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Human integrin alphavbeta5: homology modeling and ligand binding.

The recently reported crystal structures of the extracellular domains of the alphavbeta3 integrin in its unligated state and in complex with the peptide cyclo(-RGDf[NMe]V-) have dramatically increased our understanding of ligand binding to integrins. Nonetheless, ligand selectivity toward different integrin subtypes is still a challenging problem complicated by the fact that 3D structures of most of the integrin subtypes remain unknown. In this study, a three-dimensional model for the human alphavbeta5 integrin was obtained using homology modeling based on the crystal coordinates of alphavbeta3 in its bound conformation as template. The modeled receptor was refined using energy minimization and molecular dynamics simulations in explicit solvent. The refined alphavbeta5 model was used to explore the interactions between this integrin and alphavbeta3/alphavbeta5 dual and alphavbeta3-selective ligands in the attempt to provide a preliminary rationalization, at the molecular level, of ligand selectivity toward the two alphav integrins. It was found that, in the RGD binding site of the alphavbeta5 receptor, a partial "roof" composed mainly of the SDL residues Tyr179 and Lys180 is present and hampers the binding of compounds containing bulky substituents in the proximity of the carboxylate group. This study provides a testable hypothesis for alphav integrins subtype ligand binding selectivity, in line with both mutagenesis data and SARs studies.

Amino Acid Sequence↗

Homology modeling and substrate binding study of Nudix hydrolase Ndx1 from Thermos thermophilus HB8.

With homology modeling techniques, molecular mechanics, and molecular dynamics methods, a 3D structure model of Ndx1 is created and refined. This model is further assessed by Profile-3D and ProStat, which confirm that the refined model is reliable. With this model, a flexible docking study is performed and the result indicates that Glu46, Arg88, and Glu90 are three important determinant residues in binding, as they have strong hydrogen bonding interactions and electrostatic interactions with Ap6A. In addition, we further find that three residues, Ser38, Leu39 and Glu46, coordinate enzyme-bound Mg2+ ions in complex N-A. The Glu46 is consistent with the experimental results by Iwai et al., and the other four residues mentioned above may also play vital roles in catalysis of Ndx1.

Amino Acid Sequence↗

Performance of 3D-database molecular docking studies into homology models.

The performance of docking studies into protein active sites constructed by homology model building was investigated using CDK2 and factor VIIa screening data sets. When the sequence identity between model and template near the binding site area is greater than approximately 50%, roughly 5 times more active compounds are identified than would be found randomly. This performance is comparable to docking to crystal structures.

Binding Sites↗

Homology modelling of human cytochromes P450 involved in xenobiotic metabolism and rationalization of substrate selectivity.

Molecular modelling of human cytochrome P450 (CYP) isoforms is described, based on amino acid sequence homology with a unique bacterial P450 (CYP102) of known crystal structure. It is found that for the human hepatic P450s involved in the metabolism of xenobiotics, ie. CYPIA2, CYP 1A6, CYP2B6, CYP2C9, CYP2C 19, CYP2D6, CYP2E1 and CYP3A4, there is a satisfactory agreement between specific substrate characteristics and topographical features of the putative active sites, including complementarity with key amino acid residues in the P450 haem environments. A combination of homology model interactions with substrates and certain molecular properties of the compounds themselves provides a methodology for the evaluation of potential P450 selectivity in new chemical entities (NCEs).

Amino Acid Sequence↗

Homology modeling of glycosyl hydrolase family 18 enzymes and proteins.

Using state-of-the-art homology modeling methods, three-dimensional coordinates for three family 18 glycosyl hydrolases were determined. The structures for Gp39, Brp39, and chitotriosidase were computer determined using the X-ray coordinates from SmChiA. The results of the modeling efforts are assessed, and comparison of the modeled structures to other known family 18 members is made.

Acetylglucosaminidase↗

Homology modelling of human CYP1A2 based on the CYP2C5 crystallographic template structure.

1. The results of homology modelling of human cytochrome P4501A2 (CYP1A2) based on the CYP2C5 crystal structure are reported. It exhibits improved sequence homology relative to that of CYP102. 2. It was demonstrated that many selective substrates for CYP1A2 could fit within the putative active site of the enzyme, and in orientations which agree with documented evidence for CYP1A2-mediated metabolism. 3. Furthermore, a number of amino acid residues lining the haem pocket have been shown, via site-directed mutagenesis, to have an influence on substrate metabolism, and these experimental findings from the literature are consistent with the modelled interactions for selective substrates. 4. The binding affinities of several CYP1A2 substrates have also been calculated from the CYP1A2 active site interactions and they agree closely with experimental values.

