The open structure of a multi-drug-resistant HIV-1 protease is stabilized by crystal packing contacts

The introduction of HIV-1 protease (HIV-PR) inhibitors has led to a dramatic increase in patient survival; however, these gains are threatened by the emergence of multi-drug-resistant strains. Design of inhibitors that overcome resistance would be greatly facilitated by deeper insight into the mechanistic events associated with binding of substrates and inhibitors, as well as an understanding of the effects of resistance mutations on the structure and dynamic behavior of HIV-PR. We previously reported a series of simulations that provide a model for HIV-PR dynamics, with spontaneous conversions between the bound and unbound crystal forms upon addition or removal of an inhibitor. Importantly, the unbound protease transiently sampled a third fully open state that permits entry to the active site, unlike both crystallographic forms. Recently, a crystal structure of unbound HIV-PR was reported for the MDR 769 isolate (PDB: 1TW7); unlike all previous experimental structures, the binding pocket is open. It is suggested that drug resistance in this strain arises at least in part from the inability of inhibitors to induce closing. We carried out simulations of the MDR 769 HIV-PR mutant and observed that the reported structure is unstable in solution and rapidly adopts the semi-open conformation observed for the unbound wild-type protease in solution. Further analysis suggests that the wide-open structure observed for MDR 769 arises not from sequence variation, but instead is an artifact from crystal packing. Thus, despite being the first experimental structure to reveal flap opening sufficient for substrate access to the active site, this structure may not be directly relevant to studies of inhibitor entry or to the cause of HIV-PR drug resistance.     

Insights into the dynamics of HIV-1 protease: a kinetic network model constructed from atomistic simulations

The conformational dynamics in the flaps of HIV-1 protease plays a crucial role in the mechanism of substrate binding. We develop a kinetic network model, constructed from detailed atomistic simulations, to determine the kinetic mechanisms of the conformational transitions in HIV-1 PR. To overcome the time scale limitation of conventional molecular dynamics (MD) simulations, our method combines replica exchange MD with transition path theory (TPT) to study the diversity and temperature dependence of the pathways connecting functionally important states of the protease. At low temperatures the large-scale flap opening is dominated by a small number of paths; at elevated temperatures the transition occurs through many structurally heterogeneous routes. The expanded conformation in the crystal structure 1TW7 is found to closely mimic a key intermediate in the flap-opening pathways at low temperature. We investigated the different transition mechanisms between the semi-open and closed forms. The calculated relaxation times reveal fast semi-open ? closed transitions, and infrequently the flaps fully open. The ligand binding rate predicted from this kinetic model increases by 38-fold from 285 to 309 K, which is in general agreement with experiments. To our knowledge, this is the first application of a network model constructed from atomistic simulations together with TPT to analyze conformational changes between different functional states of a natively folded protein.     

Solution structure of HIV-1 protease flaps probed by comparison of molecular dynamics simulation ensembles and EPR experiments

The introduction of multidrug treatment regimens has dramatically prolonged the progression and survival of AIDS patients. However, the success of the long-term treatment has been hindered by strains of HIV that are increasingly resistant to inhibitors of targets such as HIV protease (HIV PR). Therefore, the need for a thorough understanding of the structure and dynamics of HIV PR and how these are altered in resistant mutants is crucial for the design of more effective treatments. Crystal structures of unbound HIV PR show significant heterogeneity and often have extensive crystal packing interactions. Recent site-directed spin labeling (SDSL) and double electron-electron resonance (DEER) spectroscopy studies characterized flap conformations in HIV-1 protease in an inhibited and uninhibited form and distinguished the extent of flap opening in an unbound form. However, the correlation between EPR-measured interspin distances and structural/dynamic features of the flaps has not been established. In this report, we link EPR-based data and 900 ns of MD simulation in explicit water to gain insight into the ensemble of conformations sampled by HIV PR flaps in solution, both in the presence and in the absence of an FDA-approved HIV PR inhibitor.     

