The open nucleotide pocket of the profilin/actin x-ray structure is unstable and closes in the absence of profilin.

The open nucleotide pocket conformation of actin in the profilin:actinCaATP x-ray structure has been hypothesized to be a crucial intermediate for nucleotide exchange in the actin depolymerization/polymerization cycle. The requirement for ancillary modification of actin for crystallization leads to ambiguities in this interpretation, however. We have used molecular dynamics simulations to model the thermodynamic properties of the actin x-ray structure, outside the crystal lattice, in an aqueous environment with profilin removed. Our simulations show that the open-nucleotide-pocket, profilin-free structure is actually unstable, and closes. The coordination of actin to the nucleotide in the molecular-dynamics-derived closed structure is virtually identical to that in the closed profilin:actinSrATP x-ray structure. Thus, there is currently no thermodynamically stable structure representing the open-nucleotide-pocket state of actin.

Computational study of protein specificity: the molecular basis of HIV-1 protease drug resistance.

Drug resistance has sharply limited the effectiveness of HIV-1 protease inhibitors in AIDS therapy. It is critically important to understand the basis of this resistance for designing new drugs. We have evaluated the free energy contribution of each residue in the HIV protease in binding to one of its substrates and to the five FDA-approved protease drugs. Analysis of these free energy profiles and the variability at each sequence position suggests: (i) single drug resistance mutations are likely to occur at not well conserved residues if they interact more favorably with drugs than with the substrate; and (ii) resistance-evading drugs should have a free energy profile similar to the substrate and interact most favorably with well conserved residues. We also propose an empirical parameter, called the free energy/variability value, which combines free energy calculation and sequence analysis to suggest possible drug resistance mutations on the protease. The free energy/variability value is defined as the product of one residue's contribution to the binding free energy and the variability of that residue. This parameter can assist in designing resistance-evading drugs for any target.

An alternative explanation for the catalytic proficiency of orotidine 5'-phosphate decarboxylase.

Orotidine 5'-phosphate decarboxylase (ODCase) is the most proficient enzyme known, enhancing the rate of decarboxylation of orotidine 5'-phosphate (OMP) by a factor of 10(17), which corresponds to a DeltaDeltaG++ of approximately 24 kcal/mol. Ground-state destabilization through local electrostatic stress has been recently proposed as the basis of catalytic rate enhancement for a mechanism that is the same as in solution. We have carried out gas-phase ab initio quantum mechanical calculations combined with a free energy method, a continuum solvent model, and molecular dynamics simulations to assess an alternative mechanism. Although we are not able to reproduce the experimentally observed DeltaDeltaG++ quantitatively, we present evidence that this DeltaDeltaG++ is very large, in the range found experimentally. We thus conclude that the preferred mechanism may well be different from that in solution, involving an equilibrium pre-protonation of OMP C5 by a catalytic lysine residue that greatly reduces the barrier to subsequent decarboxylation.

Free-energy calculations highlight differences in accuracy between X-ray and NMR structures and add value to protein structure prediction.

BACKGROUND: While X-ray crystallography structures of proteins are considerably more reliable than those from NMR spectroscopy, it has been difficult to assess the inherent accuracy of NMR structures, particularly the side chains. RESULTS: For 15 small single-domain proteins, we used a molecular mechanics-/dynamics-based free-energy approach to investigate native, decoy, and fully extended alpha conformations. Decoys were all less energetically favorable than native conformations in nine of the ten X-ray structures and in none of the five NMR structures, but short 150 ps molecular dynamics simulations on the experimental structures caused them to have the lowest predicted free energy in all 15 proteins. In addition, a strong correlation exists (r(2) = 0.86) between the predicted free energy of unfolding, from native to fully extended conformations, and the number of residues. CONCLUSIONS: This work suggests that the approximate treatment of solvent used in solving NMR structures can lead NMR model conformations to be less reliable than crystal structures. This conclusion was reached because of the considerably higher calculated free energies and the extent of structural deviation during aqueous dynamics simulations of NMR models compared to those determined by X-ray crystallography. Also, the strong correlation found between protein length and predicted free energy of unfolding in this work suggests, for the first time, that a free-energy function can allow for identification of the native state based on calculations on an extended state and in the absence of an experimental structure.

