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PubMed2002

Homology modeling of falcipain-2: validation, de novo ligand design and synthesis of novel inhibitors.

Increasing resistance of malaria parasites, in particular Plasmodium falciparum, demands a serious search for novel targets. Cysteine protease in P. falciparum, encoded by a previously unidentified gene falcipain 2, provides one such target to design chemotherapeutic agents for treatment of malaria. In fact, a few cysteine protease inhibitors have been shown to inhibit growth of cultured malarial parasites. In absence of a crystal structure for this enzyme, homology modeling proved to be a reasonable alternative to study binding requirements of the enzyme. A homology model for falcipain 2 was developed and validated by docking of known vinyl sulfone inhibitors. Further, based on the observations of these studies, novel isoquinoline inhibitors were designed and synthesized, which exhibited in vitro enzyme inhibition at micromolar concentrations.

Algorithms↗
PubMed2003

Investigation of enzyme selectivity in the human CYP2C subfamily: homology modelling of CYP2C8, CYP2C9 and CYP2C19 from the CYP2C5 crystallographic template.

Homology modelling of human CYP2C subfamily enzymes, CYP2C8, CYP2C9 and CYP2C19, based on the rabbit CYP2C5 crystal structure template is reported. The relatively high sequence homologies (75-80%) between the rabbit CYP2C5 and human CYP2C subfamily enzymes tend to indicate that the resulting structures should prove adequate models of these major catalysts of human drug metabolism. Selective substrates of all three human CYP2C enzymes are found to fit closely within the putative active sites in a manner which is consistent with site-directed mutagenesis experiments and known positions of substrate metabolism. The specific interactions between substrates and enzymes can be used to rationalize the variation in substrate binding affinity and generate QSAR models for both inhibition and metabolism via CYP2C family enzymes, yielding a generally good agreement with experimental binding data obtained from Km values, with correlation coefficients (R values) of between 0.97 and 0.99 depending on the QSAR equation produced.

Aryl Hydrocarbon Hydroxylases↗
PubMed2002

Homology modeling of the cation binding sites of Na+K+-ATPase.

Homology modeling of the alpha-subunit of Na+K+-ATPase, a representative member of P-type ion transporting ATPases, was carried out to identify the cation (three Na+ and two K+) binding sites in the transmembrane region, based on the two atomic models of Ca2+-ATPase (Ca2+-bound form for Na+, unbound form for K+). A search for potential cation binding sites throughout the atomic models involved calculation of the valence expected from the disposition of oxygen atoms in the model, including water molecules. This search identified three positions for Na+ and two for K+ at which high affinity for the respective cation is expected. In the models presented, Na+- and K+-binding sites are formed at different levels with respect to the membrane, by rearrangements of the transmembrane helices. These rearrangements ensure that release of one type of cation coordinates with the binding of the other. Cations of different radii are accommodated by the use of amino acid residues located on different faces of the helices. Our models readily explain many mutational and biochemical results, including different binding stoichiometry and affinities for Na+ and K+.

Amino Acid Sequence↗
PubMed2006

CYP2E1 active site residues in substrate recognition sequence 5 identified by photoaffinity labeling and homology modeling.

Despite its biological importance, our knowledge of active site structure and relevance of critical amino acids in CYP2E1 catalytic processes remain limited. In this study, we identified CYP2E1 active site residues using photoaffinity labeling with 7-azido-4-methylcoumarin (AzMC) coupled with a CYP2E1 homology model. In the absence of light, AzMC was an effective competitor against substrate p-nitrophenol oxidation by CYP2E1. Photoactivation of AzMC led to a concentration-dependent loss in CYP2E1 activity and structural integrity resulting from the modification of both heme and protein. The photo-labeling reaction degraded heme and produced a possible heme adduct. Probe incorporation into the protein occurred at multiple sites within substrate recognition sequence 5 (SRS-5). Based on a CYP2E1 homology model, we hypothesize AzMC labels SRS-5 residues, Leu363, Val364, and Leu368, in the active site. In addition, we propose a series of phenylalanines, especially Phe106, mediate contacts with the coumarin.

Amino Acid Sequence↗
PubMed2003

An atomic detail model for the human ATP binding cassette transporter P-glycoprotein derived from disulfide cross-linking and homology modeling.