Folding processes of the B domain of protein A to the native state observed in all-atom ab initio folding simulations

Reaching the native states of small proteins, a necessary step towards a comprehensive understanding of the folding mechanisms, has remained a tremendous challenge to ab initio protein folding simulations despite the extensive effort. In this work, the folding process of the B domain of protein A (BdpA) has been simulated by both conventional and replica exchange molecular dynamics using AMBER FF03 all-atom force field. Started from an extended chain, a total of 40 conventional (each to 1.0 micros) and two sets of replica exchange (each to 200.0 ns per replica) molecular dynamics simulations were performed with different generalized-Born solvation models and temperature control schemes. The improvements in both the force field and solvent model allowed successful simulations of the folding process to the native state as demonstrated by the 0.80 A C(alpha) root mean square deviation (RMSD) of the best folded structure. The most populated conformation was the native folded structure with a high population. This was a significant improvement over the 2.8 A C(alpha) RMSD of the best nativelike structures from previous ab initio folding studies on BdpA. To the best of our knowledge, our results demonstrate, for the first time, that ab initio simulations can reach the native state of BdpA. Consistent with experimental observations, including Phi-value analyses, formation of helix II/III hairpin was a crucial step that provides a template upon which helix I could form and the folding process could complete. Early formation of helix III was observed which is consistent with the experimental results of higher residual helical content of isolated helix III among the three helices. The calculated temperature-dependent profile and the melting temperature were in close agreement with the experimental results. The simulations further revealed that phenylalanine 31 may play critical to achieve the correct packing of the three helices which is consistent with the experimental observation. In addition to the mechanistic studies, an ab initio structure prediction was also conducted based on both the physical energy and a statistical potential. Based on the lowest physical energy, the predicted structure was 2.0 A C(alpha) RMSD away from the experimentally determined structure.     

Molecular dynamics simulations of the enzyme catechol-O-methyltransferase: methodological issues

Results from extensive 70 ns all-atom molecular dynamics simulations of catechol-O-methyltransferase (COMT) enzyme are reported. The simulations were performed with explicit TIP3P water and Mg2+ ions. Four different crystal structures of COMT, with and without different ligands, were used. These simulations are among the most extensive of their kind and as such served as a stability test for such simulations. On the methodological side we found that the initial energy minimization procedure may be a crucial step: particular hydrogen bonds may break, and this can initiate an irreversible loss of protein structure that becomes observable in longer time scales of the order of tens of nanoseconds. This has important implications for both molecular dynamics and quantum mechanics-molecular mechanics simulations.     

Computational studies of darunavir into HIV-1 protease and DMPC bilayer: necessary conditions for effective binding and the role of the flaps

Human immunodeficiency virus type 1 protease (HIV-1 PR) is one of the main targets toward AIDS therapy. We have selected the potent drug darunavir and a weak inhibitor (fullerene analog) as HIV-1 PR substrates to compare protease's conformational features upon binding. Molecular dynamics (MD), molecular mechanics Poisson-Boltzmann surface area (MM-PBSA), and quantum-mechanical (QM) calculations indicated the importance of the stability of HIV-1 PR flaps toward effective binding: a weak inhibitor may induce flexibility to the flaps, which convert between closed and semiopen states. A water molecule in the darunavir-HIV-1 PR complex bridged the two flap tips of the protease through hydrogen bonding (HB) interactions in a stable structure, a feature that was not observed for the fullerene-HIV-1 PR complex. Additionally, despite that van der Waals interactions and nonpolar contribution to solvation favored permanent fullerene entrapment into the cavity, these interactions alone were not sufficient for effective binding; enhanced electrostatic interactions as observed in the darunavir-complex were the crucial component of the binding energy. An alternative pathway to the usual way of a ligand to access the cavity was also observed for both compounds. Each ligand entered the binding cavity through an opening between the one flap of the protease and a neighboring loop. This suggested that access to the cavity is not necessarily regulated by flap opening. Darunavir exerts its biological action inside the cell, after crossing the membrane barrier. Thus, we also initiated a study on the interactions between darunavir and the DMPC bilayer to reveal that the drug was accommodated inside the bilayer in conformations that resembled its structure into HIV-1 PR, being stabilized via HBs with the lipids and water molecules.     

