Full Length Vpu from HIV-1: Combining Molecular Dynamics Simulations with NMR Spectroscopy

Abstract

Based on structures made available by solution NMR, molecular models of the protein Vpu from HIV-1 were built and refined by 6 ns MD simulations in a fully hydrated lipid bilayer. Vpu is an 81 amino acid type I integral membrane protein encoded by the human immunodeficiency virus type-1 (HIV-1) and closely related simian immunodeficiency viruses (SIVs). Its role is to amplify viral release. Upon phosphorylation, the cytoplasmic domain adopts a more compact shape with helices 2 and 3 becoming almost parallel to each other. A loss of helicity for several residues belonging to the helices adjacent to both ends of the loop region containing serines 53 and 57 is observed. A fourth helix, present in one of the NMR-based structures of the cytoplasmic domain and located near the C-terminus, is lost upon phosphorylation.     

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.     

Interaction of amiloride and one of its derivatives with Vpu from HIV-1: a molecular dynamics simulation

Vpu is an 81-residue membrane protein, with a single transmembrane segment that is encoded by HIV-1 and is involved in the enhancement of virion release via formation of an ion channel. Cyclohexamethylene amiloride (Hma) has been shown to inhibit ion channel activity. In the present 12-ns simulation study a putative binding site of Hma blockers in a pentameric model bundle built of parallel aligned helices of the first 32 residues of Vpu was found near Ser-23. Hma orientates along the channel axis with its alkyl ring pointing inside the pore, which leads to a blockage of the pore.     

Structure and dynamics of Mycobacterium tuberculosis truncated hemoglobin N: insights from NMR spectroscopy and molecular dynamics simulations

The potent nitric oxide dioxygenase (NOD) activity (trHbN-Fe2?-O? + (?)NO → trHbN-Fe3?-OH? + NO??) of Mycobacterium tuberculosis truncated hemoglobin N (trHbN) protects aerobic respiration from inhibition by (?)NO. The high activity of trHbN has been attributed in part to the presence of numerous short-lived hydrophobic cavities that allow partition and diffusion of the gaseous substrates (?)NO and O? to the active site. We investigated the relation between these cavities and the dynamics of the protein using solution NMR spectroscopy and molecular dynamics (MD). Results from both approaches indicate that the protein is mainly rigid with very limited motions of the backbone N-H bond vectors on the picoseconds-nanoseconds time scale, indicating that substrate diffusion and partition within trHbN may be controlled by side-chains movements. Model-free analysis also revealed the presence of slow motions (microseconds-milliseconds), not observed in MD simulations, for many residues located in helices B and G including the distal heme pocket Tyr33(B10). All currently known crystal structures and molecular dynamics data of truncated hemoglobins with the so-called pre-A N-terminal extension suggest a stable α-helical conformation that extends in solution. Moreover, a recent study attributed a crucial role to the pre-A helix for NOD activity. However, solution NMR data clearly show that in near-physiological conditions these residues do not adopt an α-helical conformation and are significantly disordered and that the helical conformation seen in crystal structures is likely induced by crystal contacts. Although this lack of order for the pre-A does not disagree with an important functional role for these residues, our data show that one should not assume an helical conformation for these residues in any functional interpretation. Moreover, future molecular dynamics simulations should not use an initial α-helical conformation for these residues in order to avoid a bias based on an erroneous initial structure for the N-termini residues. This work constitutes the first study of a truncated hemoglobin dynamics performed by solution heteronuclear relaxation NMR spectroscopy.     

Supramolecular structure of membrane-associated polypeptides by combining solid-state NMR and molecular dynamics simulations

Elemental biological functions such as molecular signal transduction are determined by the dynamic interplay between polypeptides and the membrane environment. Determining such supramolecular arrangements poses a significant challenge for classical structural biology methods. We introduce an iterative approach that combines magic-angle spinning solid-state NMR spectroscopy and atomistic molecular dynamics simulations for the determination of the structure and topology of membrane-bound systems with a resolution and level of accuracy difficult to obtain by either method alone. Our study focuses on the Shaker B ball peptide that is representative for rapid N-type inactivating domains of voltage-gated K(+) channels, associated with negatively charged lipid bilayers.     