Molecular dynamics simulations of a highly charged peptide from an SH3 domain: possible sequence-function relationship.

A seven-residue peptide that is highly conserved in SH3 domains despite being far from the active site has been shown by NMR to be stable in solution. This peptide, biologically important because it is a likely folding nucleus for SH3 domains, provides a challenging subject for molecular dynamics because it is highly charged. We present stable, 10-ns simulations of both the native-like diverging turn structure and a helical model. Free energies of these two conformations, estimated through MM-PBSA analysis using several force fields, suggest a comparable free energy (DeltaDeltaG < or =6 kcal/mol) for native and helix conformations. NOE intensities calculated from the native trajectory reproduce experimental data quite well, suggesting that the conformations sampled by the trajectory reasonably represent those observed in the NMR experiment. The molecular dynamics results, as well as sequence analysis of a diverse 690-member family of SH3 domain proteins, suggest that the presence of two elements is essential for formation of the diverging turn structure: a pair of residues with low helical propensity in the turn region and, as previously recognized, two hydrophobic residues to close the end of the diverging turn. Thus, these two sequence features may form the structural basis for the function of this peptide as a folding nucleus in this family of proteins.

2.1 and 1.8 A average C(alpha) RMSD structure predictions on two small proteins, HP-36 and s15.

On two different small proteins, the 36-mer villin headpiece domain (HP-36) and the 65-mer structured region of ribosomal protein (S15), several model predictions from the ab initio approach Rosetta were subjected to molecular dynamics simulations for refinement. After clustering the resulting trajectories into conformational families, the average molecular mechanics--Poisson Boltzmann/surface area (MM-PBSA) free energies and alpha carbon (C(alpha)) RMSDs were then calculated for each family. Those conformational families with the lowest average free energies also contained the best C(alpha) RMSD structures (1.4 A for S15 and HP-36 core) and the lowest average C(alpha) RMSDs (1.8 A for S15, 2.1 A for HP-36 core). For comparison, control simulations starting with the two experimental structures were very stable, each consisting of a single conformational family, with an average C(alpha) RMSD of 1.3 A for S15 and 1.2 A for HP-36 core (1.9 A over all residues). In addition, the average free energies' ranks (Spearman rank, r(s)) correlate well with the average C(alpha) RMSDs (r(s) = 0.77 for HP-36, r(s) = 0.83 for S15). Molecular dynamics simulations combined with the MM--PBSA free energy function provide a potentially powerful tool for the protein structure prediction community in allowing for both high-resolution structural refinement and accurate ranking of model predictions. With all of the information that genomics is now providing, this methodology may allow for advances in going from sequence to structure.

An analysis of the interactions between the Sem-5 SH3 domain and its ligands using molecular dynamics, free energy calculations, and sequence analysis.

The Src-homology-3 (SH3) domain of the Caenorhabditis elegans protein Sem-5 binds proline-rich sequences. It is reported that the SH3 domains broadly accept amide N-substituted residues instead of only recognizing prolines on the basis of side chain shape or rigidity. We have studied the interactions between Sem-5 and its ligands using molecular dynamics (MD), free energy calculations, and sequence analysis. Relative binding free energies, estimated by a method called MM/PBSA, between different substitutions at sites -1, 0, and +2 of the peptide are consistent with the experimental data. A new method to calculate atomic partial charges, AM1-BCC method, is also used in the binding free energy calculations for different N-substitutions at site -1. The results are very similar to those obtained from widely used RESP charges in the AMBER force field. AM1-BCC charges can be calculated more rapidly for any organic molecule than can the RESP charges. Therefore, their use can enable a broader and more efficient application of the MM/PBSA method in drug design. Examination of each component of the free energy leads to the construction of van der Waals interaction energy profiles for each ligand as well as for wild-type and mutant Sem-5 proteins. The profiles and free energy calculations indicate that the van der Waals interactions between the ligands and the receptor determine whether an N- or a Calpha-substituted residue is favored at each site. A VC value (defined as a product of the conservation percentage of each residue and its van der Waals interaction energy with the ligand) is used to identify several residues on the receptor that are critical for specificity and binding affinity. This VC value may have a potential use in identifying crucial residues for any ligand-protein or protein-protein system. Mutations at two of those crucial residues, N190 and N206, are examined. One mutation, N190I, is predicted to reduce the selectivity of the N-substituted residue at site -1 of the ligand and is shown to bind similarly with N- and Calpha-substituted residues at that site.