The multidrug resistance P-glycoprotein mediates the extrusion of chemotherapeutic drugs from cancer cells. Characterization of the drug binding and ATPase activities of the protein have made it the paradigm ATP binding cassette (ABC) transporter. P-glycoprotein has been imaged at low resolution by electron cryo-microscopy and extensively analyzed by disulphide cross-linking, but a high resolution structure solved ab initio remains elusive. Homology models of P-glycoprotein were generated using the structure of a related prokaryotic ABC transporter, the lipid A transporter MsbA, as a template together with structural data describing the dimer interface of the nucleotide binding domains (NBDs). The first model, which maintained the NBD:transmembrane domain (TMD) interface of MsbA, did not satisfy previously published cross-linking data. This suggests that either P-glycoprotein has a very different structure from MsbA or that the published E. coli MsbA structure does not reflect a physiological state. To distinguish these alternatives, we mapped the interface between the two TMDs of P-glycoprotein experimentally by chemical cross-linking of introduced triple-cysteine residues. Based on these data, a plausible atomic model of P-glycoprotein could be generated using the MsbA template, if the TMDs of MsbA are reoriented with respect to the NBDs. This model will be important for understanding the mechanism of P-glycoprotein and other ABC transporters.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Homology modeling of an immunoglobulin-like domain in the Saccharomyces cerevisiae adhesion protein alpha-agglutinin.

The Saccharomyces cerevisiae adhesion protein alpha-agglutinin is expressed by cells of alpha mating type. On the basis of sequence similarities, alpha-agglutinin has been proposed to contain variable-type immunoglobulin-like (IgV) domains. The low level of sequence similarity to IgV domains of known structure made homology modeling using standard sequence-based alignment algorithms impossible. We have therefore developed a secondary structure-based method that allowed homology modeling of alpha-aggulutinin domain III, the domain most similar to IgV domains. The model was assessed and where necessary refined to accommodate information obtained by biochemical and molecular genetic approaches, including the positions of a disulfide bond, glycosylation sites, and proteolytic sites. The model successfully predicted surface exposure of glycosylation and proteolytic sites, as well as identifying residues essential for binding activity. One side of the domain was predicted to be covered by carbohydrate residues. Surface accessibility and volume packing analyses showed that the regions of the model that have greatest sequence dissimilarity from the IgV consensus sequence are poorly structured in the biophysical sense. Nonetheless, the utility of the model suggests that these alignment and testing techniques should be of general use for building and testing of models of proteins that share limited sequence similarity with known structures.

Agglutinins↗
PubMed2003

Homology model of the CB1 cannabinoid receptor: sites critical for nonclassical cannabinoid agonist interaction.

Association of cannabimimetic compounds such as cannabinoids, aminoalkylindoles (AAIs), and arachidonylethanolamide (anandamide) with the brain cannabinoid (CB(1)) receptor activates G-proteins and relays signals to regulate neuronal functions. A CB(1) receptor homology model was constructed using the published x-ray crystal structure of bovine rhodopsin (Palczewski et al., Science, 2000, Vol. 289, pp. 739-745) in the conformation most likely to represent the "high-affinity" state for agonist binding to G-protein coupled receptors (GPCRs). A molecular docking approach that combined Monte Carlo and molecular dynamics simulations was used to identify the putative binding conformations of nonclassical cannabinoid agonists, including AC-bicyclic CP47497 and CP55940, and ACD-tricyclic CP55244. Placement of these ligands was based upon the assumption of a critical hydrogen bond between the A-ring OH and the side chain N of Lys192 in transmembrane helix 3. We evaluated two alternative binding conformations, C3-in and C3-out, denoting the directionality of the ligand C3 side chain within the receptor with respect to the inside or the outside of the cell. Assuming both the C3-in or C3-out conformation, the calculated ligand-receptor binding energy (DeltaE(bind)) was correlated with the experimentally observed binding affinity (K(i)) for a series of nonclassical cannabinoid agonists. The C3-in conformation was marginally better than the alternative C3-out conformation in predicting the rank order of the tested nonclassical cannabinoid analogs. Adopting the C3-in conformation due to the greater number of receptor interactions with known pharmacophoric elements of the ligand, key residues were identified comprising the presumed hydrophobic pocket that interacts with the C3 side chain of cannabinoid agonists. Key hydrogen bonds would form between both K3.28(192) and E(258) and the A-ring OH, and between Q(261) and the C-ring C-12 hydroxypropyl. In summary, the present study represents one of the first attempts to construct a homology model of the CB(1) cannabinoid receptor based upon the published bovine rhodopsin x-ray crystal structure and to elucidate the putative ligand binding site for nonclassical cannabinoid agonists. We postulated sites of the CB(1) receptor critical for the ligand interaction, including the hydrophobic pocket interacting with the key pharmacophoric moiety, the C3 side chain. More work is needed to delineate between two alternative (and possibly other) binding conformations of the nonclassical cannabinoid ligands within the CB(1) receptor. The present study provides a consistent framework for further investigation of the CB(1) receptor-ligand interaction and for the study of CB(1) receptor activation.

