we describe the extension of our first trial by expanding the search database to whole heteroatom binding sites appearing within the Protein Data Bank (PDB) with the new analysis protocol. In addition, we have determined the similarity threshold line, by using 10 structure pairs with solved free and complex structures. Finally, we extensively applied our method to newly determined hypothetical proteins, including some without annotations, and evaluated the performance of our methods.     

The RESID Database of protein structure modifications and the NRL-3D Sequence-Structure Database

The RESID Database is a comprehensive collection of annotations and structures for protein post-translational modifications including N-terminal, C-terminal and peptide chain cross-link modifications. The RESID Database includes systematic and frequently observed alternate names, Chemical Abstracts Service registry numbers, atomic formulas and weights, enzyme activities, taxonomic range, keywords, literature citations with database cross-references, structural diagrams and molecular models. The NRL-3D Sequence-Structure Database is derived from the three-dimensional structure of proteins deposited with the Research Collaboratory for Structural Bioinformatics Protein Data Bank. The NRL-3D Database includes standardized and frequently observed alternate names, sources, keywords, literature citations, experimental conditions and searchable sequences from model coordinates. These databases are freely accessible through the National Cancer Institute-Frederick Advanced Biomedical Computing Center at these web sites: http://www. ncifcrf.gov/RESID, http://www.ncifcrf.gov/NRL-3D; or at these National Biomedical Research Foundation Protein Information Resource web sites: http://pir.georgetown.edu/pirwww/dbinfo/resid .html, http://pir.georgetown.edu/pirwww/dbinfo/nrl3d .html     

Crystal structure of human cytochrome P450 2D6 with prinomastat bound

Human cytochrome P450 2D6 contributes to the metabolism of >15% of drugs used in clinical practice. This study determined the structure of P450 2D6 complexed with a substrate and potent inhibitor, prinomastat, to 2.85 ? resolution by x-ray crystallography. Prinomastat binding is well defined by electron density maps with its pyridyl nitrogen bound to the heme iron. The structure of ligand-bound P450 2D6 differs significantly from the ligand-free structure reported for the P450 2D6 Met-374 variant (Protein Data Bank code 2F9Q). Superposition of the structures reveals significant differences for β sheet 1, helices A, F, F', G", G, and H as well as the helix B-C loop. The structure of the ligand complex exhibits a closed active site cavity that conforms closely to the shape of prinomastat. The closure of the open cavity seen for the 2F9Q structure reflects a change in the direction and pitch of helix F and introduction of a turn at Gly-218, which is followed by a well defined helix F' that was not observed in the 2F9Q structure. These differences reflect considerable structural flexibility that is likely to contribute to the catalytic versatility of P450 2D6, and this new structure provides an alternative model for in silico studies of substrate interactions with P450 2D6.