Use of MM-PBSA in reproducing the binding free energies to HIV-1 RT of TIBO derivatives and predicting the binding mode to HIV-1 RT of efavirenz by docking and MM-PBSA.

In this work, a new ansatz is presented that combines molecular dynamics simulations with MM-PBSA (Molecular Mechanics Poisson-Boltzmann/surface area) to rank the binding affinities of 12 TIBO-like HIV-1 RT inhibitors. Encouraging results have been obtained not only for the relative binding free energies, but also for the absolute ones, which have a root-mean-square deviation of 1.0 kcal/mol (the maximum error is 1.89 kcal/mol). Since the root-mean-square error is rather small, this approach can be reliably applied in ranking the ligands from the databases for this important target. Encouraged by the results, we decided to apply MM-PBSA combined with molecular docking to determine the binding mode of efavirenz SUSTIVA(TM) another promising HIV-1 RT inhibitor for which no ligand-protein crystal structure had been published at the time of this work. To proceed, we define the following ansatz: Five hundred picosecond molecular dynamics simulations were first performed for the five binding modes suggested by DOCK 4.0, and then MM-PBSA was carried out for the collected snapshots. MM-PBSA successfully identified the correct binding mode, which has a binding free energy about 7 kcal/mol more favorable than the second best mode. Moreover, the calculated binding free energy (-13.2 kcal/mol) is in reasonable agreement with experiment (-11.6 kcal/mol). In addition, this procedure was also quite successful in modeling the complex and the structure of the last snapshot was quite close to that of the measured 2,3 A resolution crystal (structure the root-mean-square deviation of the 54 C(alpha) around the binding site and the inhibitor is 1.1 A). We want to point out that this result was achieved without prior knowledge of the structure of the efavirenz/RT complex. Therefore, molecular docking combined with MD simulations followed by MM-PBSA analysis is an attractive approach for modeling protein complexes a priori.

Metabolism of sirolimus and its derivative everolimus by cytochrome P450 3A4: insights from docking, molecular dynamics, and quantum chemical calculations.

A combination of quantum chemical calculations and molecular simulations (DOCKing and molecular dynamics) is used to investigate the metabolism of sirolimus (rapamycin) and its derivative everolimus (SDZ-RAD) by cytochrome P450 3A4. Both molecules are drugs with high immunosuppressive activity. Our calculations yield qualitative predictions of the regiospecificities of the hydroxylations and O-dealkylations occurring in these two substrates which are in good agreement with recent experimental results. An analysis of the modeled enzyme-substrate interactions allows us to rationalize the reduced metabolic activity of the larger substrate everolimus compared to sirolimus. Moreover, our simulations suggest that hydrogen donor functionalities close to the metabolic site are important for anchoring the substrate at the active center of the enzyme. In particular, we predict that replacing one hydroxyl group by a fluorine atom should considerably suppress the major metabolic reaction in sirolimus, 39-O-demethylation.

Biomolecular simulations: recent developments in force fields, simulations of enzyme catalysis, protein-ligand, protein-protein, and protein-nucleic acid noncovalent interactions.

Computer modeling has been developed and widely applied in studying molecules of biological interest. The force field is the cornerstone of computer simulations, and many force fields have been developed and successfully applied in these simulations. Two interesting areas are (a) studying enzyme catalytic mechanisms using a combination of quantum mechanics and molecular mechanics, and (b) studying macromolecular dynamics and interactions using molecular dynamics (MD) and free energy (FE) calculation methods. Enzyme catalysis involves forming and breaking of covalent bonds and requires the use of quantum mechanics. Noncovalent interactions appear ubiquitously in biology, but here we confine ourselves to review only noncovalent interactions between protein and protein, protein and ligand, and protein and nucleic acids.

