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PubMed2003

Protein-based virtual screening of chemical databases. II. Are homology models of G-Protein Coupled Receptors suitable targets?

The aim of the current study is to investigate whether homology models of G-Protein-Coupled Receptors (GPCRs) that are based on bovine rhodopsin are reliable enough to be used for virtual screening of chemical databases. Starting from the recently described 2.8 A-resolution X-ray structure of bovine rhodopsin, homology models of an "antagonist-bound" form of three human GPCRs (dopamine D3 receptor, muscarinic M1 receptor, vasopressin V1a receptor) were constructed. The homology models were used to screen three-dimensional databases using three different docking programs (Dock, FlexX, Gold) in combination with seven scoring functions (ChemScore, Dock, FlexX, Fresno, Gold, Pmf, Score). Rhodopsin-based homology models turned out to be suitable, indeed, for virtual screening since known antagonists seeded in the test databases could be distinguished from randomly chosen molecules. However, such models are not accurate enough for retrieving known agonists. To generate receptor models better suited for agonist screening, we developed a new knowledge- and pharmacophore-based modeling procedure that might partly simulate the conformational changes occurring in the active site during receptor activation. Receptor coordinates generated by this new procedure are now suitable for agonist screening. We thus propose two alternative strategies for the virtual screening of GPCR ligands, relying on a different set of receptor coordinates (antagonist-bound and agonist-bound states).

Adrenergic beta-2 Receptor Agonists↗
PubMed1998

Homology modeling with low sequence identity.

The field of protein structure prediction is evolving rapidly and in the last few years a number of new methods have been developed and evaluated. However, comparative modeling, or modeling by homology, is still the method of choice when the unknown protein shares any significant sequence similarity with a protein of known structure. The accuracy of the method is highly dependent on the degree of similarity between the target protein and that used as a template. Nevertheless, careful consideration of all the steps performed in the modeling procedure allows useful information to be obtained also from a model based on very low sequence identity.

Models, Molecular↗
PubMed2002

A structural model of pestivirus E(rns) based on disulfide bond connectivity and homology modeling reveals an extremely rare vicinal disulfide.

E(rns) is a pestivirus envelope glycoprotein and is the only known viral surface protein with RNase activity. E(rns) is a disulfide-linked homodimer of 100 kDa; it is found on the surface of pestivirus-infected cells and is secreted into the medium. In this study, the disulfide arrangement of the nine cysteines present in the mature dimer was established by analysis of the proteolytically cleaved protein. Fragments were obtained after digestion with multiple proteolytic enzymes and subsequently analyzed by liquid chromatography-electrospray ionization mass spectrometry. The analysis demonstrates which cysteine is involved in dimerization and reveals an extremely rare vicinal disulfide bridge of unknown function. With the assistance of the disulfide arrangement, a three-dimensional model was built by homology modeling based on the alignment with members of the Rh/T2/S RNase family. Compared to these other RNase family members, E(rns) shows an N-terminal truncation, a large insertion of a cystine-rich region, and a C-terminal extension responsible for membrane translocation. The homology to mammalian RNase 6 supports a possible role of E(rns) in B-cell depletion.

Amino Acid Sequence↗
PubMed1993

Structural relationships of homologous proteins as a fundamental principle in homology modeling.

Protein structure prediction is based mainly on the modeling of proteins by homology to known structures; this knowledge-based approach is the most promising method to date. Although it is used in the whole area of protein research, no general rules concerning the quality and applicability of concepts and procedures used in homology modeling have been put forward yet. Therefore, the main goal of the present work is to provide tools for the assessment of accuracy of modeling at a given level of sequence homology. A large set of known structures from different conformational and functional classes, but various degrees of homology was selected. Pairwise structure superpositions were performed. Starting with the definition of the structurally conserved regions and determination of topologically correct sequence alignments, we correlated geometrical properties with sequence homology (defined by the 250 PAM Dayhoff Matrix) and identity. It is shown that both the topological differences of the protein backbones and the relative positions of corresponding side chains diverge with decreasing sequence identity. Below 50% identity, the deviation in regions that are structurally not conserved continually increases, thus implying that with decreasing sequence identity modeling has to take into account more and more structurally diverging loop regions that are difficult to predict.

