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.     

Solution structure of the mature HIV-1 protease monomer: insight into the tertiary fold and stability of a precursor

We present the first solution structure of the HIV-1 protease monomer spanning the region Phe1-Ala95 (PR1-95). Except for the terminal regions (residues 1-10 and 91-95) that are disordered, the tertiary fold of the remainder of the protease is essentially identical to that of the individual subunit of the dimer. In the monomer, the side chains of buried residues stabilizing the active site interface in the dimer, such as Asp25, Asp29, and Arg87, are now exposed to solvent. The flap dynamics in the monomer are similar to that of the free protease dimer. We also show that the protease domain of an optimized precursor flanked by 56 amino acids of the N-terminal transframe region is predominantly monomeric, exhibiting a tertiary fold that is quite similar to that of PR1-95 structure. This explains the very low catalytic activity observed for the protease prior to its maturation at its N terminus as compared with the mature protease, which is an active stable dimer under identical conditions. Adding as few as 2 amino acids to the N terminus of the mature protease significantly increases its dissociation into monomers. Knowledge of the protease monomer structure and critical features of its dimerization may aid in the screening and design of compounds that target the protease prior to its maturation from the Gag-Pol precursor.     

Comprehensive mutagenesis of HIV-1 protease: a computational geometry approach

A computational geometry technique based on Delaunay tessellation of protein structure, represented by C(alpha) atoms, is used to study effects of single residue mutations on sequence-structure compatibility in HIV-1 protease. Profiles of residue scores derived from the four-body statistical potential are constructed for all 1881 mutants of the HIV-1 protease monomer and compared with the profile of the wild-type protein. The profiles for an isolated monomer of HIV-1 protease and the identical monomer in a dimeric state with an inhibitor are analyzed to elucidate changes to structural stability. Protease residues shown to undergo the greatest impact are those forming the dimer interface and flap region, as well as those known to be involved in inhibitor binding.     

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.     

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     

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.     

The folding and dimerization of HIV-1 protease: evidence for a stable monomer from simulations

HIV-1 protease (PR) is a major drug target in combating AIDS, as it plays a key role in maturation and replication of the virus. Six FDA-approved drugs are currently in clinical use, all designed to inhibit enzyme activity by blocking the active site, which exists only in the dimer. An alternative inhibition mode would be required to overcome the emergence of drug-resistance through the accumulation of mutations. This might involve inhibiting the formation of the dimer itself. Here, the folding of HIV-1 PR dimer is studied with several simulation models appropriate for folding mechanism studies. Simulations with an off-lattice Gō-model, which corresponds to a perfectly funneled energy landscape, indicate that the enzyme is formed by association of structured monomers. All-atom molecular dynamics simulations strongly support the stability of an isolated monomer. The conjunction of results from a model that focuses on the protein topology and a detailed all-atom force-field model suggests, in contradiction to some reported equilibrium denaturation experiments, that monomer folding and dimerization are decoupled. The simulation result is, however, in agreement with the recent NMR detection of folded monomers of HIV-1 PR mutants with a destabilized interface. Accordingly, the design of dimerization inhibitors should not focus only on the flexible N and C termini that constitute most of the dimer interface, but also on other structured regions of the monomer. In particular, the relatively high phi values for residues 23-35 and 79-87 in both the folding and binding transition states, together with their proximity to the interface, highlight them as good targets for inhibitor design.     

Autoprocessing of HIV-1 protease is tightly coupled to protein folding.

In the Gag-Pol polyprotein of HIV-1, the 99-amino acid protease is flanked at its N-terminus by a transframe region (TFR) composed of the transframe octapeptide (TFP) and 48 amino acids of the p6pol, separated by a protease cleavage site. The intact precursor (TFP-p6pol-PR) has very low dimer stability relative to that of the mature enzyme and exhibits negligible levels of stable tertiary structure. Thus, the TFR functions by destabilizing the native structure, unlike proregions found in zymogen forms of monomeric aspartic proteases. Cleavage at the p6pol-PR site to release a free N-terminus of protease is concomitant with the appearance of enzymatic activity and formation of a stable tertiary structure that is characteristic of the mature protease as demonstrated by nuclear magnetic resonance. The release of the mature protease from the precursor can either occur in two steps at pH values of 4 to 6 or in a single step above pH 6. The mature protease forms a dimer through a four-stranded beta-sheet at the interface. Residues 1-4 of the mature protease from each subunit constitute the outer strands of the beta-sheet, and are essential for maintaining the stability of the free protease but are not a prerequisite for the formation of tertiary structure and catalytic activity. Our experimental results provide the basis for the model proposed here for the regulation of the HIV-1 protease in the viral replication cycle.     