Hydrophobic core flexibility modulates enzyme activity in HIV-1 protease

Human immunodeficiency virus Type-1 (HIV-1) protease is crucial for viral maturation and infectivity. Studies of protease dynamics suggest that the rearrangement of the hydrophobic core is essential for enzyme activity. Many mutations in the hydrophobic core are also associated with drug resistance and may modulate the core flexibility. To test the role of flexibility in protease activity, pairs of cysteines were introduced at the interfaces of flexible regions remote from the active site. Disulfide bond formation was confirmed by crystal structures and by alkylation of free cysteines and mass spectrometry. Oxidized and reduced crystal structures of these variants show the overall structure of the protease is retained. However, cross-linking the cysteines led to drastic loss in enzyme activity, which was regained upon reducing the disulfide cross-links. Molecular dynamics simulations showed that altered dynamics propagated throughout the enzyme from the engineered disulfide. Thus, altered flexibility within the hydrophobic core can modulate HIV-1 protease activity, supporting the hypothesis that drug resistant mutations distal from the active site can alter the balance between substrate turnover and inhibitor binding by modulating enzyme activity.     

Replica exchange Monte-Carlo simulations of helix bundle membrane proteins: rotational parameters of helices

We propose a united-residue model of membrane proteins to investigate the structures of helix bundle membrane proteins (HBMPs) using coarse-grained (CG) replica exchange Monte-Carlo (REMC) simulations. To demonstrate the method, it is used to identify the ground state of HBMPs in a CG model, including bacteriorhodopsin (BR), halorhodopsin (HR), and their subdomains. The rotational parameters of transmembrane helices (TMHs) are extracted directly from the simulations, which can be compared with their experimental measurements from site-directed dichroism. In particular, the effects of amphiphilic interaction among the surfaces of TMHs on the rotational angles of helices are discussed. The proposed CG model gives a reasonably good structure prediction of HBMPs, as well as a clear physical picture for the packing, tilting, orientation, and rotation of TMHs. The root mean square deviation (RMSD) in coordinates of C_α atoms of the ground state CG structure from the X-ray structure is 5.03 Å for BR and 6.70 Å for HR. The final structure of HBMPs is obtained from the all-atom molecular dynamics simulations by refining the predicted CG structure, whose RMSD is 4.38 Å for BR and 5.70 Å for HR.     

Molecular dynamics simulations of the enzyme Catechol-O-Methyltransferase: methodological issues1

Results from extensive 70 ns all-atom molecular dynamics simulations of catechol-O-methyltransferase (COMT) enzyme are reported. The simulations were performed with explicit TIP3P water and Mg^2?+ ions. Four different crystal structures of COMT, with and without different ligands, were used. These simulations are among the most extensive of their kind and as such served as a stability test for such simulations. On the methodological side we found that the initial energy minimization procedure may be a crucial step: particular hydrogen bonds may break, and this can initiate an irreversible loss of protein structure that becomes observable in longer time scales of the order of tens of nanoseconds. This has important implications for both molecular dynamics and quantum mechanics–molecular mechanics simulations.     

All-atom structure prediction and folding simulations of a stable protein

We present results from all-atom, fully unrestrained ab initio folding simulations for a stable protein with nontrivial secondary structure elements and a hydrophobic core. The construct, "trpcage", is a 20-residue sequence optimized by the Andersen group at University of Washington and is currently the smallest protein that displays two-state folding properties. Compared over the well-defined regions of the experimental structure, our prediction has a remarkably low 0.97 A Calpha root-mean-square-deviation (rmsd) and 1.4 A for all heavy atoms. The simulated structure family displays additional features that are suggested by experimental data, yet are not evident in the family of NMR-derived structures.     

Molecular dynamics simulation of papain-E-64 (N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]agmatine) complex

To investigate the possible binding mode of E-64 (N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]agmatine), a potent cysteine protease inhibitor, to papain active site, molecular dynamics simulations were applied to two complex forms: R- and S- configurational forms of E-64 C2 atom for the covalent bond formation with the papain Cys-25 SH group. The tertiary structures of the papain-E-64 complexes were built by visual interactive modelling and the energy minimization technique, and were subjected to the dynamics simulations of 10 ps. Although no significant difference was observed between the potential energies of energy-minimized R- and S-complex forms, the molecular dynamics simulations suggested that the hydrogen bonding mode of the former form is more advantageous than that of the latter one. Comparing with the hydrogen bonds observed in the papain-E-64 complex crystal, it could be concluded that the present molecular dynamics simulation reflects well the three-dimensional structure concerning the interaction of E-64 with the papain active site. The conformational characteristics of E-64 and its possible interaction mode with papain were also discussed.     