Bacterial Proteins↗
PubMed2006

Comparison of homology models and X-ray structures of the nuclear receptor CAR: assessing the structural basis of constitutive activity.

The constitutive androstane receptor (CAR) possesses an intrinsic basal activity whose structural basis has been analysed during the last decade. Recently, we published a homology model of the CAR ligand binding domain (LBD) based on the X-ray structures of the closely related pregnane X (PXR) and vitamin D (VDR) receptor. A detailed analysis of the homology model and molecular dynamics (MD) simulations afforded us to propose a potential mechanism underlying the constitutive activity of CAR. Almost simultaneously, X-ray structures of human and mouse CAR LBD were released. In the present study, a detailed analysis and comparison of homology model and X-ray structures is carried out in order to evaluate the quality and reliability of our homology modelling procedure. The hypothesis of the constitutive activity which we proposed on the basis of our modelling results was tested for consistency with the crystal structures. In addition, the features stated to be essential for the basal activity based on the X-ray data were investigated by means of molecular dynamics simulations. Our results show that the homology modelling procedure was able to predict the CAR LBD structure with high accuracy. Structural features that have been revealed as critical for constitutive activity in the model are also observed in the X-ray structures. Furthermore, the MD simulations of the CAR X-ray structures and a detailed analysis of other NRs clarify the role of distinct structural features that have been assigned an important role for the constitutive activity.

Amino Acid Sequence↗
PubMed1995

Homology modeling of histidine-containing phosphocarrier protein and eosinophil-derived neurotoxin: construction of models and comparison with experiment.

Homology modeling methods have been used to construct models of two proteins--the histidine-containing phosphocarrier protein (HPr) from Mycoplasma capricolum and human eosinophil-derived neurotoxin (EDN). Comparison of the models with the subsequently determined X-ray crystal structures indicates that the core regions of both proteins are reasonably well reproduced, although the template structures are closer to the X-ray structures in these regions--possible enhancements are discussed. The conformations of most of the side chains in the core of HPr are well reproduced in the modeled structure. As expected, the conformations of surface side chains in this protein differ significantly from the X-ray structure. The loop regions of EDN were incorrectly modeled--reasons for this and possible enhancements are discussed.

Algorithms↗
PubMed1994

Homology modeling of divergent proteins.

A method is presented for homology modeling of proteins bearing weak sequence identity to proteins of known tertiary structure. To accommodate non-identical amino acids in the core region, the backbone of the structurally conserved core of the model protein is allowed to deviate from that of the template protein. We have expanded FOLDER, a distance geometry-based homology modeling method, to allow for such displacements in the structurally conserved core. Models are built by rigidly constraining the interatomic distances within a structurally conserved segment and by allowing the interatomic distances between these segments to vary by a "divergence factor". We test this method by simulating models of the beta-barrel domain D1 of CD4 and a four-helix bundle protein cytochrome b562 using the crystal structures of Bence-Jones protein and cytochrome c' as templates, respectively. In both cases, previously published structure-based sequence alignments were used for simulating models. The root-mean-square (r.m.s.) deviation of the backbone atoms in the common core between the templates and models was found to be a function of the imposed divergence factor. Our results demonstrate that this r.m.s. deviation results from the relative displacements of structurally conserved segments to accommodate the amino acid replacements in the core of the model protein. To test the integrity of the simulated structures we compared them with their respective crystal structures. The r.m.s. deviation of the backbone atoms in the core regions of the simulated models and their respective crystal structures is approximately 1.4 A. The r.m.s. deviation for all the backbone atoms in the models, including those in the structurally variable regions, which are modeled de novo, is 2.4 A for CD4 and 3.2 A for cytochrome b562 when compared with their respective X-ray structures.