Amino Acid Sequence↗
PubMed2003

Prediction of 5-HT3 receptor agonist-binding residues using homology modeling.

5-HT(3) receptors demonstrate significant structural and functional homology to other members of the Cys-loop ligand-gated ion channel superfamily. The extracellular domains of these receptors share similar sequence homology (approximately 20%) with Limnaea acetylcholine binding protein, for which an x-ray crystal structure is available. We used this structure as a template for computer-based homology modeling of the 5-HT(3) receptor extracellular domain. AutoDock software was used to dock 5-HT into the putative 5-HT(3) receptor ligand-binding site, resulting in seven alternative energetically favorable models. Residues located no more than 5 A from the docked 5-HT were identified for each model; of these, 12 were found to be common to all seven models with five others present in only certain models. Some docking models reflected the cation-pi interaction previously demonstrated for W183, and data from these and other studies were used to define our preferred models.

Amino Acid Sequence↗
PubMed2004

Homology modelling of CYP3A4 from the CYP2C5 crystallographic template: analysis of typical CYP3A4 substrate interactions.

1. The results of homology modelling of cytochrome P4503A4 (CYP3A4), which is a human enzyme of major importance for the Phase 1 metabolism of drug substrates, from the CYP2C5 crystal structure is reported. 2. The overall homology between the two protein sequences was generally good (46%) with 24% of amino acid residues being identical and a 22% similarity between matched pairs in the CYP3A4 and CYP2C5 aligned sequences, thus indicating that CYP2C5 represents a viable template for modelling CYP3A4 by homology. 3. The CYP3A4 model appears to show consistency with the reported findings from the extensive site-directed mutagenesis studies already published. 4. Typical CYP3A4 substrates, such as midazolam, testosterone, nifedipine and verapamil, are shown to fit the putative active site of the enzyme structure in a manner consistent with their known positions of metabolism.

Amino Acid Sequence↗
PubMed1998

Homology models for the PERB11 multigene family.

BACKGROUND: PERB11 is a multicopy polymorphic gene family found in association with HLA Class I genes within the major histocompatibility complex (MHC). Although its function is unknown, PERB11 has sequence similarities to HLA Class I and other related proteins. To explore the possible functional roles for PERB11, homology models have been constructed using both HLA Class I and Class I-like protein structures as templates. RESULTS: The models show that PERB11.1 appears to have an unusual distribution of charged residues that potentially give the molecule a distinct polarity. Furthermore, a cluster of negatively charged residues in the traditional P2 site may form a novel binding site for a positively charged ligand such as a metal ion or complex. Other charged residues line the floor and walls of the cleft and are able to form salt bridges, reminiscent of the closed cleft of the Class I-like mouse neonatal Fc receptor structure. The closely related PERB11.2 family has a different arrangement of charged residues in the cleft, but these residues are still able to form salt bridges. Unlike HLA Class I, the majority of polymorphic positions in the PERB11 family occur outside the cleft and on the surface of the molecule. CONCLUSIONS: Homology models for PERB11 suggest that the structure is capable of associating with beta2 microglobulin or a similar molecule. Furthermore, not all of the potential glycosylation sites suggested by the PERB11 sequences appear viable. Importantly, the models suggest that the molecule has a less accessible cleft than HLA Class I and is not, therefore, able to bind peptides. Other small ligands, including metal ions, might be bound, however.

Amino Acid Sequence↗
PubMed2005

A two-state homology model of the hERG K+ channel: application to ligand binding.

Homology models based on available K+ channel structures have been used to construct a multiple state representation of the hERG cardiac K+ channel. These states are used to capture the flexibility of the channel. We show that this flexibility is essential in order to correctly model the binding affinity of a set of diverse ligands. Using this multiple state approach, a binding affinity model was constructed for set of known hERG channel binders. The predicted pIC50s are in good agreement with experiment (RMSD: 0.56 kcal/mol). In addition, these calculations provide structures for the bound ligands that are consistent with published mutation studies. These computed ligand bound complex structures can be used to guide synthesis of analogs with reduced hERG liability.

ERG1 Potassium Channel↗
PubMed1999

Modularity and homology: modelling of the type II module family from titin.