Amino Acid Sequence↗

Bioinformatic comparison of structures and homology-models of matrix metalloproteinases.

The entire family of human matrix metalloproteinases (MMPs) was investigated using phylogenetic trees and homology modeling. The phylogenetic analysis indicates that individual domains of each MMP have evolved in a correlated manner. Despite their high sequence similarity, the phylogenetic tree of the catalytic domains already allows functional (e.g., linked to regulation and substrate recognition) homologies between different MMPs to be identified. The same pattern of functional homologies is confirmed by the phylogenetic analysis of the mature proteins. Structural models were built for the catalytic domains of the entire MMP family, for twelve hemopexin domains and for twelve mature proteins. The surface properties around the active site cleft of the modeled and experimental structures are quite conserved, whereas the hemopexin domains are more differentiated, possibly indicating a role in determining substrate specificity. The analysis of mature MMPs showed that the area of the interface between the catalytic and hemopexin domains is essentially conserved, with both hydrophobic and hydrophilic amino acids at the interface. The absence of specific conserved interdomain contacts suggests that the interface is tolerant to amino acid replacements, and that there may be a certain degree of plasticity with respect to the reciprocal orientation of the two domains.

Binding Sites↗

Homology modelling and binding site mapping of the human histamine H1 receptor.

Three-dimensional model of the human histamine H1 receptor was developed by homology modelling using the high resolution structure of bovine rhodopsin as template. Genetic algorithm based docking calculations were used to identify the role of several amino acids having an effect on agonist or antagonist binding. Binding mode analyses of mepyramine, desloratidine, loratidine and acrivastine allowed us to rationalise their binding affinity. Binding site mapping resulted in seven new potential aromatic interaction points (Tyr 108, Phe 184, Phe 190, Phe 199, Phe 424, Trp 428, Tyr 431), that took part in forming the lipophilic pocket of the antagonist binding cavity.

Algorithms↗

Homology modelling of the enzyme P450 17 alpha-hydroxylase/17,20-lyase--a target for prostate cancer chemotherapy--from the crystal structure of P450BM-3.

Using homology modelling techniques the structure of cytochrome P450 17 alpha-hydroxylase/17-20 lyase (P450c17) has been predicted from the crystal structure of P450BM-3. The resulting structure has been compared to a previous model, built on the basis of homology to P450cam. Despite considerable structural differences between the two template structures, the two derived models for P450c17 show a high degree of similarity in the active-site region. As before, a bilobal active-site cavity is predicted, and we hypothesize that binding of the steroid in one lobe of the active site is associated with the hydroxylase activity of the enzyme, whilst binding in the other is associated with the lyase reaction.

Amino Acid Sequence↗

All are not equal: a benchmark of different homology modeling programs.

Modeling a protein structure based on a homologous structure is a standard method in structural biology today. In this process an alignment of a target protein sequence onto the structure of a template(s) is used as input to a program that constructs a 3D model. It has been shown that the most important factor in this process is the correctness of the alignment and the choice of the best template structure(s), while it is generally believed that there are no major differences between the best modeling programs. Therefore, a large number of studies to benchmark the alignment qualities and the selection process have been performed. However, to our knowledge no large-scale benchmark has been performed to evaluate the programs used to transform the alignment to a 3D model. In this study, a benchmark of six different homology modeling programs- Modeller, SegMod/ENCAD, SWISS-MODEL, 3D-JIGSAW, nest, and Builder-is presented. The performance of these programs is evaluated using physiochemical correctness and structural similarity to the correct structure. From our analysis it can be concluded that no single modeling program outperform the others in all tests. However, it is quite clear that three modeling programs, Modeller, nest, and SegMod/ ENCAD, perform better than the others. Interestingly, the fastest and oldest modeling program, SegMod/ ENCAD, performs very well, although it was written more than 10 years ago and has not undergone any development since. It can also be observed that none of the homology modeling programs builds side chains as well as a specialized program (SCWRL), and therefore there should be room for improvement.

Amino Acid Sequence↗

A homology model of the pore region of HCN channels.

