Biomedical subjects

R C Wade

Publications and source records attributed to R C Wade.

At least 19 recordsLinked to original sources

PubMed2006

ProSAT2--Protein Structure Annotation Server.

ProSAT2 is a server to facilitate interactive visualization of sequence-based, residue-specific annotations mapped onto 3D protein structures. As the successor of ProSAT (Protein Structure Annotation Tool), it includes its features for visualizing SwissProt and PROSITE functional annotations. Currently, the ProSAT2 server can perform automated mapping of information on variants and mutations from the UniProt KnowledgeBase and the BRENDA enzyme information system onto protein structures. It also accepts and maps user-prepared annotations. By means of an annotation selector, the user can interactively select and group residue-based information according to criteria such as whether a mutation affects enzyme activity. The visualization of the protein structures is based on the WebMol Java molecular viewer and permits simultaneous highlighting of annotated residues and viewing of the corresponding descriptive texts. ProSAT2 is available at http://projects.villa-bosch.de/mcm/database/prosat2/.

Amino Acids↗

PubMed2005

Issues in high-throughput comparative modelling: a case study using the ubiquitin E2 conjugating enzymes.

Sequences of the ubiquitin-conjugating enzyme (UBC or E2) family were used as a test set to investigate issues associated with the high-throughput comparative modelling of protein structures. A semi-automatic method was initially developed with particular emphasis on producing models of a quality suitable for structural comparison. Structural and sequence features of the E2 family were used to improve the sequence alignment and the quality of the structural templates. Initially, failure to correct for subtle structural inconsistencies between templates lead to problems in the comparative analysis of the UBC electrostatic potentials. Modelling of known UBC structures using Modeller 4.0 showed that multiple templates produced, on average, no better models than the use of just one template, as judged by the root-mean-squared deviation between the comparative model and crystal structure backbones. Using four different quality-checking methods, for a given target sequence, it was not possible to distinguish the model most similar to the experimental structure. The UBC models were thus finally modelled using only the crystal structure template with the highest sequence identity to the target to be modelled, and producing only one model solution. Quality checking was used to reject models with obvious structural anomalies (e.g., bad side-chain packing). The resulting models have been used for a comparison of UBC structural features and of their electrostatic potentials. The work was extended through the development of a fully automated pipeline that identifies E2 sequences in the sequence databases, aligns and models them, and calculates the associated electrostatic potential.

Amino Acid Motifs↗

PubMed2004

Computational approaches to structural and functional analysis of plastocyanin and other blue copper proteins.

Computational techniques are becoming increasingly important in structural and functional biology, in particular as tools to aid the interpretation of experimental results and the design of new systems. This review reports on recent studies employing a variety of computational approaches to unravel the microscopic details of the structure-function relationships in plastocyanin and other proteins belonging to the blue copper superfamily. Aspects covered include protein recognition, electron transfer and protein-solvent interaction properties of the blue copper protein family. The relevance of integrating diverse computational approaches to address the analysis of a complex protein system, such as a cupredoxin metalloprotein, is emphasized.

Algorithms↗

PubMed2001

Comparative binding energy analysis of the substrate specificity of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10.

