A diverse view of protein dynamics from NMR studies of HIV-1 protease flaps

Internal motion in proteins fulfills a multitude of roles in biological processes. NMR spectroscopy has been applied to elucidate protein dynamics at the atomic level, albeit at a low resolution, and is often complemented by molecular dynamics simulation. However, it is critical to justify the consistency between simulation results and conclusions often drawn from multiple experiments in which uncertainties arising from assumed motional models may not be explicitly evaluated. To understand the role of the flaps of HIV-1 protease dimer in substrate recognition and protease function, many molecular dynamics simulations have been performed. The simulations have resulted in various proposed models of the flap dynamics, some of which are more consistent than others with our working model previously derived from experiments. However, using the working model to discriminate among the simulation results is not straightforward because the working model was derived from a combination of NMR experiments and crystal structure data. In this study, we use the NMR chemical shifts and relaxation data of the protease "monomer" rather than structural data to narrow down the possible conformations of the flaps of the "dimer". For the first time, we show that the tips of the flaps in the unliganded protease dimer interact with each other in solution. Accordingly, we discuss the consistency of the simulations with the model derived from all experimental data.     

HIV-1 protease substrate binding and product release pathways explored with coarse-grained molecular dynamics

We analyze the encounter of a peptide substrate with the native HIV-1 protease, the mechanism of substrate incorporation in the binding cleft, and the dissociation of products after substrate hydrolysis. To account for the substrate, we extend a coarse-grained model force field, which we previously developed to study the flap opening dynamics of HIV-1 protease on a microsecond timescale. Molecular and Langevin dynamics simulations show that the flaps need to open for the peptide to bind and that the protease interaction with the substrate influences the flap opening frequency and interval. On the other hand, release of the products does not require flap opening because they can slide out from the binding cleft to the sides of the enzyme. Our data show that in the protease-substrate complex the highest fluctuations correspond to the 17- and 39-turns and the substrate motion is anticorrelated with the 39-turn. Moreover, the active site residues and the flap tips move in phase with the peptide. We suggest some mechanistic principles for how the flexibility of the protein may be involved in ligand binding and release.     

Relation between sequence and structure of HIV-1 protease inhibitor complexes: a model system for the analysis of protein flexibility.

The flexibility of different regions of HIV-1 protease was examined by using a database consisting of 73 X-ray structures that differ in terms of sequence, ligands or both. The root-mean-square differences of the backbone for the set of structures were shown to have the same variation with residue number as those obtained from molecular dynamics simulations, normal mode analyses and X-ray B-factors. This supports the idea that observed structural changes provide a measure of the inherent flexibility of the protein, although specific interactions between the protease and the ligand play a secondary role. The results suggest that the potential energy surface of the HIV-1 protease is characterized by many local minima with small energetic differences, some of which are sampled by the different X-ray structures of the HIV-1 protease complexes. Interdomain correlated motions were calculated from the structural fluctuations and the results were also in agreement with molecular dynamics simulations and normal mode analyses. Implications of the results for the drug-resistance engendered by mutations are discussed briefly.     

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.     

Structural insights into the South African HIV-1 subtype C protease: impact of hinge region dynamics and flap flexibility in drug resistance

The HIV protease plays a major role in the life cycle of the virus and has long been a target in antiviral therapy. Resistance of HIV protease to protease inhibitors (PIs) is problematic for the effective treatment of HIV infection. The South African HIV-1 subtype C protease (C-SA PR), which contains eight polymorphisms relative to the consensus HIV-1 subtype B protease, was expressed in Escherichia coli, purified, and crystallized. The crystal structure of the C-SA PR was resolved at 2.7?Å, which is the first crystal structure of a HIV-1 subtype C protease that predominates in Africa. Structural analyses of the C-SA PR in comparison to HIV-1 subtype B proteases indicated that polymorphisms at position 36 of the homodimeric HIV-1 protease may impact on the stability of the hinge region of the protease, and hence the dynamics of the flap region. Molecular dynamics simulations showed that the flap region of the C-SA PR displays a wider range of movements over time as compared to the subtype B proteases. Reduced stability in the hinge region resulting from the absent E35-R57 salt bridge in the C-SA PR, most likely contributes to the increased flexibility of the flaps which may be associated with reduced susceptibility to PIs.