The influence of macromolecular crowding on HIV-1 protease internal dynamics

High macromolecular concentrations, or crowded conditions, have been shown to affect a wide variety of molecular processes, including diffusion, association and dissociation, and protein folding and stability. Here, we model the effect of macromolecular crowding on the internal dynamics of a protein, HIV-1 protease, using Brownian dynamics simulations. HIV-1 protease possesses a pair of flaps which are postulated to open in the early stages of its catalytic mechanism. Compared to low concentrations, close-packed concentrations of repulsive crowding agents are found to significantly reduce the fraction of time that the protease flaps are open. Macromolecular crowding is likely to have a major effect on in vivo enzyme activity, and may play an important regulatory role in the viral life cycle.     

Implementation of a protein reduced point charge model toward molecular dynamics applications

A reduced point charge model was developed in a previous work from the study of extrema in smoothed charge density distribution functions generated from the Amber99 molecular electrostatic potential. In the present work, such a point charge distribution is coupled with the Amber99 force field and implemented in the program TINKER to allow molecular dynamics (MD) simulations of proteins. First applications to two polypeptides that involve α-helix and β-sheet motifs are analyzed and compared to all-atom MD simulations. Two types of coarse-grained (CG)-based trajectories are generated using, on one hand, harmonic bond stretching terms and, on the other hand, distance restraints. Results show that the use of the unrestrained CG conditions are sufficient to preserve most of the secondary structure characteristics but restraints lead to a better agreement between CG and all-atom simulation results such as rmsd, dipole moment, and time-dependent mean square deviation functions.     

Binding of low molecular weight inhibitors promotes large conformational changes in the dengue virus NS2B-NS3 protease: fold analysis by pseudocontact shifts

The two-component dengue virus NS2B-NS3 protease (DEN NS2B-NS3pro) is an established drug target, but inhibitor design is hampered by the lack of a crystal structure of the protease in its fully active form. In solution and without inhibitors, the functionally important C-terminal segment of the NS2B cofactor is dissociated from DEN NS3pro ("open state"), necessitating a large structural change to produce the "closed state" thought to underpin activity. We analyzed the fold of DEN NS2B-NS3pro in solution with and without bound inhibitor by nuclear magnetic resonance (NMR) spectroscopy. Multiple paramagnetic lanthanide tags were attached to different sites to generate pseudocontact shifts (PCS). In the face of severe spectral overlap and broadening of many signals by conformational exchange, methods for assignment of (15)N-HSQC cross-peaks included selective mutation, combinatorial isotope labeling, and comparison of experimental PCSs and PCSs back-calculated for a structural model of the closed conformation built by using the structure of the related West Nile virus (WNV) protease as a template. The PCSs show that, in the presence of a positively charged low-molecular weight inhibitor, the enzyme assumes a closed state that is very similar to the closed state previously observed for the WNV protease. Therefore, a model of the protease built on the closed conformation of the WNV protease is a better template for rational drug design than available crystal structures, at least for positively charged inhibitors. To assess the open state, we created a binding site for a Gd(3+) complex and measured paramagnetic relaxation enhancements. The results show that the specific open conformation displayed in the crystal of DEN NS2B-NS3pro is barely populated in solution. The techniques used open an avenue to the fold analysis of proteins that yield poor NMR spectra, as PCSs from multiple sites in combination with model building generate powerful information even from incompletely assigned (15)N-HSQC spectra.     

All-atom modeling of anisotropic atomic fluctuations in protein crystal structures

The accurate modeling of protein dynamics in crystalline states is essential for the development of computational techniques for simulating protein dynamics under physiological conditions. Following a previous coarse-grained modeling study of atomic fluctuations in protein crystal structures, we have refined our modeling with all-atom representation and force field. We have calculated the anisotropic atomic fluctuations of a protein structure interacting with its crystalline environment either explicitly (by including neighboring proteins into modeling) or implicitly (by adding harmonic restraints to surface atoms involved in crystal contacts). The modeling results are assessed in comparison with the experimental anisotropic displacement parameters (ADP) determined by X-ray crystallography. For a list of 40 high-resolution protein crystal structures, we have found that the optimal modeling of ADPs is achieved when the protein-environment interactions are much weaker than the internal interactions within a protein structure. Therefore, the intrinsic dynamics of a protein structure is only weakly perturbed by crystal packing. We have also found no noticeable improvement in the accuracy of ADP modeling by using all-atom over coarse-grained representation and force field, which justifies the use of coarse-grained modeling to investigate protein dynamics with both efficiency and accuracy.     

