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PubMed2005

Homology modeling and SAR analysis of Schistosoma japonicum cathepsin D (SjCD) with statin inhibitors identify a unique active site steric barrier with potential for the design of specific inhibitors.

Proteases that digest the blood-meal of the parasitic fluke Schistosoma are potential targets for therapy of schistosomiasis, a disease of chronic morbidity in humans. We generated a three-dimensional model of the cathepsin D target protease of Schistosoma japonicum (SjCD) utilizing the crystal structure of human cathepsin D (huCD) in complex with pepstatin as template. A homology model was also generated for the related secreted aspartic protease 2 (SAP2) of the pathogenic yeast, Candida albicans . An initial panel of seven statin inhibitors, originally designed for huCD [Majer et al., Protein Sci. 6 (1997), pp. 1458-1466], was tested against the two pathogen proteases. One inhibitor showed poor reactivity with SjCD. Examination of the SjCD active-site cleft revealed that the poor inhibition was due to a unique steric barrier situated between the S2 and S4 subsites. An in silico screen of 20 potential statin scaffolds with the SjCD model and incorporating the steric barrier constraint was performed. Four inhibitors (SJ1-SJ4) were eventually synthesized and tested with SjCD, bovine CD and SAP2. Of these, SJ2 and SJ3 proved moderately more specific for SjCD over bovine CD, with IC 50 values of 15 and 60 nM, respectively. The unique steric barrier identified here provides a structural focus for further development of more specific SjCD inhibitors.

Amino Acid Sequence↗

PubMed2006

Defining the nucleotide binding sites of P2Y receptors using rhodopsin-based homology modeling.

Ongoing efforts to model P2Y receptors for extracellular nucleotides, i.e., endogenous ADP, ATP, UDP, UTP, and UDP-glucose, were summarized and correlated for the eight known subtypes. The rhodopsin-based homology modeling of the P2Y receptors is supported by a growing body of site-directed mutagenesis data, mainly for P2Y(1) receptors. By comparing molecular models of the P2Y receptors, it was concluded that nucleotide binding could occur in the upper part of the helical bundle, with the ribose moiety accommodated between transmembrane domain (TM) 3 and TM7. The nucleobase was oriented towards TM1, TM2, and TM7, in the direction of the extracellular side of the receptor. The phosphate chain was oriented towards TM6, in the direction of the extracellular loops (ELs), and was coordinated by three critical cationic residues. In particular, in the P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptors the nucleotide ligands had very similar positions. ADP in the P2Y(12) receptor was located deeper inside the receptor in comparison to other subtypes, and the uridine moiety of UDP-glucose in the P2Y(14) receptor was located even deeper and shifted toward TM7. In general, these findings are in agreement with the proposed binding site of small molecules to other class A GPCRs.

Amino Acid Sequence↗

PubMed2006

Physically realistic homology models built with ROSETTA can be more accurate than their templates.

We have developed a method that combines the ROSETTA de novo protein folding and refinement protocol with distance constraints derived from homologous structures to build homology models that are frequently more accurate than their templates. We test this method by building complete-chain models for a benchmark set of 22 proteins, each with 1 or 2 candidate templates, for a total of 39 test cases. We use structure-based and sequence-based alignments for each of the test cases. All atoms, including hydrogens, are represented explicitly. The resulting models contain approximately the same number of atomic overlaps as experimentally determined crystal structures and maintain good stereochemistry. The most accurate models can be identified by their energies, and in 22 of 39 cases a model that is more accurate than the template over aligned regions is one of the 10 lowest-energy models.

Models, Theoretical↗

PubMed2001

Homology modeling of wild type and pyrimethamine/cycloguanil-cross resistant mutant type Plasmodium falciparum dihydrofolate reductase. A model for antimalarial chemotherapy resistance.

We propose a low-resolution model for both the wild type and the pyrimethamine (Pyr)/cycloguanil (Cyc) cross-resistant mutant type Plasmodium falciparum DHFR (PfDHFR), based on homology modeling using chicken liver DHFR as a template. The built models contain five alpha-helices, eight beta-sheets, eight tight turns and several loops. The Ramachandran plot for the models shows 95.3 and 100% of the amino acid residues in the favorable regions for the whole enzymes and for the active sites, respectively. Furthermore, we made a preliminary analysis of the complexes Pyr/Cyc-wild DHFR and Pyr/Cyc-mutant DHFR in order to explain the probable mechanism of resistance. Our results show that the steric factor may be the main structural cause of P. falciparum resistance toward antifolate drugs.