We report the homology modelling of the structures of the 162 type II modules from the giant multi-domain protein titin (also known as connectin). The package MODELLER was used and implemented in an automated fashion using four experimentally determined structures as templates. Validation of the models was assessed in terms of divergence from the templates and consensus of the alignments. The homology within the whole family of type II modules as well as with the templates is relatively high (20-35% identity and ca 50% similarity). Comparison between the models of domains for which an NMR structure has been solved and the experimental solution gives an estimate of the quality of the modelling. Our results allow us to distinguish between a set of structurally relevant residues, which are conserved throughout the whole family and buried in the hydrophobic core, from the residues that are conserved and exposed. These latter residues are potentially functionally important. Comparison of exposed conserved patches for modules in different regions of the titin molecule suggests potential interaction surfaces. Our results may be tested directly for those modules whose binding partner is known.

Amino Acid Sequence↗
PubMed2003

Bioinformatics of granzymes: sequence comparison and structural studies on granzyme family by homology modeling.

Cytotoxic lymphocytes (CTLs), the key players of cell mediated immunity, induce apoptosis by engaging death receptors or through exocytosis of cytolytic granules containing granzyme (proteases) and pore-forming protein (perforin). The crystal structure of granzyme B from human (B(h)) and rat (B(r)), as well as that of pro-granzyme K (K(h)) has been reported recently. In the present communication, we describe the homology modeling of granzyme family (in particular Gzm A(h), M(h), B(m), and C(m) from human and mouse) based on the crystal structural coordinates of trypsin, granzyme K (K(h)), and granzyme B (B(h)). These models have been used for establishing phylogenetic relationship as well as identifying characteristic features for designing specific inhibitors. The paper also highlights key residues at the S1, S2, and S2(') binding subsites in all granzyme, which may be involved in the structure-function relationship of this enzyme family. The predicted 3D homology models show a conserved two similar domain structure, i.e., an N-terminal domain and a C-terminal domain comprising predominantly of beta-sheet structure with a little alpha-helical content. Micro-heterogeneities have been observed in the vicinity of the active site in all granzymes as compared to granzyme B(h). For example, in granzyme M(h), valine is present at the S1 subsite instead of arginine. Similarly differences at S2 (Leu-->Phe), S3 (Ser-->Gly), and S4 (Arg-->Asn) subsites are quite apparent and appear to hold the potential for selective designing of inhibitors for possible therapeutic applications. Furthermore, analysis of the electrostatic surface potential on the shape of granzyme-inhibitor binding groove reveals clear differences at the reactive site. Additionally the different posttranslational modification sites such as phosphorylation (e.g., in granzyme M Thr101, Ser109), myristoylation (Gly22, 117, and 131), and glycosylation (Ser160) have been identified, as very little is known about the functional significance of these modifications in the granzyme family. Thus, glycosylation at Ser160 in granzyme M may influence the net charge of the enzyme, resulting in altered substrate binding as compared to granzyme B. Also this modification may influence the rate of complexation and binding affinity with proteoglycans. These studies are expected to contribute towards the basic understanding of functional associations of the granzymes with other molecules and their possible role in apoptosis.

Amino Acid Sequence↗
PubMed2003

Homology modelling of the nuclear receptors: human oestrogen receptorbeta (hERbeta), the human pregnane-X-receptor (PXR), the Ah receptor (AhR) and the constitutive androstane receptor (CAR) ligand binding domains from the human oestrogen receptor alpha (hERalpha) crystal structure, and the human peroxisome proliferator activated receptor alpha (PPARalpha) ligand binding domain from the human PPARgamma crystal structure.

We have generated by homology the three-dimensional structures of the ligand binding domain (LBD) of several interrelated human steroid hormone receptors (SHRs). These are the oestrogen receptor beta (hERbeta), the pregnane-X-receptor (PXR), the Ah receptor (AhR) and the constitutive androstane receptor (CAR). They were produced by homology modelling from the human oestrogen receptor alpha (hERalpha) crystallographic coordinates [Nature 389 (1997) 753] as a template together with the amino acid sequences for hERbeta [FEBS Lett. 392 (1996) 49], PXR [J. Clin. Invest. 102 (1998) 1016], AhR [Proc. Natl. Acad. Sci. U.S.A. 89 (1992) 815] and CAR [Nature 395 (1998) 612; Mol. Cell. Biol. 14 (1994) 1544], respectively. The selective endogenous ligand, in each case, was docked interactively within the putative ligand binding site using the position of oestradiol in hERalpha as a guide, and the total energy was calculated. In each receptor model a number of different ligands known to fit closely within the ligand binding site were interactively docked and binding interactions noted. Specific binding interactions included combinations of hydrogen bonding and hydrophobic contacts with key amino acid sidechains, which varied depending on the nature of the ligand and receptor concerned. We also produced the human peroxisome proliferator activated receptor alpha (PPARalpha) by homology modelling using the human PPARgamma (hPPARgamma) LBD crystallographic coordinates summarised in [Toxicol. In Vitro 12 (1998) 619] as a template together with the amino acid sequence for hPPARalpha [Toxicol. In Vitro 12 (1998) 619; Nature 395 (1998) 137]. The models will provide a useful tool in unravelling the complexity in the physiologic response to xenobiotics by examining the ligand binding interactions and differences between the steroid hormone receptors activation or inactivation by their ligands.

