Molecular dynamics simulations on the first two helices of Vpu from HIV-1

Vpu is an 81 amino acid protein of HIV-1 with two phosphorylation sites. It consists of a short N-terminal end traversing the bilayer and a longer cytoplasmic part. The dual functional role of Vpu is attributed to these topological distinct regions of the protein. The first 52 amino acids of Vpu (HV1H2) have been simulated, which are thought to be embedded in a fully hydrated lipid bilayer and to consist of a transmembrane helix (helix-1) connected via a flexible linker region, including a Glu-Tyr-Arg (EYR) motif, with a second helix (helix-2) residing with its helix long axis on the bilayer surface. Repeated molecular dynamics simulations show that Glu-28 is involved in salt bridge formation with Lys-31 and Arg-34 establishing a kink between the two helices. Helix-2 remains in a helical conformation indicating its stability and function as a "peptide float," separating helix-1 from the rest of the protein. This leads to the conclusion that Vpu consists of three functional modules: helix-1, helix-2, and the remaining residues toward the C-terminal end.     

Molecular dynamics of HIV-1 protease.

Molecular dynamics simulations have been carried out based on the GROMOS force field on the aspartyl protease (PR) of the human immunodeficiency virus HIV-1. The principal simulation treats the HIV-1 PR dimer and 6990 water molecules in a hexagonal prism cell under periodic boundary conditions and was carried out for a trajectory of 100 psec. Corresponding in vacuo simulations, i.e., treating the isolated protein without solvent, were carried out to study the influence of solvent on the simulation. The results indicate that including waters explicitly in the simulation results in a model considerably closer to the crystal structure than when solvent is neglected. Detailed conformational and helicoidal analysis was performed on the solvated form to determine the exact nature of the dynamical model and the exact points of agreement and disagreement with the crystal structure. The calculated dynamical model was further elucidated by means of studies of the time evolution of the cross-correlation coefficients for atomic displacements of the atoms comprising the protein backbone. The cross-correlation analysis revealed significant aspects of structure originating uniquely in the dynamical motions of the molecule. In particular, an unanticipated through-space, domain-domain correlation was found between the mobile flap region covering the active site and a remote regions of the structure, which collectively act somewhat like a molecular cantilever. The significance of these results is discussed with respect to the inactivation of the protease by site-specific mutagenesis, and in the design of inhibitors.     

Molecular dynamics simulations on HIV-1 Tat

Molecular dynamics simulations are used to investigate dynamics and intramolecular interactions of the HIV-1 transactivator (Tat) in aqueous solution. The calculations are based on the AMBER force field with particle mesh Ewald treatment for long-range electrostatics. The Tat structure exhibits a large flexibility, consistent with its absence of secondary structure elements. From an analysis of the correlation matrix and of electrostatic interactions we suggest that segments expressed by the two exons (amino acids 1-72 and 73-86, respectively) exhibit rather separated dynamic and energetic properties. We also identify intramolecular interactions of importance for structure stabilization. In particular, significant electrostatic interactions are recognized between the N-terminus and the basic domain of the protein, consistent with site-directed mutagenesis performed in this work.     

The role of hydrogen bonding in the enzymatic reaction catalyzed by HIV-1 protease

The hydrogen-bond network in various stages of the enzymatic reaction catalyzed by HIV-1 protease was studied through quantum-classical molecular dynamics simulations. The approximate valence bond method was applied to the active site atoms participating directly in the rearrangement of chemical bonds. The rest of the protein with explicit solvent was treated with a classical molecular mechanics model. Two possible mechanisms were studied, general-acid/general-base (GA/GB) with Asp 25 protonated at the inner oxygen, and a direct nucleophilic attack by Asp 25. Strong hydrogen bonds leading to spontaneous proton transfers were observed in both reaction paths. A single-well hydrogen bond was formed between the peptide nitrogen and outer oxygen of Asp 125. The proton was diffusely distributed with an average central position and transferred back and forth on a picosecond scale. In both mechanisms, this interaction helped change the peptide-bond hybridization, increased the partial charge on peptidyl carbon, and in the GA/GB mechanism, helped deprotonate the water molecule. The inner oxygens of the aspartic dyad formed a low-barrier, but asymmetric hydrogen bond; the proton was not positioned midway and made a slightly elongated covalent bond, transferring from one to the other aspartate. In the GA/GB mechanism both aspartates may help deprotonate the water molecule. We observed the breakage of the peptide bond and found that the protonation of the peptidyl amine group was essential for the peptide-bond cleavage. In studies of the direct nucleophilic mechanism, the peptide carbon of the substrate and oxygen of Asp 25 approached as close as 2.3 A.     