Amino Acid Sequence↗

PubMed2006

Towards structural biology in the genus Mytilus: on-line homology modeling with Geno3D.

Seven structures from the genus Mytilus exist in the protein data bank (PDB) but > 1000 Mytilus protein sequences are known. Sequences (NCBI) were used as targets for on-line homology modeling with Geno3D as an alternative route to structure. Quality comparators include root mean square deviation (RMSD) of target from template, chain geometry and Ramachandran diagram analysis. We modeled 17 Mytilus structures for mainly stress-response proteins relevant to biomonitoring. These model files are available as a first step towards a structural resource for Mytilus. Analysis suggests that they are structurally plausible with low RMSDs relative to targets. Ramachandran analysis suggests a small percentage of disallowed dihedral angles (in the range 0-6.86% for 15 of 17 models).

Animals↗

PubMed1999

Homology model of the dengue 2 virus NS3 protease: putative interactions with both substrate and NS2B cofactor.

The crystal structure coordinates of the hepatitis C virus NS3 protease (HCVpro) were used to develop an homology model of the dengue 2 virus NS3 protease (DEN2pro). The amino acid sequence of DEN2pro accommodates the same alpha-helices, beta-sheets and protein-binding domains as its HCVpro counterpart, but the model predicts a number of significant differences for DEN2pro and its interactions with substrates and cofactor. Whereas HCVpro contains a Zn2+-binding site, there is no equivalent metal-binding motif in DEN2pro. It is possible that the structural role played by the zinc ion may be provided by a salt bridge between Glu93 and Lys145. The two-component viral protease comprises NS3 and a virus-encoded cofactor, NS4A for HCV and NS2B for DEN2. Previous studies have identified a central 40 amino acid cofactor domain of the dengue virus NS2B that is required for protease activity. Modelling of the putative interactions between DEN2pro and its cofactor suggests that a 12 amino acid hydrophobic region within this sequence (70GSSPILSITISE81) may associate directly with NS3. Modelling also suggests that the substrate binds in an extended conformation to the solvent-exposed surface of the protease, with a P1-binding site that is significantly different from its HCV counterpart. The model described in this study not only reveals unique features of the flavivirus protease but also provides a structural basis for both cofactor and substrate binding that should prove useful in the early design and development of inhibitors.

Amino Acid Sequence↗

PubMed1999

Classification of protein sequences by homology modeling and quantitative analysis of electrostatic similarity.

Protein electrostatics plays a key role in ligand binding and protein-protein interactions. Therefore, similarities or dissimilarities in electrostatic potentials can be used as indicators of similarities or dissimilarities in protein function. We here describe a method to compare the electrostatic properties within protein families objectively and quantitatively. Three-dimensional structures are built from database sequences by comparative modeling. Molecular potentials are then computed for these with a continuum solvation model by finite difference solution of the Poisson-Boltzmann equation or analytically as a multipole expansion that permits rapid comparison of very large datasets. This approach is applied to 104 members of the Pleckstrin homology (PH) domain family. The deviation of the potentials of the homology models from those of the corresponding experimental structures is comparable to the variation of the potential in an ensemble of structures from nuclear magnetic resonance data or between snapshots from a molecular dynamics simulation. For this dataset, the results for analysis of the full electrostatic potential and the analysis using only monopole and dipole terms are very similar. The electrostatic properties of the PH domains are generally conserved despite the extreme sequence divergence in this family. Notable exceptions from this conservation are seen for PH domains linked to a Db1 homology (DH) domain and in proteins with internal PH domain repeats.

Data Interpretation, Statistical↗

PubMed2005

Homology modelling and protein structure based functional analysis of five cucumovirus coat proteins.