Cryo-EM structure of monomeric photosystem II at 2.78 Å resolution reveals factors important for the formation of dimer

Photosystem II (PSII) functions mainly as a dimer to catalyze the light energy conversion and water oxidation reactions. However, monomeric PSII also exists and functions in vivo in some cases. The crystal structure of monomeric PSII has been solved at 3.6 Å resolution, but it is still not clear which factors contribute to the formation of the dimer. Here, we solved the structure of PSII monomer at a resolution of 2.78 Å using cryo-electron microscopy (cryo-EM). From our cryo-EM density map, we observed apparent differences in pigments and lipids in the monomer-monomer interface between the PSII monomer and dimer. One β-carotene and two sulfoquinovosyl diacylglycerol (SQDG) molecules are found in the monomer-monomer interface of the dimer structure but not in the present monomer structure, although some SQDG and other lipid molecules are found in the analogous region of the low-resolution crystal structure of the monomer, or cryo-EM structure of an apo-PSII monomer lacking the extrinsic proteins from Synechocystis sp. PCC 6803. In the current monomer structure, a large part of the PsbO subunit was also found to be disordered. These results indicate the importance of the β-carotene, SQDG and PsbO in formation of the PSII dimer.     

Insight into the activity of SARS main protease: Molecular dynamics study of dimeric and monomeric form of enzyme

The phenomenon that SARS coronavirus main protease (SARS M(pro)) dimer is the main functional form has been confirmed by experiment. However, because of the absence of structural information of the monomer, the reasons for this remain unknown. To investigate it, two molecular dynamics (MD) simulations in water for dimer and monomer models have been carried out, using the crystal structure of protomer A of the dimer as the starting structure for the monomer. During the MD simulation of dimer, three interest phenomena of protomer A have been observed: (i) the distance between NE2 of His41 and SG of Cys145 averages 3.72 A, which agrees well with the experimental observations made by X-ray crystallography; (ii) His163 and Glu166 form the "tooth" conformational properties, resulting in the specificity for glutamine at substrate P1 site; and (iii) the substrate-binding pocket formed by loop 140-146 and loop 184-197 is large enough to accommodate the substrate analog. However, during the MD simulation of the monomer complex, the three structural characteristics are all absent, which results directly in the inactivation of the monomer. Throughout the MD simulation of the dimer, the N-terminus of protomer B forms stable hydrogen bonds with Phe140 and Glu166, through which His163, Glu166, and loop 140-146 are kept active form. Furthermore, a water-bridge has been found between the N-terminus of protomer B and Gly170, which stabilizes His172 and avoids it moving toward Tyr161 to disrupt the H-bond between Tyr161 and His163, stabilizing the conformation of His163. The interactions between the N-terminus and another monomer maintain the activity of dimer.     

High resolution X-ray structure of yeast hexokinase,an allosteric protein exhibiting a non-symmetric arrangement of subunits

The structure determination of yeast hexokinase has been extended to 3.5 Å resolution for the dimer and to 2.7 Å resolution for the monomer using multiple isomorphous replacement. The electron density maps of both the monomer and dimer crystal forms have been substantially improved by an averaging procedure. From these maps the course of the polypeptide backbone and some aspects of the dimer interaction have been established.The hexokinase subunit arrangement is contrary to a major tenet of the Monod et al. (1965) theory of allosteric proteins which postulated that only symmetric or isologous interactions of subunits would occur in oligomeric proteins. One subunit of the dimer is related to the other by a 156 ° rotation about and a 13.8 Å translation along a molecular screw axis. In the hexokinase dimer the set of residues in one subunit that is interacting with the other subunit is different from the set of residues in the second subunit that is interacting with the first subunit. This heterologous or non-symmetric interaction of subunits is associated with some small differences in the structure of the two subunits, particularly at the subunit interface, and accounts for some of this enzyme's non-symmetric interactions with substrates and activators. Indeed, the non-symmetric subunit association may play an important role in the control of this enzyme's activity.The overall structure of hexokinase is considerably different than the known structures of the other enzymes in the glycolytic pathway. Although there is a striking similarity between the domain of hexokinase that binds AMP and the domain of lactate dehydrogenase that binds NAD, the former structure contains both antiparallel and parallel β-pleated strands, while the latter contains only parallel β-structure. In an attempt to assess the significance of this structural similarity, the structure of the nucleotide binding domains of hexokinase and lactate dehydrogenase are compared to a portion of carboxypeptidase A. The observed similarities among these structures suggests that a central β-pleated sheet flanked by α-helices is a common supersecondary structure that probably arose by convergent as well as divergent evolution. Thus, there appears to be no compelling evidence at this time to support the hypothesis that a part of hexokinase has evolved from the same gene as the dinucleotide binding domain of lactate dehydrogenase.     