Amino Acid Sequence↗
PubMed2000

Linking CoMFA and protein homology models of enzyme-inhibitor interactions: an application to non-steroidal aromatase inhibitors.

An approach to compare quantitatively a ligand-based (CoMFA) model and an enzyme active site model was investigated. The active site of the cytochrome P450 human aromatase was constructed by homology modeling techniques and two structurally different non-steroidal aromatase inhibitors were docked into it. A CoMFA model was then developed on a related series of non-steroidal inhibitors by correlating their inhibitory activity (expressed as -log IC50 values) versus only 11 steric descriptors (i.e. Csp3-ligand steric interaction energies). The resulting 3D-QSAR coefficients (11) and the steric field values of the aromatase active site calculated at the same points of the CoMFA lattice (i.e. eleven Csp3-protein steric interaction energies) were pair-wise compared. Specifically, when a positive coefficient was associated with a negative or low ( < 5 kcal/mol) value of the protein steric field or, alternatively, a negative coefficient was associated with a large positive value of the protein steric field we recorded as many matches. When a 3D-QSAR coefficient did not correspond to the protein steric potential in the sense described above we considered that point as a mis-matching point. In our view, in spite of several limitations, such a comparison represents a valuable criterion to evaluate quantitatively how convergent are the results from a 3D-QSAR CoMFA model and a homology-built protein 3D structure.

Amino Acid Sequence↗
PubMed2005

Homology modeling revealed more than 20,000 rRNA internal transcribed spacer 2 (ITS2) secondary structures.

Structural genomics meets phylogenetics and vice versa: Knowing rRNA secondary structures is a prerequisite for constructing rRNA alignments for inferring phylogenies, and inferring phylogenies is a precondition to understand the evolution of such rRNA secondary structures. Here, both scientific worlds go together. The rRNA internal transcribed spacer 2 (ITS2) region is a widely used phylogenetic marker. Because of its high variability at the sequence level, correct alignments have to take into account structural information. In this study, we examine the extent of the conservation in structure. We present (1) the homology modeled secondary structure of more than 20,000 ITS2 covering about 14,000 species; (2) a computational approach for homology modeling of rRNA structures, which additionally can be applied to other RNA families; and (3) a database providing about 25,000 ITS2 sequences with their associated secondary structures, a refined ITS2 specific general time reversible (GTR) substitution model, and a scoring matrix, available at http://its2.bioapps.biozentrum.uni-wuerzburg.de.

Animals↗
PubMed2006

A three-dimensional homology model of the O-acetylserine sulfhydrylase-B from Salmonella typhimurium.

O-acetylserine sulfhydrylase (OASS) catalyzes the last step in the cysteine biosynthetic pathway in enteric bacteria and plants. The overall pathway involves the substitution of the beta-acetoxy group of O-acetyl-L-serine with inorganic bisulfide. Two isozymes are present in S. typhimurium, the A- and B-isozymes, expressed under aerobic and anaerobic conditions, respectively. No crystal structure is presently available for the B-isozyme. Kinetic data indicate the catalytic mechanism of OASS-B is ping-pong, as found for the A-isozyme, but kinetic parameters and substrate specificity differ. In order to estimate whether structural differences may be responsible for the kinetic differences, a homology model was built using the structure of OASS-A as the template for the OASS-B model. The beta-subunit of tryptophan synthase and cystathionine beta-synthase were used for comparison. Differences between the OASS-A structure and the homology model for OASS-B are discussed.

Amino Acid Sequence↗
PubMed2004

Automated protein structure homology modeling: a progress report.

Understanding the molecular function of proteins is greatly enhanced by insights gained from their three-dimensional structures. Since experimental structures are only available for a small fraction of proteins, computational methods for protein structure modeling play an increasingly important role. Comparative protein structure modeling is currently the most accurate method, yielding models suitable for a wide spectrum of applications, such as structure-guided drug development or virtual screening. Stable and reliable automated prediction pipelines have been developed to apply large-scale comparative modeling to whole genomes or entire sequence databases. Model repositories give access to these annotated and evaluated models. In this review, we will discuss recent developments in automated comparative modeling and provide selected examples illustrating the use of homology models.

Animals↗