Comparative binding energy (COMBINE) analysis was conducted for 18 substrates of the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA): 1-chlorobutane, 1-chlorohexane, dichloromethane, 1,2-dichloroethane, 1,2-dichloropropane, 2-chloroethanol, epichlorohydrine, 2-chloroacetonitrile, 2-chloroacetamide, and their brominated analogues. The purpose of the COMBINE analysis was to identify the amino acid residues determining the substrate specificity of the haloalkane dehalogenase. This knowledge is essential for the tailoring of this enzyme for biotechnological applications. Complexes of the enzyme with these substrates were modeled and then refined by molecular mechanics energy minimization. The intermolecular enzyme-substrate energy was decomposed into residue-wise van der Waals and electrostatic contributions and complemented by surface area dependent and electrostatic desolvation terms. Partial least-squares projection to latent structures analysis was then used to establish relationships between the energy contributions and the experimental apparent dissociation constants. A model containing van der Waals and electrostatic intermolecular interaction energy contributions calculated using the AMBER force field explained 91% (73% cross-validated) of the quantitative variance in the apparent dissociation constants. A model based on van der Waals intermolecular contributions from AMBER and electrostatic interactions derived from the Poisson-Boltzmann equation explained 93% (74% cross-validated) of the quantitative variance. COMBINE models predicted correctly the change in apparent dissociation constants upon single-point mutation of DhlA for six enzyme-substrate complexes. The amino acid residues contributing most significantly to the substrate specificity of DhlA were identified; they include Asp124, Trp125, Phe164, Phe172, Trp175, Phe222, Pro223, and Leu263. These residues are suitable targets for modification by site-directed mutagenesis.

Binding Sites↗

PubMed2001

Comparative binding energy (COMBINE) analysis of influenza neuraminidase-inhibitor complexes.

Neuraminidase is a surface glycoprotein of influenza viruses that cleaves terminal sialic acids from carbohydrates. It is critical for viral release from infected cells and facilitates viral spread in the respiratory tract. The catalytic active site of neuraminidase is highly conserved in all type A and B influenza viruses, making it an excellent target for antiinfluenza drug design. Indeed, neuraminidase inhibitors have recently become available in the clinic for the treatment of influenza. Here, we describe the use of 3D structures of neuraminidase-inhibitor complexes to derive quantitative structure-activity relationships (QSARs) to aid understanding of the mechanism of inhibition and the discovery of new inhibitors. Crystal structures of neuraminidase-inhibitor complexes were used alongside modeled complexes to derive QSAR models by COMparative BINding Energy (COMBINE) analysis (Ortiz, A. R.; Pisabarro, M. T.; Gago, F.; Wade, R. C. J. Med. Chem. 1995, 38, 2681-2691). The neuraminidase proteins studied include type A subtypes N2 and N9 (which have ca. 50% sequence identity) and an active site mutant of the N9 subtype. The inhibitors include sialic acid and benzoic acid analogues with diverse frameworks and substitution groups. By considering the contributions of the protein residues and a key water molecule to the electrostatic and van der Waals intermolecular interaction energies, a predictive and robust QSAR model for binding to type A neuraminidase was obtained. In this QSAR model, 12 protein residues and 1 bound water molecule are highlighted as particularly important for inhibitory activity. This QSAR model provides guidelines for structural modification of current inhibitors and the design of novel inhibitors in order to optimize inhibitory activity.

Algorithms↗

PubMed2001

Protein-protein association: investigation of factors influencing association rates by brownian dynamics simulations.

The rate of protein-protein association limits the response time due to protein-protein interactions. The bimolecular association rate may be diffusion-controlled or influenced, and in such cases, Brownian dynamics simulations of protein-protein diffusional association may be used to compute association rates. Here, we report Brownian dynamics simulations of the diffusional association of five different protein-protein pairs: barnase and barstar, acetylcholinesterase and fasciculin-2, cytochrome c peroxidase and cytochrome c, the HyHEL-5 antibody and hen egg lysozyme (HEL), and the HyHEL-10 antibody and HEL. The same protocol was used to compute the diffusional association rates for all the protein pairs in order to assess, by comparison to experimentally measured rates, whether the association of these proteins can be explained solely on the basis of diffusional encounter. The simulation protocol is similar to those previously derived for simulation of the association of barnase and barstar, and of acetylcholinesterase and fasciculin-2; these produced results in excellent agreement with experimental data for these protein pairs, with changes in association rate due to mutations reproduced within the limits of expected computational and modeling errors. Here, we find that for all protein pairs, the effects of mutations can be well reproduced by the simulations, even though the degree of the electrostatic translational and orientational steering varies widely between the cases. However, the absolute values of association rates for the acetylcholinesterase: fasciculin-2 and HyHEL-10 antibody: HEL pairs are overestimated. Comparison of bound and unbound protein structures shows that this may be due to gating resulting from protein flexibility in some of the proteins. This may lower the association rates compared to their bimolecular diffusional encounter rates.