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.     

Role of invariant Thr80 in human immunodeficiency virus type 1 protease structure, function, and viral infectivity

Sequence variability associated with human immunodeficiency virus type 1 (HIV-1) is useful for inferring structural and/or functional constraints at specific residues within the viral protease. Positions that are invariant even in the presence of drug selection define critically important residues for protease function. While the importance of conserved active-site residues is easily understood, the role of other invariant residues is not. This work focuses on invariant Thr80 at the apex of the P1 loop of HIV-1, HIV-2, and simian immunodeficiency virus protease. In a previous study, we postulated, on the basis of a molecular dynamics simulation of the unliganded protease, that Thr80 may play a role in the mobility of the flaps of protease. In the present study, both experimental and computational methods were used to study the role of Thr80 in HIV protease. Three protease variants (T80V, T80N, and T80S) were examined for changes in structure, dynamics, enzymatic activity, affinity for protease inhibitors, and viral infectivity. While all three variants were structurally similar to the wild type, only T80S was functionally similar. Both T80V and T80N had decreased the affinity for saquinavir. T80V significantly decreased the ability of the enzyme to cleave a peptide substrate but maintained infectivity, while T80N abolished both activity and viral infectivity. Additionally, T80N decreased the conformational flexibility of the flap region, as observed by simulations of molecular dynamics. Taken together, these data indicate that HIV-1 protease functions best when residue 80 is a small polar residue and that mutations to other amino acids significantly impair enzyme function, possibly by affecting the flexibility of the flap domain.     

Whiskers-less HIV-protease: a possible way for HIV-1 deactivation

Background

Among viral enzymes, the human HIV-1 protease comprises the most interesting target for drug discovery. There are increasing efforts focused on designing more effective inhibitors for HIV-1 protease in order to prevent viral replication in AIDS patients. The frequent and continuous mutation of HIV-1 protease gene creates a formidable obstacle for enzyme inhibition which could not be overcome by the traditional single drug therapy. Nowadays, in vitro and in silico studies of protease inhibition constitute an advanced field in biological researches. In this article, we tried to simulate protease-substrate complexes in different states; a native state and states with whiskers deleted from one and two subunits. Molecular dynamic simulations were carried out in a cubic box filled with explicit water at 37°C and in 1atomsphere of pressure.

Results

Our results showed that whisker truncation of protease subunits causes the dimer structure to decrease in compactness, disrupts substrate-binding site interactions and changes in flap status simultaneously.

Conclusions

Based on our findings we claim that whisker truncation even when applied to a single subunit, threats dimer association which probably leads to enzyme inactivation. We may postulate that inserting a gene to express truncated protease inside infected cells can interfere with protease dimerization. The resulted proteases would presumably have a combination of native and truncated subunits in their structures which exert no enzyme activities as evidenced by the present work. Our finding may create a new field of research in HIV gene therapy for protease inhibition, circumventing problems of drug resistance.     

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.     

Studies on adaptability of binding residues and flap region of TMC-114 resistance HIV-1 protease mutants

Drug resistant mutations have severely restricted the success of HIV therapy. 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, which are able to produce significant resistance against the anti-HIV drug TMC-114. We provide insight into the molecular basis of TMC-114 resistance major flap mutations (I50V and I54M) in HIV-1 protease. It reports the shape complementarity and receptor-ligand interaction analysis supported by unrestrained all-atom molecular dynamics simulations of wild and major flap mutants of HIV-1 protease that sample large conformational changes of the flaps and active site binding residues. Both resistant flap mutants showed less atomic interaction toward TMC-114 and more structural deviation compared to wild HIV-protease. It is due to increasing flexibility at TMC-114 binding cavity and deviation of binding residues in 3-D space. Distortion in binding cavity and deviation in binding residues are the result of alteration in hydrogen bonding. Flap region also exhibited similar behaviour due to changes in number of hydrogen bonds during simulations.     