HCN channels are activated by membrane hyperpolarization and regulated by cyclic nucleotides, such as cyclic adenosine-mono-phosphate (cAMP). Here we present structural models of the pore region of these channels obtained by using homology modeling and validated against spatial constraints derived from electrophysiological experiments. For the construction of the models we make two major assumptions, justified by electrophysiological observations: i), in the closed state, the topology of the inner pore of HCN channels is similar to that of K(+) channels. In particular, the orientation of the S5 and S6 helices of HCN channels is very similar to that of the corresponding helices of the K(+) KcsA and K(+) KirBac1.1 channels. Thus, we use as templates the x-ray structure of these K(+) channels. ii), In the open state, the S6 helix is bent further than it is in the closed state, as suggested (but not proven) by experimental data. For this reason, the template of the open conformation is the x-ray structure of the MthK channel. The structural models of the closed state turn out to be consistent with all the available electrophysiological data. The model of the open state turned out to be consistent with all the available electrophysiological data in the filter region, including additional experimental data performed in this work. However, it required the introduction of an appropriate, experimentally derived constraint for the S6 helix. Our modeling provides a structural framework for understanding several functional properties of HCN channels: i), the cysteine ring at the inner mouth of the pore may act as a sensor of the intracellular oxidizing/reducing conditions; ii), the bending amplitude of the S6 helix upon gating appears to be significantly smaller than that found in MthK channels; iii), the reduced ionic selectivity of HCN channels, relative to that of K(+) channels, may be caused, at least in part, by the larger flexibility of the inner pore of HCN channels.

Amino Acid Sequence↗

Mechanistic study of proton transfer and hysteresis in catalytic antibody 16E7 by site-directed mutagenesis and homology modeling.

Antibody 16E7 catalyzes the carbon protonation of enol ether 2 to hemiacetal 3, and the carbon deprotonation of benzisoxazole 7 to phenol 8. This antibody shows an extreme case of hysteresis, requiring several hours to reach full activity. Antibody 16E7 was expressed as recombinant chimeric Fab in Escherichia coli. A model for the three-dimensional structure was produced by homology modeling and used for a docking procedure to obtain models for antibody-ligand complexes. Site-direct mutagenesis of GluL39, identified as a possible catalytic residue by the model, to either glutamine or alanine abolished catalysis, showing that both the protonation reaction of enol ether 2 and the deprotonation of benzisoxazole 7 are promoted by the same residue. The model furthermore suggested that substrate access to the catalytic site might be hindered by a flexible HCDR3 loop held in closed position by a hydrogen bond between SerH99 and GluL39, which could explain the observed hysteresis effect. In agreement with this model, mutagenesis of SerH99 to alanine, or deletion of this residue, was found to reduce hysteresis by approximately 50%.

Antibodies, Catalytic↗

Completion and refinement of 3-D homology models with restricted molecular dynamics: application to targets 47, 58, and 111 in the CASP modeling competition and posterior analysis.

A method is presented to refine models built by homology by the use of restricted molecular dynamics (MD) techniques. The basic idea behind this method is the use of structure validation software to determine for each residue the likelihood that it is modeled correctly. This information is used to determine constraints and restraints in an MD simulation including explicit solvent molecules, which is used for model refinement. The procedure is based on the idea that residues that the validation software identifies as correctly positioned should be strongly constrained or restrained in the MD simulations, whereas residues that are likely to be positioned wrongly should move freely. Two different protocols are compared: one (applied to CASP3 target T58) using full structural constraints with separate optimization of each short fragment and the other (applied to T47) allowing some freedom using harmonic restraining potentials, with automatic optimization of the whole molecule. Structures along the MD trajectory that scored best in structural checks were selected for the construction of models that appeared to be successful in the CASP3 competition. Model refinement with MD in general leads to a model that is less like the experimental structure (Levitt et al. Nature Struct Biol 1999;6:108-111). Actually, refined T47 was slightly improved compared to the starting model; changes in model T58 led not to further enhancement. After the X-ray structure of the modeled proteins became known, the procedure was evaluated for two targets (T47 and the CASP4 target T111) by comparing a long simulation in water with the experimental target structures. It was found that structural improvements could be obtained on a nanosecond time scale by allowing appropriate freedom in the simulation. Structural checks applied to fast fluctuations do not appear to be informative for the correctness of the structure. However, both a simple hydrogen bond count and a simple compactness measure, if averaged over times of typically 300 ps, correlate well with structural correctness and we suggest that criteria based on these properties may be used in computational folding strategies.

Amino Acid Sequence↗

A self consistent mean field approach to simultaneous gap closure and side-chain positioning in homology modelling.