Molecular dynamics and binding specificity analysis of the bovine immunodeficiency virus BIV Tat-TAR complex.

We have performed molecular dynamics (MD) simulations, with particle-mesh Ewald, explicit waters, and counterions, and binding specificity analyses using combined molecular mechanics and continuum solvent (MM-PBSA) on the bovine immunodeficiency virus (BIV) Tat peptide-TAR RNA complex. The solution structure for the complex was solved independently by Patel and co-workers and Puglisi and co-workers. We investigated the differences in both structures and trajectories, particularly in the formation of the U-A-U base triple, the dynamic flexibility of the Tat peptide, and the interactions at the binding interface. We observed a decrease in RMSD in comparing the final average RNA structures and initial RNA structures of both trajectories, which suggests the convergence of the RNA structures to a MD equilibrated RNA structure. We also calculated the relative binding of different Tat peptide mutants to TAR RNA and found qualitative agreement with experimental studies.

State of the art in studying protein folding and protein structure prediction using molecular dynamics methods.

This study presents an overview of the state of the art in using molecular dynamics methods to simulate protein folding and in the end game of protein structure prediction. In principle, these methods should allow the highest level of detail possible and the highest accuracy, but they are limited by both the accuracy of the force field used in the simulation and the sampling possible in the available computer time. We describe current capabilities in running the simulations longer and more efficiently.

Does a diol cyclic urea inhibitor of HIV-1 protease bind tighter than its corresponding alcohol form? A study by free energy perturbation and continuum electrostatics calculations.

The cyclic urea inhibitors of HIV-1 protease generally have two hydroxyl groups on the seven-membered ring. In this study, free energy perturbation and continuum electrostatic calculations were used to study the contributions of the two hydroxyl groups to the binding affinity and solubility of a cyclic urea inhibitor DMP323. The results indicated that the inhibitor with one hydroxyl group has better binding affinity and solubility than the inhibitor with two hydroxyl groups. Therefore, removal of one hydroxyl group from DMP323 may help to improve the properties of DMP323. This is also likely to be true for other cyclic urea inhibitors. The study also illustrated the difficulty in accurate modeling of the binding affinities of HIV-1 protease inhibitors, which involves many possible protonation states of the two catalytic aspartic acids in the active site of the enzyme.

Elucidating the nature of enzyme catalysis utilizing a new twist on an old methodology: quantum mechanical-free energy calculations on chemical reactions in enzymes and in aqueous solution.

How do enzymes achieve very large rate enhancements compared to corresponding uncatalyzed reactions in solution? We present a computational approach which combines high-level ab initio quantum mechanical calculations with classical free energy calculations to address this question. Our calculations lead to accurate estimates of DeltaG for both trypsin and catechol O-methyltransferase-catalyzed and reference uncatalyzed reactions and give new insights into the nature of enzyme catalysis. The same methodology applied to steps in the catalytic mechanism of citrate synthase further supports the conclusion that one need not invoke special concepts such as "low-barrier hydrogen bonds" or "pK(a) matching" to explain enzyme catalysis.

Computed free energy differences between point mutations in a collagen-like peptide.

We studied the results of mutating alanine --> glycine at three positions of a collagen-like peptide in an effort to develop a computational method for predicting the energetic and structural effects of a single point genetic mutation in collagen, which is associated with the clinical diagnosis of Osteogenesis Imperfecta (OI). The differences in free energy of denaturation were calculated between the collagen-like peptides [(POG)(4)(POA)(POG)(4)](3) and [(POG)(10)](3) (POG: proline-hydroxyproline-glycine).* Our computational results, which suggest significant destabilization of the collagen-like triple-helix upon the glycine --> alanine mutations, correlate very well with the experimental free energies of denaturation. The robustness of our collagen-like peptide model is shown by its reproduction of experimental results with both different simulation paths and different lengths of the model peptide. The individual free energy for each alanine --> glycine mutation (and the reverse free energy, glycine --> alanine mutation) in the collagen-like peptide has been calculated. We find that the first alanine introduced into the triple helix causes a very large destabilization of the helix, but the last alanine introduced into the same position of an adjacent chain causes a very small change in the peptide stability. Thus, our results demonstrate that each mutation does not contribute equally to the free energy. We find that the sum of the calculated individual residues' free energy can accurately model the experimental free energy for the whole peptide.