Molecular dynamics of sickle and normal hemoglobins

Molecular dynamics (MD) simulations have been carried out for 62.5 ps on crystal structures of deoxy sickle cell hemoglobin (HbS) and normal deoxy hemoglobin (HbA) using the CHARMM MD algorithm, with a time step of 0.001 ps. In the trajectory analysis of the 12.5–62.5 (50 ps) simulation, oscillations of the radius of gyration and solvent-accessible surface area were calculated. HbS exhibited a general contraction during the simulation, while HbA exhibited a nearly constant size. The average deviations of simulated structures from the starting structures were found to be 1.8 Å for HbA and 2.3 Å for HbS. The average rms amplitudes of atomic motions (atomic flexibility) were about 0.7 Å for HbA and about 1.0 Å for HbS. The amplitudes of backbone motion correlate well with temperature factors derived from x-ray crystallography. A comparison of flexibility between the α- and β-chains in both HbA and HbS indicates that the β-chains generally exhibited greater flexibility than the α-chains, and that the HbS β-chains exhibit greater flexibility in the N-terminal and D- and F-helix regions than do those of HbA. The average amplitude of backbone torsional oscillations was about 9°, a value comparable with that of other simulations, with enhanced torsional oscillation occurring primarily at the ends of helices or in loop regions between helices. Comparison of atomic flexibility and torsional oscillation results suggests that the increased β-chain flexibility results from relatively concerted motions of secondary structure elements. The increased flexibility may play an important role in HbS polymerization. Time course analysis of conformational energy of association, hydrogen bonding and hydrophobic bonding (as calculated from solvent accessibility) shows that all three of these factors contribute to the stability of subunit association for both hemoglobins. © 1993 John Wiley & Sons, Inc.     

Curling of flap tips in HIV-1 protease as a mechanism for substrate entry and tolerance of drug resistance

BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) protease is an essential viral protein that is a major drug target in the fight against Acquired Immune Deficiency Syndrome (AIDS). Access to the active site of this homodimeric enzyme is gained when two large flaps, one from each monomer, open. The flap movements are therefore central to the function of the enzyme, yet determining how these flaps move at an atomic level has not been experimentally possible. RESULTS: In the present study, we observe the flaps of HIV-1 protease completely opening during a 10 ns solvated molecular dynamics simulation starting from the unliganded crystal structure. This movement is on the time scale observed by Nuclear Magnetic Resonance (NMR) relaxation data. The highly flexible tips of the flaps, with the sequence Gly-Gly-Ile-Gly-Gly, are seen curling back into the protein and thereby burying many hydrophobic residues. CONCLUSIONS: This curled-in conformational change has never been previously described. Previous models of this movement, with the flaps as rigid levers, are not consistent with the experimental data. The residues that participate in this hydrophobic cluster as a result of the conformational change are highly sensitive to mutation and often contribute to drug resistance when they do change. However, several of these residues are not part of the active site cavity, and their essential role in causing drug resistance could possibly be rationalized if this conformational change actually occurs. Trapping HIV-1 protease in this inactive conformation would provide a unique opportunity for future drug design.     

Solvation influences flap collapse in HIV-1 protease

HIV-1 protease (HIVp) is an important target for the development of therapies to treat AIDS and is one of the classic examples of structure-based drug design. The flap region of HIVp is known to be highly flexible and undergoes a large conformational change upon binding a ligand. Accurately modeling the inherent flexibility of the HIVp system is critical for developing new methods for structure-based drug design. We report several 3-ns molecular dynamics simulations investigating the role of solvation in HIVp flap rearrangement. Using an unliganded crystal structure of HIVp, other groups have observed flap reorganization on the nanosecond timescale. We have also observed rapid, initial flap movement, but we propose that it may be caused by system setup. The initial solvation of the system creates vacuum regions around the protein that may encourage large conformational deformities. By reducing the vacuum space created by the solvation routine, the observed flap collapse is attenuated. Also, a more thorough equilibration procedure preserves a more stable protein conformation over the course of the simulation.