Coat proteins (CP) of five cucumovirus isolates, Cucumber mosaic virus (CMV) strains R, M and Trk7, Tomato aspermy virus (TAV) strain P and Peanut stunt virus (PSV) strain Er, were constructed by homology modelling. The X-ray structure of the Fny-CMV CP subunit B was used as a template. Models of cucumovirus CPs were built by the MODELLER program. Model refinements were carried out using the Kollman molecular mechanical force field. Models were analyzed by the PROCHECK programs. Electrostatic potential calculations were applied to all models and functional site search was performed with the PROSITE software, a web based tool for searching biologically significant sites. Symptom determinants published up to the present were compared with the PROSITE hits in the light of 3D models and electrostatic information. In all cases, we analyzed the effect of mutations on the structure, electrostatic potential patterns and function of CPs, respectively. We found that high flexibility of the betaE-alphaEF loop starting with the residue 129 is required, but it is not sufficient for the symptom appearance. Furthermore, phosphorylation of the CP is prospective to be important in the host response mechanism. All analyzed mutations were related to the modifications of the predicted phosphorylation sites. Based on our conclusions we predicted the infectivity of the examined viruses.

Amino Acid Sequence↗

PubMed2006

Homology modeling and molecular docking study of translationally controlled tumor protein and artemisinin.

Translationally controlled tumor protein (TCTP), also known as histamine releasing factor (HRF), is found abundantly in different eukaryotic cell types. The sequence homology of TCTP between different species is very high, belonging to the MSS4/DSS4 superfamily of proteins. TCTP is involved in both cell growth and human late allergy reaction, as well as having a calcium binding property; however, its primary biological functions remain to be clearly elucidated. In regard to many possible functions, the TCTP of Plasmodium falciparum (Pf) is known to bind with an antimalarial agent, artemisinin, which is activated by heme. It is assumed that the endoperoxide-bridge of artemisinin is opened up by heme to form a free radical, which then eventually alkylates, probably to the Cys14 of PfTCTP. Study of the docking of artemisinin with heme, and subsequently with PfTCTP, was carried out to verify the above hypothesis on the basis of structural interactions. The three dimensional (3D) structure of PfTCTP was built by homology modeling, using the NMR structure of the TCTP of Schizosaccharomyces pombe as a template. The quality of the model was examined based on its secondary structure and biological function, as well as with the use of structure evaluating programs. The interactions between artemisinin, heme and PfTCTP were then studied using the docking program, FlexiDock. The center of the peroxide bond of artemisinin and the Fe of heme were docked within a short distance of 2.6A, implying the strong possibility of an interaction between the two molecules, as proposed. When the activated form of artemisinin was docked on the PfTCTP, the C4-radical of the drug faced towards the sulfur of Cys14 within a distance of 2.48A, again suggesting the possibility of alkylation having occurred. These results confirm the proposed mechanism of the antimalarial effect of artemisinin, which will provide a reliable method for establishing the mechanism of its biological activity using a molecular modeling study.

Antimalarials↗

PubMed1999

Cysteine mutagenesis and homology modeling of the ligand-binding site of a kainate-binding protein.

Glutamate receptors comprise the most abundant group of neurotransmitter receptors in the vertebrate central nervous system. Cysteine mutagenesis in combination with homology modeling has been used to study the determinants of kainate binding in a glutamate receptor subtype, a low molecular weight goldfish kainate-binding protein, GFKARbeta. A construct of GFKARbeta with no cysteines in the extracellular domain was produced, and single cysteine residues were introduced at selected positions. N-Ethylmaleimide or derivatized methanethiosulfonate reagents (neutral or charged) were used to modify the introduced cysteines covalently, and the effect on [(3)H]kainate binding was determined. In addition, cysteine mutants of GFKARbeta transiently expressed in HEK293 cells were labeled with a membrane-impermeable biotinylating reagent followed by precipitation with streptavidin beads and specific detection of GFKARbeta by Western blot analysis. The results are consistent with the proposal that the energy driving kainate binding is contributed both from residues within the binding site and from interactions between two regions (i.e. two lobes) of the protein that are brought into contact upon ligand binding in a manner analogous to that seen in bacterial amino acid-binding proteins.

Binding Sites↗

PubMed1998

Selecting near-native conformations in homology modeling: the role of molecular mechanics and solvation terms.