Animals↗
PubMed2003

A homology model of penicillin acylase from Alcaligenes faecalis and in silico evaluation of its selectivity.

A three-dimensional model of the relatively unknown penicillin acylase from Alcaligenes faecalis (PA-AF) was built up by means of homology modeling based on three different crystal structures of penicillin acylase from various sources. An in silico selectivity study was performed to compare this homology model to the structure of the Escherichia coli enzyme (PA-EC) in order to find any selectivity differences between the two enzymes. The program GRID was applied in combination with the principal component analysis technique to identify the regions of the active sites where the PAs potentially engage different interactions with ligands. These differences were further analyzed and confirmed by molecular docking simulations. The PA-AF homology model provided the structural basis for the explanation of the different enantioselectivities of the enzymes previously demonstrated experimentally and reported in the literature. Different substrate selectivities were also predicted for PA-AF compared to PA-EC. Since no crystallographic data are available for PA-AF to date, the three-dimensional homology model represents a useful and efficient tool for fully exploiting this attractive and efficient biocatalyst, particularly in enantioselective acylations of amines.

Alcaligenes faecalis↗
PubMed2004

Successful virtual screening for a submicromolar antagonist of the neurokinin-1 receptor based on a ligand-supported homology model.

The neurokinin-1 (NK1) receptor belongs to the family of G-protein-coupled receptors (GPCRs), which represents one of the most relevant target families in small-molecule drug design. In this paper, we describe a homology modeling of the NK1 receptor based on the high-resolution X-ray structure of rhodopsin and the successful virtual screening based on this protein model. The NK1 receptor model has been generated using our new MOBILE (modeling binding sites including ligand information explicitly) approach. Starting with preliminary homology models, it generates improved models of the protein binding pocket together with bound ligands. Ligand information is used as an integral part in the homology modeling process. For the construction of the NK1 receptor, antagonist CP-96345 was used to restrain the modeling. The quality of the obtained model was validated by probing its ability to accommodate additional known NK1 antagonists from structurally diverse classes. On the basis of the generated model and on the analysis of known NK1 antagonists, a pharmacophore model was deduced, which subsequently guided the 2D and 3D database search with UNITY. As a following step, the remaining hits were docked into the modeled binding pocket of the NK1 receptor. Finally, seven compounds were selected for biochemical testing, from which one showed affinity in the submicromolar range. Our results suggest that ligand-supported homology models of GPCRs may be used as effective platforms for structure-based drug design.

Amino Acid Sequence↗
PubMed2004

A set of homology models of pore loop domain of six eukaryotic voltage-gated potassium channels Kv1.1-Kv1.6.

Homology models of the pore loop domain of six eukaryotic potassium channels, Kv1.1-Kv1.6, were generated based on the crystallographic structure of KcsA. The results of amino acid sequence alignment indicate that these Kv channels are composed of two structurally and functionally independent domains: the N-terminal 'voltage sensor' domain and the C-terminal 'pore loop' domain. The homology models reveal that the pore loop domains of these Kv channels exhibit similar folds to those of KcsA. The structural features and specific packing of aromatic residues around the selectivity filter of these Kv channels are nearly identical to those of KcsA, whereas most of the structural variations occur in the turret as well as in the inner and outer helices. The distribution of polar and nonpolar side chains on the surfaces of the KcsA and Kv channels reveals that they exhibit a segregation of side chains common to most integral membrane proteins. As the hydrogen bond between Glu71 and Asp80 in KcsA plays an important role in stabilizing the channel, the substituted Val residue in the Kv family corresponding to Glu71 of KcsA stabilizes the channel by making hydrophobic contact with Tyr residue from the signature sequence of the selectivity filter. The homology models of these Kv channels provide particularly attractive subjects for further structure-based studies.