Molecular dynamics study of the energetic, mechanistic, and structural implications of a closed phosphate tube in ncd.

The switch 1 region of myosin forms a lid over the nucleotide phosphates as part of a structure known as the phosphate-tube. The homologous region in kinesin-family motors is more open, not interacting with the nucleotide. We used molecular dynamics (MD) simulations to examine a possible displacement of switch 1 of the microtubule motor, ncd, from the open conformation to the closed conformation seen in myosin. MD simulations were done of both the open and the closed conformations, with either MgADP or MgATP at the active site. All MD structures were stable at 300 K for 500 ps, implying that the open and closed conformers all represented local minima on a global free energy surface. Free energy calculations indicated that the open structure was energetically favored with MgADP at the active site, suggesting why only the open structure has been captured in crystallographic work. With MgATP, the closed and open structures had roughly equal energies. Simulated annealing MD showed the transformation from the closed phosphate-tube ncd structure to an open configuration. The MD simulations also showed that the coordination of switch 1 to the nucleotide dramatically affected the position of both the bound nucleotide and switch 2 and that a closed phosphate-tube may be necessary for catalysis.

Molecular recognition by beta-cyclodextrin derivatives: FEP vs MM/PBSA study.

The complexation of p-tert-butylphenyl p-tert-butylbenzoate, N-(p-tert-butylphenyl)-p-tert-butylbenzamide and a bisadamantyl-phosphate derivative with a beta-cyclodextrin derivative formed by two cyclodextrin units linked by a disulfide bridge on one of the C6 atoms have been studied by computational methods (free energy perturbation (FEP) and Molecular Mechanics/Poisson Bolzmann Surface Area (MM/PBSA)). The calculated relative free energies of the amide and ester are in good agreement with experiment only for MM/PBSA and not for FEP. Only MM/PBSA was applied to the bisadamantyl-phosphate complex and its calculated association free energy was calculated to be similar to that of the ester, which is consistent with the experimental tendencies.

Molecular dynamics in the endgame of protein structure prediction.

In order adequately to sample conformational space, methods for protein structure prediction make necessary simplifications that also prevent them from being as accurate as desired. Thus, the idea of feeding them, hierarchically, into a more accurate method that samples less effectively was introduced a decade ago but has not met with more than limited success in a few isolated instances. Ideally, the final stages should be able to identify the native state, show a good correlation with native similarity in order to add value to the selection process, and refine the structures even further. In this work, we explore the possibility of using state-of-the-art explicit solvent molecular dynamics and implicit solvent free energy calculations to accomplish all three of those objectives on 12 small, single-domain proteins, four each of alpha, beta and mixed topologies. We find that this approach is very successful in ranking the native and also enhances the structure selection of predictions generated from the Rosetta method.

Molecular modeling of nucleic acid structure: setup and analysis.

The last in a set of units by these authors, this unit addresses some important remaining questions about molecular modeling of nucleic acids. It describes how to choose an appropriate molecular mechanics force field; how to set up and equilibrate the system for accurate simulation of a nucleic acid in an explicit solvent by molecular dynamics or Monte Carlo simulation; and how to analyze molecular dynamics trajectories.

Molecular modeling of nucleic acid structure.

This unit is the first in a series of four units covering the analysis of nucleic acid structure by molecular modeling. This unit provides an overview of computer simulation of nucleic acids. Topics include the static structure model, computational graphics and energy models, generation of an initial model, and characterization of the overall three-dimensional structure.