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 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.     

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 of the first steps of the reaction catalyzed by HIV-1 protease

The mechanism of the first steps of the reaction catalyzed by HIV-1 protease was studied through molecular dynamics simulations. The potential energy surface in the active site was generated using the approximate valence bond method. The approximate valence bond (AVB) method was parameterized based on density functional calculations. The surrounding protein and explicit water environment was modeled with conventional, classical force field. The calculations were performed based on HIV-1 protease complexed with the MVT-101 inhibitor that was modified to a model substrate. The protonation state of the catalytic aspartates was determined theoretically. Possible reaction mechanisms involving the lytic water molecule are accounted for in this study. The modeled steps include the dissociation of the lytic water molecule and proton transfer onto Asp-125, the nucleophilic attack followed by a proton transfer onto peptide nitrogen. The simulations show that in the active site most preferable energetically are structures consisting of ionized or polarized molecular fragments that are not accounted for in conventional molecular dynamics. The mobility of the lytic water molecule, the dynamics of the hydrogen bond network, and the conformation of the aspartates in the active center were analyzed.     

Interchain hydrophobic clustering promotes rigidity in HIV-1 protease flap dynamics: new insights from Molecular Dynamics

The dynamics of HIV-1 protease (HIV-pr), a drug target for HIV infection, has been studied extensively by both computational and experimental methods. The flap dynamics of HIV-pr is considered to be more important for better ligand binding and enzymatic actions. Moreover, it has been demonstrated that the drug-induced mutations can change the flap dynamics of HIV-pr affecting the binding affinity of the ligands. Therefore, detailed understanding of flap dynamics is essential for designing better inhibitors. Previous computational investigations observed significant variation in the flap opening in nanosecond time scale indicating that the dynamics is highly sensitive to the simulation protocols. To understand the sensitivity of the flap dynamics on the force field and simulation protocol, molecular dynamics simulations of HIV-pr have been performed with two different AMBER force fields, ff99 and ff02. Two different trajectories (20?ns each) were obtained using the ff99 and ff02 force field. The results showed polarizable force field (ff02) make the flap tighter than the nonpolarizable force field (ff99). Some polar interactions and hydrogen bonds involving flap residues were found to be stronger with ff02 force field. The formation of interchain hydrophobic cluster (between flap tip of one chain and active site wall of another chain) was found to be dominant in the semi-open structures obtained from the simulations irrespective of the force field. It is proposed that an inhibitor, which will promote this interchain hydrophobic clustering, may make the flaps more rigid, and presumably the effect of mutation would be small on ligand binding.     

Closing of the flaps of HIV-1 protease induced by substrate binding: a model of a flap closing mechanism in retroviral aspartic proteases

The active site of aspartic proteases is covered by one or more flaps, which control access to the active site and play a significant role in the binding of the substrate. An extensive conformational change of the flaps takes place upon binding of substrate to the active site. A long molecular dynamics simulation was performed on the complex consisting of a peptide (CA-p2) from a natural substrate cleavage site of the gag/pol polyprotein placed in the active site of HIV-1 protease (PR) with an open flap conformation. During the simulation, the substrate induced the closing of the flaps into the closed conformation in an asymmetrical way through a hydrophobic intermediate state cluster. The nature of the residues of HIV-1 PR identified to be important in the flap closing mechanism is conserved across known structures of retroviral aspartic proteases family. The flap closing mechanism described in HIV-1 PR is proposed to be a general model for flap closing in retroviral aspartic proteases.     

X-ray crystallographic structure of ABT-378 (lopinavir) bound to HIV-1 protease

The crystal structure of ABT-378 (lopinavir), bound to the active site of HIV-1 protease is described. A comparison with crystal structures of ritonavir, A-78791, and BILA-2450 shows some analogous features with previous reported compounds. A cyclic urea unit in the P(2) position of ABT-378 is novel and makes two bidentate hydrogen bonds with Asp 29 of HIV-1 protease. In addition, a previously unreported shift in the Gly 48 carbonyl position is observed. A discussion of the structural features responsible for its high potency against wild-type HIV protease is given along with an analysis of the effect of active site mutations on potency in in vitro assays.     

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.     

X-ray structure and conformational dynamics of the HIV-1 protease in complex with the inhibitor SDZ283-910: agreement of time-resolved spectroscopy and molecular dynamics simulations

Based on the X-ray structure of the human immunodeficiency virus type-1 (HIV-1) protease in complex with the statine-derived inhibitor SDZ283-910, a 542 ps molecular dynamics trajectory was computed. For comparison with the 805 ps trajectory obtained for the uncomplexed enzyme, the theoretical fluorescence anisotropy decay of the unliganded protease and the inhibitor complex was calculated from the trajectories of the Trp6A/Trp6B and Trp42A/Trp42B transition dipole moments. This enabled us to directly compare the simulated data with the experimental picosecond time-resolved fluorescence data. Fitting both experimental and simulated data to the Kohlrausch-Williams-Watts (KWW) function exp(-t/tauk)beta revealed a very good agreement for the uncomplexed protease as well as for the SDZ283-910 complex. Binding of the inhibitor induced a faster decay of both the experimental and the computed protease fluorescence anisotropy decay. By this integrative approach, the atomic detail of inhibitor-induced changes in the conformational dynamics of the HIV-1 protease was experimentally verified and will be used for further inhibitor optimisation.     