Acetylcholinesterase↗

PubMed2001

Electrostatic analysis and Brownian dynamics simulation of the association of plastocyanin and cytochrome f.

The oxidation of cytochrome f by the soluble cupredoxin plastocyanin is a central reaction in the photosynthetic electron transfer chain of all oxygenic organisms. Here, two different computational approaches are used to gain new insights into the role of molecular recognition and protein-protein association processes in this redox reaction. First, a comparative analysis of the computed molecular electrostatic potentials of seven single and multiple point mutants of spinach plastocyanin (D42N, E43K, E43N, E43Q/D44N, E59K/E60Q, E59K/E60Q/E43N, Q88E) and the wt protein was carried out. The experimentally determined relative rates (k(2)) for the set of plastocyanin mutants are found to correlate well (r(2) = 0.90 - 0.97) with the computed measure of the similarity of the plastocyanin electrostatic potentials. Second, the effects on the plastocyanin/cytochrome f association rate of these mutations in the plastocyanin "eastern site" were evaluated by simulating the association of the wild type and mutant plastocyanins with cytochrome f by Brownian dynamics. Good agreement between the computed and experimental relative rates (k(2)) (r(2) = 0.89 - 0.92) was achieved for the plastocyanin mutants. The results obtained by applying both computational techniques provide support for the fundamental role of the acidic residues at the plastocyanin eastern site in the association with cytochrome f and in the overall electron-transfer process.

Computer Simulation↗

PubMed2000

How do substrates enter and products exit the buried active site of cytochrome P450cam? 1. Random expulsion molecular dynamics investigation of ligand access channels and mechanisms.

Cytochrome P450s form a ubiquitous protein family with functions including the synthesis and degradation of many physiologically important compounds and the degradation of xenobiotics. Cytochrome P450cam from Pseudomonas putida has provided a paradigm for the structural understanding of cytochrome P450s. However, the mechanism by which camphor, the natural substrate of cytochrome P450cam, accesses the buried active site is a long-standing puzzle. While there is recent crystallographic and simulation evidence for opening of a substrate-access channel in cytochrome P450BM-3, for cytochrome P450cam, no such conformational changes have been observed either in different crystal structures or by standard molecular dynamics simulations. Here, a novel simulation method, random expulsion molecular dynamics, is presented, in which substrate-exit channels from the buried active site are found by imposing an artificial randomly oriented force on the substrate, in addition to the standard molecular dynamics force field. The random expulsion molecular dynamics method was tested in simulations of the substrate-bound structure of cytochrome P450BM-3, and then applied to complexes of cytochrome P450cam with different substrates and with product. Three pathways were identified, one of which corresponds to a channel proposed earlier on the basis of crystallographic and site-directed mutagenesis data. Exit via the water-filled channel, which was previously suggested to be a product exit channel, was not observed. The pathways obtained by the random expulsion molecular dynamics method match well with thermal motion pathways obtained by an analysis of crystallographic B-factors. In contrast to large backbone motions (up to 4 A) observed in cytochrome P450BM-3 for the exit of palmitoleic acid, passage of camphor through cytochrome P450cam only requires small backbone motions (less than 2.4 A) in conjunction with side-chain rotations. Concomitantly, in almost all the exit trajectories, salt-links that have been proposed to act as ionic tethers between secondary structure elements of the protein, are perturbed.

Bacterial Proteins↗

PubMed2000

How do substrates enter and products exit the buried active site of cytochrome P450cam? 2. Steered molecular dynamics and adiabatic mapping of substrate pathways.