Restrained molecular dynamics simulations of HIV-1 protease: the first step in validating a new target for drug design

To test the anticorrelated relationship that was recently displayed in conventional molecular dynamics (MD) simulations, several different restrained MD simulations on a wild type and on the V82F/I84V drug-resistant mutant of HIV-1 protease were performed. This anticorrelated relationship refers to the observation that compression of the peripheral ear-to-cheek region of HIV protease (i.e., the elbow of the flap to the fulcrum and the cantilever) occurred as the active site flaps were opening, and, conversely, expansion of that ear-to-cheek region occurred as both flaps were closing. An additional examination of this anticorrelated relationship was necessary to determine whether it can be harnessed in a useful manner. Consequently, six different MD experiments were performed that incorporated pairwise distance restraints in that ear-to-cheek region (i.e., the distance between the alpha-carbons of Gly40 and Gln61 was restrained to either 7.7 or 10.5 A, in both monomers). Pushing the backbones of the ear and the cheek regions away from each other slightly did force the flaps that guard the active site to remain closed in both the wild type and the mutant systems-even though there were no ligands in the active sites. Thus, these restrained MD simulations provided evidence that the anticorrelated relationship can be exploited to affect the dynamic behavior of the flaps that guard the active site of HIV-1 protease. These simulations supported our hypothesis of the mechanism governing flap motion, and they are the first step towards validating that peripheral surface as a new target for drug design.     

Molecular dynamics simulations of HIV-1 protease monomer: Assembly of N-terminus and C-terminus into beta-sheet in water solution

HIV-1 protease (HIV-PR) consists of two identical subunits that are united together through a four-stranded antiparallel beta-sheet formed of the peptide termini of each monomer. Since the active site exists only in the dimer, a strategy that is attracting more and more attention in inhibitor design and which may overcome the serious drug resistance caused by competitive inhibitors is to block the peptide termini of the monomer, thereby interfering with formation of the active dimer. In the present work, we performed several extensive molecular dynamics (MD) simulations of the HIV-PR monomer in water to illustrate its solvated conformation and dynamics behavior. We found that the peptide termini usually assembled into beta-sheet after several nanoseconds' simulation, and became much less flexible. This beta-sheet is stabilized by intramolecular interactions and is not easily disaggregated under the present MD simulation conditions. This transformation may be an important transition during the relaxing and equilibrating of the HIV-PR monomer in aqueous solution, and the terminal beta-sheet may be one of the major conformations of the solvated HIV-PR monomer termini in water. This work may provide new insights into the dynamics behavior and dimerization mechanism of HIV-PR, and more significantly, offer a more rational receptor model for the design and discovery of novel dimerization inhibitors than crystalline structures.     