Amino Acid Sequence↗
PubMed2004

Utility of homology models in the drug discovery process.

Advances in bioinformatics and protein modeling algorithms, in addition to the enormous increase in experimental protein structure information, have aided in the generation of databases that comprise homology models of a significant portion of known genomic protein sequences. Currently, 3D structure information can be generated for up to 56% of all known proteins. However, there is considerable controversy concerning the real value of homology models for drug design. This review provides an overview of the latest developments in this area and includes selected examples of successful applications of the homology modeling technique to pharmaceutically relevant questions. In addition, the strengths and limitations of the application of homology models during all phases of the drug discovery process are discussed.

Animals↗
PubMed2005

Binding modes of dihydroquinoxalinones in a homology model of bradykinin receptor 1.

We report the first homology model of human bradykinin receptor B1 generated from the crystal structure of bovine rhodopsin as a template. Using an automated docking procedure, two B1 receptor antagonists of the dihydroquinoxalinone structural class were docked into the receptor model. Site-directed mutagenesis data of the amino acid residues in TM1, TM3, TM6, and TM7 were incorporated to place the compounds in the binding site of the homology model of the human B1 bradykinin receptor. The best pose in agreement with the mutation data was selected for detailed study of the receptor-antagonist interaction. To test the model, the calculated antagonist-receptor binding energy was correlated with the experimentally measured binding affinity (K(i)) for nine dihydroquinoxalinone analogs. The model was used to gain insight into the molecular mechanism for receptor function and to optimize the dihydroquinoxalinone analogs.

Amino Acid Sequence↗
PubMed1994

Comparison of homology models with the experimental structure of a novel serine protease.

A model structure of the human complement enzyme factor D was built based on homology with related serine proteases. A molecular-replacement solution of the factor D crystal structure employing the homology model refined without manual intervention to an R factor of 0.249 with 2.4 A native diffraction data. A multiple isomorphous replacement (MIR) electron-density map was subsequently produced, leading to a model refined at 2.0 A resolution to an R factor of 0.188. A homology model built with commercial modeling software was subjected to the same procedure. Comparisons of the homology models with the final refined MIR structure are presented. Major discrepancies were found in critical active-site regions.

Journal Article↗
PubMed2005

Homology modelling and active-site-mutagenesis study of the catalytic domain of the pneumococcal phosphorylcholine esterase.

Streptococcus pneumoniae is among the major human pathogens. Several interactions of this bacterium with its host appear to have been mediated by bacterial cell wall components. Specifically, phosphorylcholine residues covalently attached to teichoic and lipoteichoic acids serve as anchors for many surface-located proteins (choline-binding proteins CBPs), including cell-adhesion and virulence factors, and are also recognized by host response components through choline-binding receptors. In this study, we have performed modelling of the catalytic domain of pneumococcal phosphorylcholine esterase (Pce), a modular enzyme that is capable of removing phosphorycholine residues from teichoic and lipoteichoic acids, remodelling their distribution on the bacterial envelope. We wish to contribute to the structural knowledge of Pce. In this pursuit, 3D models of Pce have been established by homology modelling, using the X-ray structure of enzymes from the alpha/beta metallo-lactamase family fold as templates. Theoretical models of pneumococcal phosphorylcholine esterase (Pce) catalytic modules obtained by homology modelling, and corresponding docking studies employed to find out the residues involved in the binding of Zn ions, are discussed according to mutational studies and ab initio calculations. The presence of a binuclear Zn cluster in the catalytic domain of Pce and a likely coordination model are proposed.

Amino Acid Sequence↗
PubMed2005

Homology models of the tetramerization domain of six eukaryotic voltage-gated potassium channels Kv1.1-Kv1.6.