Three possible channels by which substrates and products can exit from the buried active site of cytochrome P450cam have been identified by means of random expulsion molecular dynamics simulations. In the investigation described here, we computed estimates of the relative probabilities of ligand passage through the three channels using steered molecular dynamics and adiabatic mapping. For comparison, the same techniques are also applied to investigate substrate egress from cytochrome P450-BM3. The channel in cytochrome P450cam, for which there is the most supporting evidence from experiments (which we name pathway 2a), is computed to be the most probable ligand exit channel. It has the smallest computed unbinding work and force. For this channel, the ligand exits between the F/G loop and the B' helix. Two mechanistically distinct, but energetically similar routes through this channel were observed, showing that multiple pathways along one channel are possible. The probability of ligand exit via the next most probable channel (pathway 3), which is located between the I helix and the F and G helices, is estimated to be less than 1/10 of the probability of exit along pathway 2a. Low-frequency modes of the protein extracted from an essential dynamics analysis of a 1 ns duration molecular dynamics simulation of cytochrome P450cam with camphor bound, support the opening of pathway 2a on a longer timescale. On longer timescales, it is therefore expected that this pathway becomes more dominant than estimated from the present computations.

Bacterial Proteins↗

PubMed2000

Multicopy molecular dynamics simulations suggest how to reconcile crystallographic and product formation data for camphor enantiomers bound to cytochrome P-450cam.

Multiple ligand binding modes are possible in many enzyme active sites; their presence in cytochrome P450cam (P450cam) is evident from crystallographic studies of the binding of thiocamphor and phenylimidazoles. Here, we use multicopy molecular dynamics simulations to compare the binding modes of (1R)- and (1S)-camphor in the active site of P450cam. Simulations with (1R)-camphor, the natural substrate, serve to calibrate our protocol: 19 out of 20 copies of (1R)-camphor converged to coordinates very close to those observed for (1R)-camphor in its crystallographic complex with P450cam during the simulations. Simulations with the (1S)-camphor enantiomer showed greater mobility of the substrate, consistent with spectroscopic data, and resulted in 3 major binding modes. One of these is similar to the major conformation (of the two conformations assigned) in a recently determined crystal structure, but this conformation is not correctly oriented for regiospecific hydroxylation at C-5. The simulations, however, provide evidence for reorientation of (1S)-camphor upon formation of the reactive Fe-O intermediate to an orientation suitable for hydroxylation. The simulations thus permit rationalisation of the apparent inconsistency between the crystal structure and the reaction products.

Binding Sites↗

PubMed2000

pK(a) calculations for class C beta-lactamases: the role of Tyr-150.

The Poisson-Boltzmann method was used to compute the pK(a) values of titratable residues in a set of class C beta-lactamases. In these calculations, the pK(a) of the phenolic group of residue Tyr150 is the only one to stand out with an abnormally low value of 8.3, more than one pK(a) unit lower than the measured reference value for tyrosine in solution. Other important residues of the catalytic pocket, such as the conserved Lys67, Lys315, His314, and Glu272 (hydrogen-bonded to the ammonium group of Lys315), display normal protonation states at neutral pH. pK(a) values were also computed in catalytically impaired beta-lactamase mutants. Comparisons between the relative k(cat) values and the Tyr150 pK(a) value in these mutants revealed a striking correlation. In active enzymes, this pK(a) value is always lower than the solution reference value while it is close to normal in inactive enzymes. These results thus support the hypothesis that the phenolate form of Tyr150 is responsible for the activation of the nucleophilic serine. The possible roles of Lys67 and Lys315 during catalysis are also discussed.

Catalytic Domain↗

PubMed2000

Nuclear receptor-DNA binding specificity: A COMBINE and Free-Wilson QSAR analysis.