Generalized-ensemble simulations of the human parathyroid hormone fragment PTH(1-34)

A generalized-ensemble technique, multicanonical sampling, is used to study the folding of a 34-residue human parathyroid hormone fragment. An all-atom model of the peptide is employed and the protein-solvent interactions are approximated by an implicit solvent. Our results demonstrate that generalized-ensemble simulations are well suited to sample low-energy structures of such large polypeptides. Configurations with a root-mean-square deviation to the crystal structure of less than 1 A are found. Finally, we discuss limitations of our implicit solvent model.     

Q-Dock: Low-resolution flexible ligand docking with pocket-specific threading restraints

The rapidly growing number of theoretically predicted protein structures requires robust methods that can utilize low-quality receptor structures as targets for ligand docking. Typically, docking accuracy falls off dramatically when apo or modeled receptors are used in docking experiments. Low-resolution ligand docking techniques have been developed to deal with structural inaccuracies in predicted receptor models. In this spirit, we describe the development and optimization of a knowledge-based potential implemented in Q-Dock, a low-resolution flexible ligand docking approach. Self-docking experiments using crystal structures reveals satisfactory accuracy, comparable with all-atom docking. All-atom models reconstructed from Q-Dock's low-resolution models can be further refined by even a simple all-atom energy minimization. In decoy-docking against distorted receptor models with a root-mean-square deviation, RMSD, from native of approximately 3 A, Q-Dock recovers on average 15-20% more specific contacts and 25-35% more binding residues than all-atom methods. To further improve docking accuracy against low-quality protein models, we propose a pocket-specific protein-ligand interaction potential derived from weakly homologous threading holo-templates. The success rate of Q-Dock employing a pocket-specific potential is 6.3 times higher than that previously reported for the Dolores method, another low-resolution docking approach.     

Molecular dynamics with the united-residue model of polypeptide chains. II. Langevin and Berendsen-bath dynamics and tests on model alpha-helical systems

The implementation of molecular dynamics (MD) with our physics-based protein united-residue (UNRES) force field, described in the accompanying paper, was extended to Langevin dynamics. The equations of motion are integrated by using a simplified stochastic velocity Verlet algorithm. To compare the results to those with all-atom simulations with implicit solvent in which no explicit stochastic and friction forces are present, we alternatively introduced the Berendsen thermostat. Test simulations on the Ala(10) polypeptide demonstrated that the average kinetic energy is stable with about a 5 fs time step. To determine the correspondence between the UNRES time step and the time step of all-atom molecular dynamics, all-atom simulations with the AMBER 99 force field and explicit solvent and also with implicit solvent taken into account within the framework of the generalized Born/surface area (GBSA) model were carried out on the unblocked Ala(10) polypeptide. We found that the UNRES time scale is 4 times longer than that of all-atom MD simulations because the degrees of freedom corresponding to the fastest motions in UNRES are averaged out. When the reduction of the computational cost for evaluation of the UNRES energy function is also taken into account, UNRES (with hydration included implicitly in the side chain-side chain interaction potential) offers about at least a 4000-fold speed up of computations relative to all-atom simulations with explicit solvent and at least a 65-fold speed up relative to all-atom simulations with implicit solvent. To carry out an initial full-blown test of the UNRES/MD approach, we ran Berendsen-bath and Langevin dynamics simulations of the 46-residue B-domain of staphylococcal protein A. We were able to determine the folding temperature at which all trajectories converged to nativelike structures with both approaches. For comparison, we carried out ab initio folding simulations of this protein at the AMBER 99/GBSA level. The average CPU time for folding protein A by UNRES molecular dynamics was 30 min with a single Alpha processor, compared to about 152 h for all-atom simulations with implicit solvent. It can be concluded that the UNRES/MD approach will enable us to carry out microsecond and, possibly, millisecond simulations of protein folding and, consequently, of the folding process of proteins in real time.