The homology models of the tetramerization (T1) domain of six eukaryotic potassium channels, Kv1.1-Kv1.6, were constructed based on the crystal structure of the Shaker T1 domain. The results of amino acid sequence alignment indicate that the T1 domains of these K+ channels are highly conserved, with the similarities varying from 77% between Shaker and Kv1.6 to 93% between Kv1.2 and Kv1.3. The homology models reveal that the T1 domains of these Kv channels exhibit similar folds as those of Shaker K+ channel. These models also show that each T1 monomer consists of three distinct layers, with N-terminal layer 1 and C-terminal layer 3 facing the cytoplasm and the membrane, respectively. Layer 2 exhibits the highest structural conservation because it is located around the central hydrophobic core. For each Kv channel, four identical subunits assemble into the homotetramer architecture around a four-fold axis through the hydrogen bonds and salt bridges formed by 15 highly conserved polar residues. The narrowest opening of the pore is formed by the four conserved residues corresponding to R115 of the Shaker T1 domain. The homology models of these Kv T1 domains provide particularly attractive targets for further structure-based studies.

Amino Acid Sequence↗
PubMed2003

First X-ray cocrystal structure of a bacterial FabH condensing enzyme and a small molecule inhibitor achieved using rational design and homology modeling.

The first cocrystal structure of a bacterial FabH condensing enzyme and a small molecule inhibitor is reported. The inhibitor was obtained by rational modification of a high throughput screening lead with the aid of a S. pneumoniae FabH homology model. This homology model was used to design analogues that would have both high affinity for the enzyme and appropriate aqueous solubility to facilitate cocrystallization studies.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed1995

A model for vicilin solubility at mild acidic pH, based on homology modelling and electrostatics calculations.

The crystallographic structures of jack bean canavalin and French bean phaseolin have been used to construct a homology model of the storage vicilin of cocoa. Reported molecular weights for cocoa storage protein subunits correlate with proteolysis at the site of a large hydrophilic insert in the mature protein. Burial of the hydrophobic amino acids on trimer formation is a strongly conserved feature in the vicilin family. Histidine residues also sit at the monomer-monomer interfaces of the trimer and are likely to contribute to the decreased solubility of cocoa vicilin at mild acidic pH, which is generally considered to be caused solely by aggregation near to the isoelectric point. Electrostatic calculations suggest that such an arrangement of histidine residues in the absence of specific counterion binding will not favour the particular geometry of trimer formation below neutral pH. Higher order aggregates that do not exclude histidine charge from the solvent may be favoured, aiding the precipitation of cocoa vicilin at mild acidic pH. This suggestion is considered for the vicilin family. The hypothesis could contribute to an understanding of the pH and ionic strength dependence of vicilin solubility in vitro, and possibly of the behaviour of vicilins in the seed storage environment.

Amino Acid Sequence↗
PubMed1997

Homology modeling of rabbit prolactin hormone complexed with its receptor.

The pituitary hormone prolactin (prl) is implicated in a number of biological functions, especially lactation, which is mediated through specific lactogenic receptors (PrlR). Human growth hormone (hGH) is also a pituitary hormone responsible for linear growth. While the growth hormone receptor (hGHR) binds only hGH, hPrlR can interact with both hGH and hPrl. Using structural information from the human growth hormone (hGH)/receptor (hGHR) complex, we modeled by homology a complex between rabbit prolactin hormone (rbPrl) and its receptor (rbPrlR). While the somatogenic hormone/somatogenic receptor (hGH/hGHR) and somatogenic hormone/lactogenic receptor (hGH/hPrlR) interactions are now known and well studied, here we propose a model for the interaction of the lactogenic hormone with its receptor (rbPrl/rbPrlR), and compare these three kinds of ligand/receptor interaction. We identified residues contributing to the active site and tested the potential dimerization of the receptor. Biochemical studies and information deduced from the modeled complex do not exclude a homodimeric form but point to a functional heterodimeric complex.

Amino Acid Sequence↗