A free energy function, combining molecular mechanics energy with empirical solvation and entropic terms, is used for ranking near-native conformations that occur in the conformational search steps of homology modeling, i.e., side-chain search and loop closure calculations. Correlations between the free energy and RMS deviation from the X-ray structure are established. It is shown that generally both molecular mechanics and solvation/entropic terms should be included in the potential. The identification of near-native backbone conformations is accomplished primarily by the molecular mechanics term that becomes the dominant contribution to the free energy if the backbone is even slightly strained, as frequently occurs in loop closure calculations. Both terms become equally important if a sufficiently accurate backbone conformation is found. Finally, the selection of the best side-chain positions for a fixed backbone is almost completely governed by the solvation term. The discriminatory power of the combined potential is demonstrated by evaluating the free energies of protein models submitted to the first meeting on Critical Assessment of techniques for protein Structure Prediction (CASP1), and comparing them to the free energies of the native conformations.

Bacterial Proteins↗

PubMed2006

Analysis and prediction of helix shift errors in homology modeling.

High sequence identity between two proteins (e.g. > 60%) is a strong evidence for high structural similarity. However, internal shifts in one of the two proteins can sometimes give rise to unexpectedly high structural differences. This, in turn, causes unreliable structure predictions when two such proteins are used in homology modeling. Here, we perform a computational analysis of helix shifts and we show that their occurrence can be predicted with statistical learning methods. Our results indicate that helix shifts increase the RMS error by factor 2.6 compared to those protein pairs without a helix shift. Although helix shifts are rare (1.6% of helices and a commensurately higher number of proteins are affected), they therefore pose a significant problem for reliable structure prediction systems. In this paper, we prototype a new approach for model quality assessment and demonstrate that it can successfully warn against helix shifts. A support vector machine trained on a wide range of sequence and structure properties predicts the occurrence of helix shifts with a sensitivity of 74.2% and a specificity of 83.6%. On an equalized test dataset, this corresponds to an accuracy of 78.9%. Projected to the full dataset, it translates to an accuracy of 83.4%. Our analysis shows that helix shift detection is a valuable building block for highly reliable structure prediction systems. Furthermore, the statistical learning based approach to helix shift detection that we employ here is orthogonal to well-established model quality assessment methods (which use geometric constraint checking or mean force potentials). Therefore, a further increase of prediction accuracy is expected from the combination of these methods.

Computational Biology↗

PubMed2004

Homology modeling and molecular dynamics studies of a novel C3-like ADP-ribosyltransferase.

The novel C3-like ADP-ribosyltransferase is produced by a Staphylococcus aureus strain that especially ADP-ribosylates RhoE/Rnd3 subtype proteins, and its three-dimensional (3D) structure has not known. In order to understand the catalytic mechanism, the 3D structure of the protein is built by using homology modeling based on the known crystal structure of exoenzyme C3 from Clostridium botulinum (1G24). Then the model structure is further refined by energy minimization and molecular dynamics methods. The putative nicotinamide adenine dinucleotide (NAD(+))-binding pocket of exoenzyme C3(Stau) is determined by Binding-Site Search module. The NAD(+)-enzyme complex is developed by molecular dynamics simulation and the key residues involved in the combination of enzyme binding to the ligand-NAD(+) are determined, which is helpful to guide the experimental realization and the new mutant designs as well. Our results indicated that the key binding-site residues of Arg48, Glu180, Ser138, Asn134, Arg85, and Gln179 play an important role in the catalysis of exoenzyme C3(Stau), which is in consistent with experimental observation.

ADP Ribose Transferases↗

PubMed2005

Toward a blueprint for UDP-glucose pyrophosphorylase structure/function properties: homology-modeling analyses.

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme of synthesis of sucrose, cellulose, and several other polysaccharides in all plants. The protein is evolutionarily conserved among eukaryotes, but has little relation, aside from its catalytic reaction, to UGPases of prokaryotic origin. Using protein homology modeling strategy, 3D structures for barley, poplar, and Arabidopsis UGPases have been derived, based on recently published crystal structure of human UDP-N-acetylglucosamine pyrophosphorylase. The derived 3D structures correspond to a bowl-shaped protein with the active site at a central groove, and a C-terminal domain that includes a loop (I-loop) possibly involved in dimerization. Data on a plethora of earlier described UGPase mutants from a variety of eukaryotic organisms have been revisited, and we have, in most cases, verified the role of each mutation in enzyme catalysis/regulation/structural integrity. We have also found that one of two alternatively spliced forms of poplar UGPase has a very short I-loop, suggesting differences in oligomerization ability of the two isozymes. The derivation of the structural model for plant UGPase should serve as a useful blueprint for further function/structure studies on this protein.