An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:36     

HIV-1 protease molecular dynamics of a wild-type and of the V82F/I84V mutant: possible contributions to drug resistance and a potential new target site for drugs

The protease from type 1 human immunodeficiency virus (HIV-1) is a critical drug target against which many therapeutically useful inhibitors have been developed; however, the set of viral strains in the population has been shifting to become more drug-resistant. Because indirect effects are contributing to drug resistance, an examination of the dynamic structures of a wild-type and a mutant could be insightful. Consequently, this study examined structural properties sampled during 22 nsec, all atom molecular dynamics (MD) simulations (in explicit water) of both a wild-type and the drug-resistant V82F/I84V mutant of HIV-1 protease. The V82F/I84V mutation significantly decreases the binding affinity of all HIV-1 protease inhibitors currently used clinically. Simulations have shown that the curling of the tips of the active site flaps immediately results in flap opening. In the 22-nsec MD simulations presented here, more frequent and more rapid curling of the mutant's active site flap tips was observed. The mutant protease's flaps also opened farther than the wild-type's flaps did and displayed more flexibility. This suggests that the effect of the mutations on the equilibrium between the semiopen and closed conformations could be one aspect of the mechanism of drug resistance for this mutant. In addition, correlated fluctuations in the active site and periphery were noted that point to a possible binding site for allosteric inhibitors.     

Cooperative fluctuations of unliganded and substrate-bound HIV-1 protease: a structure-based analysis on a variety of conformations from crystallography and molecular dynamics simulations

The dynamics of HIV-1 protease, both in unliganded and substrate-bound forms have been analyzed by using an analytical method, Gaussian network model (GNM). The method is applied to different conformations accessible to the protein backbone in the native state, observed in crystal structures and snapshots from fully atomistic molecular dynamics (MD) simulation trajectories. The modes of motion obtained from GNM on different conformations of HIV-1 protease are conserved throughout the MD simulations. The flaps and 40's loop of the unliganded HIV-1 protease structure are identified as the most mobile regions. However, in the liganded structure these flaps lose mobility, and terminal regions of the monomers become more flexible. Analysis of the fast modes shows that residues important for stability are in the same regions of all the structures examined. Among these, Gly86 appears to be a key residue for stability. The contribution of residues in the active site region and flaps to the stability is more pronounced in the substrate-bound form than in the unliganded form. The convergence of modes in GNM to similar regions of HIV-1 protease, regardless of the conformation of the protein, supports the robustness of GNM as a potentially useful and predictive tool.     

HIV-1 dynamics at different time scales under antiretroviral therapy

We exploit a model that considers three compartments: blood plasma (BP), lymphoid tissue-interstitial spaces (LT-IS), and follicular dendritic cells (FDC), for the HIV-1 dynamics under the application of highly active antiretroviral therapy (HAART) which allowed us to unravel distinct viral dynamics occurring in short- (2 days), middle- (21 days), and long-term (183 days) time scales. The different time scales are determined by the viral clearance rate, the ratio of productively infected CD4⁺ T cells to chronically infected cells, and the dissociation rate of HIV-1 complexes from FDC. This generates a scenario in which, after an initial transient stage, the viral BP dynamics decouples and becomes governed by the lymphoid tissue (LT) dynamics; in a later stage, a new decoupling occurs in which the LT-IS dynamics is slaved to that of the FDC dynamics. We observed an initial increase in the viremia after HAART in a patient who did not receive protease inhibitors (PI). By means of the above-mentioned model we were able to highlight the relevant parameters which need to be estimated at three different time scales after HAART.     

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.     

The denatured state of HIV-1 protease under native conditions

The denatured state of several proteins has been shown to display transient structures that are relevant for folding, stability, and aggregation. To detect them by nuclear magnetic resonance (NMR) spectroscopy, the denatured state must be stabilized by chemical agents or changes in temperature. This makes the environment different from that experienced in biologically relevant processes. Using high-resolution heteronuclear NMR spectroscopy, we have characterized several denatured states of a monomeric variant of HIV-1 protease, which is natively structured in water, induced by different concentrations of urea, guanidinium chloride, and acetic acid. We have extrapolated the chemical shifts and the relaxation parameters to the denaturant-free denatured state at native conditions, showing that they converge to the same values. Subsequently, we characterized the conformational properties of this biologically relevant denatured state under native conditions by advanced molecular dynamics simulations and validated the results by comparison to experimental data. We show that the denatured state of HIV-1 protease under native conditions displays rich patterns of transient native and non-native structures, which could be of relevance to its guidance through a complex folding process.     

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.     

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.     

Insights into the functional role of protonation states in the HIV-1 protease-BEA369 complex: molecular dynamics simulations and free energy calculations

The molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method combined with molecular dynamics (MD) simulations were used to investigate the functional role of protonation in human immunodeficiency virus type 1 (HIV-1) protease complexed with the inhibitor BEA369. Our results demonstrate that protonation of two aspartic acids (Asp25/Asp25′) has a strong influence on the dynamics behavior of the complex, the binding free energy of BEA369, and inhibitor–residue interactions. Relative binding free energies calculated using the MM-PBSA method show that protonation of Asp25 results in the strongest binding of BEA369 to HIV-1 protease. Inhibitor–residue interactions computed by the theory of free energy decomposition also indicate that protonation of Asp25 has the most favorable effect on binding of BEA369. In addition, hydrogen-bond analysis based on the trajectories of the MD simulations shows that protonation of Asp25 strongly influences the water-mediated link of a conserved water molecule, Wat301. We expect that the results of this study will contribute significantly to binding calculations for BEA369, and to the design of high affinity inhibitors.     