Atomistic simulations of the HIV-1 protease folding inhibition

Biochemical experiments have recently revealed that the p-S8 peptide, with an amino-acid sequence identical to the conserved fragment 83-93 (S8) of the HIV-1 protease, can inhibit catalytic activity of the enzyme by interfering with protease folding and dimerization. In this study, we introduce a hierarchical modeling approach for understanding the molecular basis of the HIV-1 protease folding inhibition. Coarse-grained molecular docking simulations of the flexible p-S8 peptide with the ensembles of HIV-1 protease monomers have revealed structurally different complexes of the p-S8 peptide, which can be formed by targeting the conserved segment 24-34 (S2) of the folding nucleus (folding inhibition) and by interacting with the antiparallel termini β-sheet region (dimerization inhibition). All-atom molecular dynamics simulations of the inhibitor complexes with the HIV-1 PR monomer have been independently carried out for the predicted folding and dimerization binding modes of the p-S8 peptide, confirming the thermodynamic stability of these complexes. Binding free-energy calculations of the p-S8 peptide and its active analogs are then performed using molecular dynamics trajectories of the peptide complexes with the HIV-1 PR monomers. The results of this study have provided a plausible molecular model for the inhibitor intervention with the HIV-1 PR folding and dimerization and have accurately reproduced the experimental inhibition profiles of the active folding inhibitors.     

Molecular dynamics simulations applied to the study of subtypes of HIV-1 protease common to Brazil, Africa, and Asia

Africa accounts for the majority of HIV-1 infections worldwide caused mainly by the A and C viral subtypes rather than B subtype, which prevails in the United States and Western Europe. In Brazil, B subtype is the major subtype, but F, C, and A also circulate. These non-B subtypes present polymorphisms, and some of them occur at sites that have been associated with drug resistance, including the HIV-1 protease (PR), one important drug target. Here, we report a Molecular Dynamics study of the B and non-B PR complexed with the inhibitor ritonavir to delineate the behavior of each subtype. We compare root mean squared deviation, binding free energy by linear interaction energy approach, hydrogen bonds, and intermolecular contact surface area between inhibitor and PR. From our results, we can provide a basis to understand the molecular mechanism of drug resistance in non-B subtypes. In this sense, we found a decrease of approx 4 kcal/mol in ΔG of binding between B and non-B subtypes. This corresponds to the loss of one hydrogen bond, which is in agreement with our H-bond analysis. Previous experimental affinity studies reported analogous results with inhibition constant values for non-B PR.     

Molecular dynamics studies on HIV-1 protease drug resistance and folding pathways

Drug resistance to HIV-1 protease involves the accumulation of multiple mutations in the protein. We investigate the role of these mutations by using molecular dynamics simulations that exploit the influence of the native-state topology in the folding process. Our calculations show that sites contributing to phenotypic resistance of FDA-approved drugs are among the most sensitive positions for the stability of partially folded states and should play a relevant role in the folding process. Furthermore, associations between amino acid sites mutating under drug treatment are shown to be statistically correlated. The striking correlation between clinical data and our calculations suggest a novel approach to the design of drugs tailored to bind regions crucial not only for protein function, but for folding as well.     

Gated binding of ligands to HIV-1 protease: Brownian dynamics simulations in a coarse-grained model

The internal motions of proteins may serve as a "gate" in some systems, which controls ligand-protein association. This study applies Brownian dynamics simulations in a coarse-grained model to study the gated association rate constants of HIV-1 proteases and drugs. The computed gated association rate constants of three protease mutants, G48V/V82A/I84V/L90M, G48V, and L90M with three drugs, amprenavir, indinavir, and saquinavir, yield good agreements with experiments. The work shows that the flap dynamics leads to "slow gating". The simulations suggest that the flap flexibility and the opening frequency of the wild-type, the G48V and L90M mutants are similar, but the flaps of the variant G48V/V82A/I84V/L90M open less frequently, resulting in a lower gated rate constant. The developed methodology is fast and provides an efficient way to predict the gated association rate constants for various protease mutants and ligands.     

Molecular recognition of macrocyclic peptidomimetic inhibitors by HIV-1 protease.