A new computational procedure which simultaneously provides gap closure and side-chain positioning in homology modelling is described. It uses a database search scheme to generate fragments to model gaps, a rotamer library to define side-chain conformations, and iteratively refines a conformational matrix CM, such that its elements CM(i,j,o) and CM(i,j,k) give the probabilities that the backbone of residue i adopts the conformation described by fragment j and that its side-chain adopts the conformation of its possible rotamer k. Each residue experiences the average of all possible environments, weighted by their respective probabilities. The method converges, thereby deserving the name of 'self consistent mean field' approach.

Algorithms↗

Proposal of a new binding orientation for non-peptide AT1 antagonists: homology modeling, docking and three-dimensional quantitative structure-activity relationship analysis.

A three-dimensional model of the AT1 receptor was constructed by means of a homology modeling procedure, using the X-ray structure of bovine rhodopsin as the initial template and taking into account the available site-directed mutagenesis data. The docking of losartan and its active metabolite EXP3174, followed by 1 ns of molecular dynamics (MD) simulation inserted into the phospholipid bilayer, suggested a different binding orientation for these antagonists from those previously proposed. Furthermore, the docking of several non-peptide antagonists was used as an alignment tool for the development of a three-dimensional quantitative structure-activity relationship (3D-QSAR) model, and the good results confirmed our binding hypothesis and the reliability of the model.

Amino Acid Sequence↗

Analysis of selective regions in the active sites of human cytochromes P450, 2C8, 2C9, 2C18, and 2C19 homology models using GRID/CPCA.

This study demonstrates a selectivity analysis using the GRID/CPCA strategy on four human cytochrome P450 2C homology models (CYP2C8, 2C9, 2C18, and 2C19). Although the four enzymes share more than 80% amino acid sequence identity, the substrate specificity differs. To investigate the selectivity of the enzymes and the amino acids that determine the specificity of each CYP2C enzyme, a selectivity analysis was made using GRID/CPCA. In the GRID calculations 10 probes were used covering hydrophobic, steric, and hydrogen bond acceptor and donor interactions. The selectivity analysis showed that the most important determinants of selectivity among the CYP2C models are the geometrical features of the active sites and the hydrophobic interactions. The selectivity analysis singled out CYP2C8 as the most different of the four CYP2C enzymes with amino acids with distinct properties in positions 114, 205, and 476 (Ser, Phe, and Ile, respectively) compared to the other enzymes. An inverse pharmacophore model for CYP2C9 was constructed from the selective regions, and the model agreed with the docking of diclofenac where the properties of the ligand overlapped with the pharmacophoric points in the model.

Amino Acid Sequence↗

Homology modeling of the Abl-SH3 domain.

A tertiary structure model of the Abl-SH3 domain is predicted by using homology modeling techniques coupled to molecular dynamics simulations. Two template proteins were used, Fyn-SH3 and Spc-SH3. The refined model was extensively checked for errors using criteria based on stereochemistry, packing, solvation free-energy, accessible surface areas, and contact analyses. The different checking methods do not totally agree, as each one evaluates a different characteristic of protein structures. Several zones of the protein are more susceptible to incorporating errors. These include residues 13, 15, 35, 39, 45, 46, 50, and 60. An interesting finding is that the measurement of the C alpha chirality correlated well with the rest of the criteria, suggesting that this parameter might be a good indicator of correct local conformation. Deviations of more than 4 degrees may be indicative of poor local structure.

Computer Simulation↗

Homology modelling of transferrin-binding protein A from Neisseria meningitidis.

Neisseria meningitidis, a causative agent of bacterial meningitis, obtains transferrin-bound iron by expressing two outer membrane located transferrin-binding proteins, TbpA and TbpB. TbpA is thought to be an integral outer membrane pore that facilitates iron uptake. Evidence suggests that TbpA is a useful antigen for inclusion in a vaccine effective against meningococcal disease, hence the identification of regions involved in ligand binding is of paramount importance to design strategies to block uptake of iron. The protein shares sequence and functional similarities to the Escherichia coli siderophore receptors FepA and FhuA, whose structures have been determined. These receptors are composed of two domains, a 22-stranded beta-barrel and an N-terminal plug region that sits within the barrel and occludes the transmembrane pore. A three-dimensional TbpA model was constructed using FepA and FhuA structural templates, hydrophobicity analysis and homology modelling. TbpA was found to possess a similar architecture to the siderophore receptors. In addition to providing insights into the highly immunogenic nature of TbpA and allowing the prediction of potentially important ligand-binding epitopes, the model also reveals a narrow channel through its entire length. The relevance of this channel and the spatial arrangement of external loops, to the mechanism of iron translocation employed by TbpA is discussed.

Amino Acid Sequence↗