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 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 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 simulations of protein-tyrosine phosphatase 1B. I. ligand-induced changes in the protein motions.

Activity of enzymes, such as protein tyrosine phosphatases (PTPs), is often associated with structural changes in the enzyme, resulting in selective and stereospecific reactions with the substrate. To investigate the effect of a substrate on the motions occurring in PTPs, we have performed molecular dynamics simulations of PTP1B and PTP1B complexed with a high-affinity peptide DADEpYL, where pY stands for phosphorylated tyrosine. The peptide sequence is derived from the epidermal growth factor receptor (EGFR988-993). Simulations were performed in water for 1 ns, and the concerted motions in the protein were analyzed using the essential dynamics technique. Our results indicate that the predominately internal motions in PTP1B occur in a subspace of only a few degrees of freedom. Upon substrate binding, the flexibility of the protein is reduced by approximately 10%. The largest effect is found in the protein region, where the N-terminal of the substrate is located, and in the loop region Val198-Gly209. Displacements in the latter loop are associated with the motions in the WPD loop, which contains a catalytically important aspartic acid. Estimation of the pKa of the active-site cysteine along the trajectory indicates that structural inhomogeneity causes the pKa to vary by approximately +/-1 pKa unit. In agreement with experimental observations, the active-site cysteine is negatively charged at physiological pH.     

Comparison of inhibitor binding in HIV-1 protease and in non-viral aspartic proteases: the role of the flap

The crystal structure of HIV-1 protease with an inhibitor has been compared with the structures of non-viral aspartic proteases complexed with inhibitors. In the dimeric HIV-1 protease, two 4-stranded beta-sheets are formed by half of the inhibitor, residues 27-29, and the flap from each monomer. In the monomeric non-viral enzyme the single flap does not form a beta-sheet with an inhibitor. The HIV-1 protease shows more interactions with a longer peptide inhibitor than are observed in non-viral aspartic protease-inhibitor complexes. This, and the large movement of the flaps, restricts the conformation of the protease cleavage sites in the retroviral polyprotein precursor.     

Molecular dynamics simulations of "loop closing" in the enzyme triose phosphate isomerase

We present molecular dynamics simulations on the active site region of dimeric triose phosphate isomerase (TIM) using the co-ordinates of native chicken muscle TIM as a starting point and performing simulations with no substrate, with dihydroxyacetone phosphate (DHAP), the natural substrate, and with dihydroxyacetone sulfate (DHAS), a substrate analog. Whereas most of the protein moves less than 1 A during the simulation, some residues in the active site loop move more than 8 A during the 10.5 picoseconds of dynamics for each of the simulations. Most interestingly, the nature of the loop motion depends on the substrate, with the largest motion found in the presence of DHAP, and only in the presence of DHAP does the loop move to "close off" the active site pocket. The final structure found for the DHAP-chicken TIM complex is qualitatively similar to that described by Alber et al. for DHAP-yeast TIM. Simulations on the monomeric protein gives insight into why the molecule is active only as a dimer.     

Molecular dynamics studies on HIV-1 protease: a comparison of the flap motions between wild type protease and the M46I/G51D double mutant

The emergence of drug-resistant mutants of HIV-1 is a tragic effect associated with conventional long-treatment therapies against acquired immunodeficiency syndrome. These mutations frequently involve the aspartic protease encoded by the virus; knowledge of the molecular mechanisms underlying the conformational changes of HIV-1 protease mutants may be useful in developing more effective and longer lasting treatment regimes. The flap regions of the protease are the target of a particular type of mutations occurring far from the active site. These mutations modify the affinity for both substrate and ligands, thus conferring resistance. In this work, molecular dynamics simulations were performed on a native wild type HIV-1 protease and on the drug-resistant M46I/G51D double mutant. The simulation was carried out for a time of 3.5 ns using the GROMOS96 force field, with implementation of the SPC216 explicit solvation model. The results show that the flaps may exist in an ensemble of conformations between a “closed” and an “open” conformation. The behaviour of the flap tips during simulations is different between the native enzyme and the mutant. The mutation pattern leads to stabilization of the flaps in a semi-open configuration.     

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.     

X-ray structure of HIV-1 protease in situ product complex

HIV-1 protease is an effective target for design of different types of drugs against AIDS. HIV-1 protease is also one of the few enzymes that can cleave substrates containing both proline and nonproline residues at the cleavage site. We report here the first structure of HIV-1 protease complexed with the product peptides SQNY and PIV derived by in situ cleavage of the oligopeptide substrate SQNYPIV, within the crystals. In the structure, refined against 2.0-A resolution synchrotron data, a carboxyl oxygen of SQNY is hydrogen-bonded with the N-terminal nitrogen atom of PIV. At the same time, this proline nitrogen atom does not form any hydrogen bond with catalytic aspartates. These two observations suggest that the protonation of scissile nitrogen, during peptide bond cleavage, is by a gem-hydroxyl of the tetrahedral intermediate rather than by a catalytic aspartic acid.     

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.