High-resolution crystal structures are described for seven macrocycles complexed with HIV-1 protease (HIVPR). The macrocycles possess two amides and an aromatic group within 15-17 membered rings designed to replace N- or C-terminal tripeptides from peptidic inhibitors of HIVPR. Appended to each macrocycle is a transition state isostere and either an acyclic peptide, nonpeptide, or another macrocycle. These cyclic analogues are potent inhibitors of HIVPR, and the crystal structures show them to be structural mimics of acyclic peptides, binding in the active site of HIVPR via the same interactions. Each macrocycle is restrained to adopt a beta-strand conformation which is preorganized for protease binding. An unusual feature of the binding of C-terminal macrocyclic inhibitors is the interaction between a positively charged secondary amine and a catalytic aspartate of HIVPR. A bicyclic inhibitor binds similarly through its secondary amine that lies between its component N-terminal and C-terminal macrocycles. In contrast, the corresponding tertiary amine of the N-terminal macrocycles does not interact with the catalytic aspartates. The amine-aspartate interaction induces a 1.5 A N-terminal translation of the inhibitors in the active site and is accompanied by weakened interactions with a water molecule that bridges the ligand to the enzyme, as well as static disorder in enzyme flap residues. This flexibility may facilitate peptide cleavage and product dissociation during catalysis. Proteases [Aba67,95]HIVPR and [Lys7,Ile33,Aba67,95]HIVPR used in this work were shown to have very similar crystal structures.     

Molecular dynamics simulations of ribonuclease T1

Molecular dynamics simulations in vacuum and with a water sphere around the active site were performed on the 2GMP-RNase T1 complex. The presence of water led to the maintenance of the 2-GMP-RNase T1 interactions as compared to the X-ray structure, including the hydrogen bonds implicated in the enzyme-inhibitor recognition process. The sidechain of His92 in the molecular dynamics water simulation, however, hydrogen bonds directly to the phosphate of 2GMP in contrast to the X-ray structure but in support of the role of that residue in the enzyme's catalytic mechanism. Fluctuations of activesite residues are not strongly influenced by water, possibly owing to the exclusion of water by the bound 2GMP, which did show an increase in mobility. Analysis of the 2GMP-RNase T1 interactions versus time reveal an equilibrium fluctuation in the presence of water, leading to a less favorable 2GMP-RNase T1 interaction energy, suggesting a possible relationship between picosecond fluctuations and inhibitor dissociation occurring in the millisecond time domain.Abbreviations RNase T1 Ribonuclease T1 (EC.3.1.27.3) - 2GMP Guanosine-2-monophosphate - SBS Stochastic Bondary Simulation - VS Vacuum Simulation - MD Molecular Dynamics     

Improved prediction of HIV-1 protease-inhibitor binding energies by molecular dynamics simulations

Background  

The accurate prediction of enzyme-substrate interaction energies is one of the major challenges in computational biology. This study describes the improvement of protein-ligand binding energy prediction by incorporating protein flexibility through the use of molecular dynamics (MD) simulations.     

Molecular dynamics simulations

Molecular dynamics simulations have become a standard tool for the investigation of biomolecules. Simulations are performed of ever bigger systems using more realistic boundary conditions and better sampling due to longer sampling times. Recently, realistic simulations of systems as complex as transmembrane channels have become feasible. Simulations aid our understanding of biochemical processes and give a dynamic dimension to structural data; for example, the transformation of harmless prion protein into the disease-causing agent has been modeled.     

Molecular dynamics simulations of 14 HIV protease mutants in complexes with indinavir

The molecular mechanisms of HIV drug resistance were studied using molecular dynamics simulations of HIV-1 protease complexes with the clinical inhibitor indinavir. One nanosecond molecular dynamics simulations were run for solvated complexes of indinavir with wild type protease, a control variant and 12 drug resistant mutants. The quality of the simulations was assessed by comparison with crystallographic and inhibition data. Molecular mechanisms that contribute to drug resistance include structural stability and affinity for inhibitor. The mutants showed a range of structural variation from 70 to 140% of the wild type protease. The protease affinity for indinavir was estimated by calculating the averaged molecular mechanics interaction energy. A correlation coefficient of 0.96 was obtained with observed inhibition constants for wild type and four mutants. Based on this good agreement, the trends in binding were predicted for the other mutants and discussed in relation to the clinical data for indinavir resistance. Figure Poincare map representation for WT protease-indinavir complex. The side chain of Tyr 59 showing the positions of hydrogen atoms.This revised version was published online in October 2004 with corrections to the Graphical Abstract.     