Alternative Splicing↗

PubMed2006

Novel human lipoxygenase inhibitors discovered using virtual screening with homology models.

We report the discovery of new, low micromolar, small molecule inhibitors of human platelet-type 12- and reticulocyte 15-lipoxygenase-1 (12-hLO and 15-hLO) using structure-based methods. Specifically, we created homology models of 12-hLO and 15-hLO, based on the structure of rabbit 15-lipoxygenase, for in silico screening of a large compound library followed by in vitro screening of 20 top scoring molecules. Eight of these compounds inhibited either 12- or 15-human lipoxygenase with lower than 100 microM affinity. Of these, we obtained IC50 values for the three best inhibitors, all of which displayed low micromolar inhibition. One compound showed specificity for 15-hLO versus 12-hLO; however, a selective inhibitor for 12-hLO was not identified. As a control we screened 20 randomly selected compounds, of which none showed low micromolar inhibition. The new low-micromolar inhibitors appear to be suitable as leads for further inhibitor development efforts against 12-hLO and 15-hLO, based on the fact their size and chemical properties are appropriate to classify them as drug-like compounds. The models of these protein-inhibitor complexes suggest strategies for future development of selective lipoxygenase inhibitors.

Animals↗

PubMed2001

Homology Modeling of Bacillus subtilis Tryptophanyl-tRNA Synthetase.

Tryptophanyl-tRNA synthetase (TrpRS) plays a pivotal role in protein synthesis. However, till now no stereostructural data of Bacillus subtilis TrpRS were reported. Here, by using the homology modeling using Bacillus stearothermophilus TrpRS as a template, it is demonstrated that the synthetase has 16alpha helices and 5beta sheets. The only tryptophan Trp(92) is located on the interface of subunits. Also the modeling presents the ligand binding site, active site and the putative binding of tRNA(Trp).

Journal Article↗

PubMed2003

Loops In Proteins (LIP)--a comprehensive loop database for homology modelling.

One of the most important and challenging tasks in protein modelling is the prediction of loops, as can be seen in the large variety of existing approaches. Loops In Proteins (LIP) is a database that includes all protein segments of a length up to 15 residues contained in the Protein Data Bank (PDB). In this study, the applicability of LIP to loop prediction in the framework of homology modelling is investigated. Searching the database for loop candidates takes less than 1 s on a desktop PC, and ranking them takes a few minutes. This is an order of magnitude faster than most existing procedures. The measure of accuracy is the root mean square deviation (RMSD) with respect to the main-chain atoms after local superposition of target loop and predicted loop. Loops of up to nine residues length were modelled with a local RMSD <1 A and those of length up to 14 residues with an accuracy better than 2 A. The results were compared in detail with a thoroughly evaluated and tested ab initio method published recently and additionally with two further methods for a small loop test set. The LIP method produced very good predictions. In particular for longer loops it outperformed other methods.

Data Interpretation, Statistical↗

PubMed2004

Homology modeling, force field design, and free energy simulation studies to optimize the activities of histone deacetylase inhibitors.

As an effort to develop therapeutics for cancer treatments, a number of effective histone deacetylase inhibitors with structural diversity have been discovered. To gain insight into optimizing the activity of an identified lead compound, a computational protocol sequentially involving homology modeling, docking experiments, molecular dynamics simulation, and free energy perturbation calculations was applied for rationalizing the relative activities of known histone deacetylase inhibitors. With the newly developed force field parameters for the coordination environment of the catalytic zinc ion in hand, the computational strategy proved to be successful in predicting the rank orders for 12 derivatives of three hydroxamate-based inhibitor scaffolds with indole amide, pyrrole, and sulfonamide moieties. The results showed that the free energy of an inhibitor in aqueous solution should be an important factor in determining the binding free energy. Hence, in order to enhance the inhibitory activity by adding or substituting a chemical group, the increased stabilization in solution due to the structural changes must be overcome by a stronger enzyme-inhibitor interaction. It was also found that to optimize inhibitor potency, the hydrophobic head of an inhibitor should be elongated or enlarged so that it can interact with Pro29 and His28 that are components of the flexible loop at the top of the active site.

Amino Acid Sequence↗