Specific binding of transcription factors to DNA is crucial for gene regulation. We derived models for the binding specificity of transcription factors of the nuclear receptor family to DNA using two QSAR methods: a Free-Wilson-like method and COMparative BINding Energy (COMBINE) analysis. The analysis is based on experimental data for the interaction of 20 mutant glucocorticoid receptor DNA-binding domains with 16 different response elements in a total of 320 complexes (Zilliacus, J.; Wright, A. P.; Carlstedt-Duke, J.; Nilsson, L.; Gustafsson, J. A. Proteins 1995, 21, 57-67). The predictive abilities of the models obtained by the two methods are similar. The COMBINE analysis indicates that the most important properties for determining binding specificity for this dataset are the changes upon binding of the solvation free energies of the bases that are mutated in the dataset and the electrostatic interactions of the mutated nucleotides with certain charged amino acids. Further important descriptors are the changes of solvation free energy and surface area of the side chain of the mutated residue. It is clear, however, that there are additional features important for the specificity of binding that are not included in the models, such as differences in interfacial hydration of the complexes.

Algorithms↗

PubMed2000

The cisproline(i - 1)-aromatic(i) interaction: folding of the Ala-cisPro-Tyr peptide characterized by NMR and theoretical approaches.

Cisproline(i - 1)-aromatic(i) interactions have been detected in several short peptides in aqueous solution by analysis of anomalous chemical shifts measured by 1H-NMR spectroscopy. This formation of local structure is of importance for protein folding and binding properties. To obtain an atomic-detail characterisation of the cisproline(i - 1)-aromatic(i) interaction in terms of structure, energetics and dynamics, we studied the minimal peptide unit, blocked Ala-cisPro-Tyr, using computational and experimental techniques. Structural database analyses and a systematic search revealed two groups of conformations displaying a cisproline(i - 1)-aromatic(i) interaction. These conformations were taken as seeds for molecular dynamics simulations in explicit solvent at 278 K. During a total of 33.6 ns of simulation, all the 'folded' conformations and some 'unfolded' states were sampled. 1H- and 13C-chemical shifts and 3J-coupling constants were measured for the Ala-Pro-Tyr peptide. Excellent agreement was found between all the measured and computed NMR properties, showing the good quality of the force field. We find that under the experimental and simulation conditions, the Ala-cisPro-Tyr peptide is folded 90% of the time and displays two types of folded conformation which we denote 'a' and 'b'. The type a conformations are twice as populated as the type b conformations. The former have the tyrosine ring interacting with the alanine alpha proton and are enthalpically stabilised. The latter have the aromatic ring interacting with the proline side chain and are entropically stabilised. The combined and complementary use of computational and experimental techniques permitted derivation of a detailed scenario of the 'folding' of this peptide.

Amino Acids↗

PubMed2000

Blue copper proteins: a comparative analysis of their molecular interaction properties.

Blue copper proteins are type-I copper-containing redox proteins whose role is to shuttle electrons from an electron donor to an electron acceptor in bacteria and plants. A large amount of experimental data is available on blue copper proteins; however, their functional characterization is hindered by the complexity of redox processes in biological systems. We describe here the application of a semiquantitative method based on a comparative analysis of molecular interaction fields to gain insights into the recognition properties of blue copper proteins. Molecular electrostatic and hydrophobic potentials were computed and compared for a set of 33 experimentally-determined structures of proteins from seven blue copper subfamilies, and the results were quantified by means of similarity indices. The analysis provides a classification of the blue copper proteins and shows that (I) comparison of the molecular electrostatic potentials provides useful information complementary to that highlighted by sequence analysis; (2) similarities in recognition properties can be detected for proteins belonging to different subfamilies, such as amicyanins and pseudoazurins, that may be isofunctional proteins; (3) dissimilarities in interaction properties, consistent with experimentally different binding specificities, may be observed between proteins belonging to the same subfamily, such as cyanobacterial and eukaryotic plastocyanins; (4) proteins with low sequence identity, such as azurins and pseudoazurins, can have sufficient similarity to bind to similar electron donors and acceptors while having different binding specificity profiles.