Molecular dynamics simulations investigate the pathway of substrate entry active site of rhomboid protease

Rhomboid proteases can catalyze peptide bond cleavage and participate in abundant biological processes encompassing all branches of life; however, the pathway for substrate entry into its active site remains ambiguous. Here, the two possible pathways are preliminarily determined through molecular dynamics: One pathway is between Tm2 and Tm5, and the other is between Loop3 and Loop5. Then, the umbrella sampling simulations are performed to investigate the more feasible pathway for substrate entry. The results show that free energy barriers along the two pathways are similar; in the pathway 1, Trp236 and Trp157 as pivotal residues are responsible for the rotation of substrate in the binding process; in the pathway 2, among some important residues, the residue His150 plays an important role in substrate entry. Further, combining with previous experiment results, it is concluded that the substrate is inclined to enter into the active site along pathway 2. Our results are important for further understanding the function and catalysis mechanism of rhomboid proteases.

Communicated by Ramaswamy H. Sarma     

Cell killing by HIV-1 protease

The human immunodeficiency virus protease (HIV-1 PR) was expressed both in the yeast Saccharomyces cerevisiae and in mammalian cells. Inducible expression of HIV-1 PR arrested yeast growth, which was followed by cell lysis. The lytic phenotype included loss of plasma membrane integrity and cell wall breakage leading to the release of cell content to the medium. Given that neither poliovirus 2A protease nor 2BC protein, both being highly toxic for S. cerevisiae, were able to produce similar effects, it seems that this lytic phenotype is specific of HIV-1 PR. Drastic alterations in membrane permeability preceded the lysis in yeast expressing HIV-1 PR. Cell killing and lysis provoked by HIV-1 PR were also observed in mammalian cells. Thus, COS7 cells expressing the protease showed increased plasma membrane permeability and underwent lysis by necrosis with no signs of apoptosis. Strikingly, the morphological alterations induced by HIV-1 PR in yeast and mammalian cells were similar in many aspects. To our knowledge, this is the first report of a viral protein with such an activity. These findings contribute to the present knowledge on HIV-1-induced cytopathogenesis.     

Molecular dynamics study of the connection between flap closing and binding of fullerene-based inhibitors of the HIV-1 protease

The complementary spatial relationship between fullerene C(60) and the hydrophobic cavity region of the human immunodeficiency virus (HIV) protease, which houses the active site of the enzyme, has led to the suggestion that fullerene-based derivatives could have potential use as effective HIV protease inhibitors. The ability of such compounds to desolvate the cavity region leads to a strong hydrophobic interaction between the C(60) moiety and residues in the cavity region. In this study, the connection between the motion of the so-called flexible flaps of the cavity and favorable binding of a fullerene-based protease inhibitor is explored using multiple-time scale molecular dynamics simulations and free energy techniques. In addition, the effect of the interaction between the C(60) moiety and the residues in the cavity region on the water content of the cavity is also investigated. Conformational free energy profiles along a suitably chosen flap opening coordinate show a considerable barrier to flap opening in the presence of the inhibitor, while no such barrier exists for the protease alone. This result is interpreted in terms of a strong hydrophobic interaction between the C(60) moiety and the flexible flaps, which cause the latter to close tightly around the inhibitor, thereby expelling water from the cavity and leading to a favorable binding interaction. This interpretation is rationalized by direct analysis of the water content in the cavity in the presence and absence of the inhibitor.     

Molecular dynamics simulations of early steps in RNA‐mediated conversion of prions

The rate‐limiting step in prion diseases is the initial transition of a prion protein from its native form into a mis‐folded state in which the protein not only forms cell‐toxic aggregates but also becomes infectious. Recent experiments implicate polyadenosine RNA as a possible agent for generating the initial seed. In order to understand the mechanism of RNA‐mediated mis‐folding and aggregation of prions, we dock polyadenosine RNA to mouse and human prion models. Changes in stability and secondary structure of the prions upon binding to polyadenosine RNA are evaluated by comparing molecular dynamics simulations of these complexes with that of the unbound prions.