Simulating nanoscale functional motions of biomolecules

We are describing efficient dynamics simulation methods for the characterization of functional motion of biomolecules on the nanometer scale. Multivariate statistical methods are widely used to extract and enhance functional collective motions from molecular dynamics (MD) simulations. A dimension reduction in MD is often realized through a principal component analysis (PCA) or a singular value decomposition (SVD) of the trajectory. Normal mode analysis (NMA) is a related collective coordinate space approach, which involves the decomposition of the motion into vibration modes based on an elastic model. Using the myosin motor protein as an example we describe a hybrid technique termed amplified collective motions (ACM) that enhances sampling of conformational space through a combination of normal modes with atomic level MD. Unfortunately, the forced orthogonalization of modes in collective coordinate space leads to complex dependencies that are not necessarily consistent with the symmetry of biological macromolecules and assemblies. In many biological molecules, such as HIV-1 protease, reflective or rotational symmetries are present that are broken using standard orthogonal basis functions. We present a method to compute the plane of reflective symmetry or the axis of rotational symmetry from the trajectory frames. Moreover, we develop an SVD that best approximates the given trajectory while respecting the symmetry. Finally, we describe a local feature analysis (LFA) to construct a topographic representation of functional dynamics in terms of local features. The LFA representations are low-dimensional, and provide a reduced basis set for collective motions, but unlike global collective modes they are sparsely distributed and spatially localized. This yields a more reliable assignment of essential dynamics modes across different MD time windows.     

Dynamic flaps in HIV-1 protease adopt unique ordering at different stages in the catalytic cycle

The flexibility of HIV-1 protease flaps is known to be essential for the enzymatic activity. Here we attempt to capture a multitude of conformations of the free and substrate-bound HIV-1 protease that differ drastically in their flap arrangements. The substrate binding process suggests the opening of active site gate in conjunction with a reversal of flap tip ordering, from the native semiopen state. The reversed-flap, open-gated enzyme readily transforms to a closed conformation after proper placement of the substrate into the binding cleft. After substrate processing, the closed state protease which possessed opposite flap ordering relative to the semiopen state, encounters another flap reversal via a second open conformation that facilitates the evolution of native semiopen state of correct flap ordering. The complicated transitional pathway, comprising of many high and low energy states, is explored by combining standard and activated molecular dynamics (MD) simulation techniques. The study not only complements the existing findings from X-ray, NMR, EPR, and MD studies but also provides a wealth of detailed information that could help the structure-based drug design process.     

A molecular dynamics study of the structural stability of HIV-1 protease under physiological conditions: the role of Na+ ions in stabilizing the active site

HIV-1 protease is most active under weakly acidic conditions (pH 3.5-6.5), when the catalytic Asp25 and Asp25' residues share 1 proton. At neutral pH, this proton is lost and the stability of the structure is reduced. Here we present an investigation of the effect of pH on the dynamics of HIV-1 protease using MD simulation techniques. MD simulations of the solvated HIV-1 protease with the Asp25/25' residues monoprotonated and deprotonated have been performed. In addition we investigated the effect of the inclusion of Na(+) and Cl(-) ions to mimic physiological salt conditions. The simulations of the monoprotonated form and deprotonated form including Na(+) show very similar behavior. In both cases the protein remained stable in the compact, "self-blocked" conformation in which the active site is blocked by the tips of the flaps. In the deprotonated system a Na(+) ion binds tightly to the catalytic dyad shielding the repulsion between the COO(-) groups. Ab initio calculations also suggest the geometry of the active site with the Na(+) bound closely resembles that of the monoprotonated case. In the simulations of the deprotonated form (without Na(+) ions), a water molecule bound between the Asp25 Asp25' side-chains. This disrupted the dimerization interface and eventually led to a fully open conformation.     

Clarifying allosteric control of flap conformations in the 1TW7 crystal structure of HIV-1 protease

The 1TW7 crystal structure of HIV-1 protease shows the flaps placed wider and more open than what is seen in other examples of the semi-open, apo form. It has been proposed that this might be experimental evidence of allosteric control, because crystal packing creates contacts to the "elbow region" of the protease, which may cause deformation of the flaps. Recent dynamics simulations have shown that the conformation seen in 1TW7 relaxes into the typical semi-open conformation in the absence of the crystal contacts, definitively showing that the crystal contacts cause the deformation (Layten et al., J Am Chem Soc 2006;128:13360-13361). However, this does not prove or disprove allosteric modulation at the elbow. In this study, we have conducted additional simulations, supplemented with experimental testing, to further probe the possibility of 1TW7 providing an example of allosteric control of the flap region. We show that the contacts are unstable and do not restrict the conformational sampling of the flaps. The deformation seen in the 1TW7 crystal structure is simply opportunistic crystal packing and not allosteric control.     

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.