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↗

PubMed1999

Computer simulation of protein-protein association kinetics: acetylcholinesterase-fasciculin.

Computer simulations were performed to investigate the role of electrostatic interactions in promoting fast association of acetylcholinesterase with its peptidic inhibitor, the neurotoxin fasciculin. The encounter of the two macromolecules was simulated with the technique of Brownian dynamics (BD), using atomically detailed structures, and association rate constants were calculated for the wild-type and a number of mutant proteins. In a first set of simulations, the ordering of the experimental rate constants for the mutant proteins was correctly reproduced, although the absolute values of the rate constants were overestimated by a factor of around 30. Rigorous calculations of the full electrostatic interaction energy between the two proteins indicate that this overestimation of association rates results at least in part from approximations made in the description of interaction energetics in the BD simulations. In particular, the initial BD simulations neglect the unfavourable electrostatic desolvation effects that result from the exclusion of high dielectric solvent that accompanies the approach of the two low dielectric proteins. This electrostatic desolvation component is so large that the overall contribution of electrostatics to the binding energy of the complex is unlikely to be strongly favourable. Nevertheless, electrostatic interactions are still responsible for increased association rates, because even if they are unfavourable in the fully formed complex, they are still favourable at intermediate protein-protein separation distances. It therefore appears possible for electrostatic interactions to promote the kinetics of binding even if they do not make a strongly favourable contribution to the thermodynamics of binding. When an approximate description of these electrostatic desolvation effects is included in a second set of BD simulations, the relative ordering of the mutant proteins is again correctly reproduced, but now association rate constants that are much closer in magnitude to the experimental values are obtained. Inclusion of electrostatic desolvation effects also improves reproduction of the experimental ionic strength dependence of the wild-type association rate.

Acetylcholinesterase↗

PubMed1999

Towards molecular dynamics simulation of large proteins with a hydration shell at constant pressure.

Molecular dynamics simulation of a large protein in explicit water with periodic boundary conditions is extremely demanding in terms of computation time. Consequently, we have sought approximations of the solvent environment that model its important features. Here, we describe our SAPHYR (Shell Approximation for Protein HYdRation) model in which the protein is surrounded by a shell of water molecules maintained at constant pressure. In addition to the usual pairwise interatomic interactions, these water molecules are subjected to forces approximating van der Waals and dipole-dipole interactions with the implicit surrounding bulk solvent. The SAPHYR model is tested for a system of one argon atom in water and for the protein ubiquitin, and then applied to cytochrome P450cam, a protein with over 400 residues. The results demonstrate that structural and dynamic properties of the simulated systems are improved by use of the SAPHYR model, and that this model provides a significant computational saving over simulations with periodic boundary conditions.

Algorithms↗

PubMed1999

Stability of the beta-sheet of the WW domain: A molecular dynamics simulation study.

The WW domain consists of approximately 40 residues, has no disulfide bridges, and forms a three-stranded antiparallel beta-sheet that is monomeric in solution. It thus provides a model system for studying beta-sheet stability in native proteins. We performed molecular dynamics simulations of two WW domains, YAP65 and FBP28, with very different stability characteristics, in order to explore the initial unfolding of the beta-sheet. The less stable YAP domain is much more sensitive to simulation conditions than the FBP domain. Under standard simulation conditions in water (with or without charge-balancing counterions) at 300 K, the beta-sheet of the YAP WW domain disintegrated at early stages of the simulations. Disintegration commenced with the breakage of a hydrogen bond between the second and third strands of the beta-sheet due to an anticorrelated transition of the Tyr-28 psi and Phe-29 phi angles. Electrostatic interactions play a role in this event, and the YAP WW domain structure is more stable when simulated with a complete explicit model of the surrounding ionic strength. Other factors affecting stability of the beta-sheet are side-chain packing, the conformational entropy of the flexible chain termini, and the binding of cognate peptide.

Adaptor Proteins, Signal Transducing↗