Full length amylin oligomer aggregation: insights from molecular dynamics simulations and implications for design of aggregation inhibitors

Amyloid oligomers are considered to play essential roles in the pathogenesis of amyloid-related degenerative diseases including type 2 diabetes. Using an explicit solvent all atomic MD simulation, we explored the stability, conformational dynamics and association force of different single-layer models of the full-length wild-type and glycine mutants of amylin (pentamer) obtained from a recent high resolution fibril model. The RMSF profile shows enhanced flexibility in the disorder (Lys1-Cys7) and turn region (Ser19-Gly23), along with smallest fluctuation at the residues (Asn14-Phe15-Leu16-Val17-His18) of β₁ region and (Ala25-Ile26-Leu27-Ser28-Ser29) of the β2 region. We obtained a significant difference in backbone RMSD between the wild-type and the mutants, indicating that mutations affected the stability of the peptide. The RMSD and RMSF profiles indicate the edge and loop residues are the primary contributors to the overall conformational changes. The degree of structural similarity between the oligomers in the simulation and the fibril conformation is proposed as the possible explanation for experimentally observed shortening of the nucleation lag phase of amylin with oligomer seeding. On the basis of structure-stability findings, the β₁ and β₂ portions are optimal target for further anti-amyloid drug design. The MM-PBSA binding energy calculation reveals the binding of amylin: amylin strands in single layer is dominated by contributions from van der Waals interactions. The non-polar solvation term is also found to be favorable. While the electrostatic interactions and polar solvation energy was found to be favorable for the interaction for the larger aggregate and unfavorable for the smaller aggregates. A per-residue decomposition of the binding free energy has been performed to identify the residues contributing most to the self-association free energy. Residues found in the β-sheet regions were found to be key residue making the largest favorable contributions to the single-layer association. The result from our simulation could be used in rational design of new amylinomimetic agent, amylin aggregation inhibitors and amylin-specific biomarkers.     

Structure and dynamics of the HIV-1 Vpu transmembrane domain revealed by solid-state NMR with magic-angle spinning

We report solid-state nuclear magnetic resonance (NMR) measurements on the peptide Vpu(1-40), comprising residues 1-40 of the 81-residue type 1 integral membrane protein Vpu encoded by the HIV-1 genome. On the basis of a combination of 13C and 15N NMR chemical shifts under magic-angle spinning (MAS), effects of local mobility on NMR signal intensities, site-specific MAS NMR line widths, and NMR-detected hydrogen-deuterium exchange, we develop a model for the structure and dynamics of the Vpu(1-40) monomer in phospholipid bilayer membranes. Our data are largely consistent with earlier structural studies of Vpu peptides by Opella and co-workers, in which solution NMR and solid-state NMR without MAS were used, but our data provide new information about local variations in the degree of mobility and structural order. In addition, our data indicate that the transmembrane alpha-helix of Vpu(1-40) extends beyond the hydrophobic core of the bilayer. We find no evidence for heterogeneity in the conformation and intermolecular contacts of the transmembrane alpha-helix, with the exception of two distinct chemical shifts observed for the C alpha and C beta atoms of A18 that may reflect distinct modes of helix-helix interaction. These results have possible implications for the supramolecular structure of Vpu oligomers that form cation-selective ion channels.     

Optimization of designed armadillo repeat proteins by molecular dynamics simulations and NMR spectroscopy

A multidisciplinary approach based on molecular dynamics (MD) simulations using homology models, NMR spectroscopy, and a variety of biophysical techniques was used to efficiently improve the thermodynamic stability of armadillo repeat proteins (ArmRPs). ArmRPs can form the basis of modular peptide recognition and the ArmRP version on which synthetic libraries are based must be as stable as possible. The 42-residue internal Arm repeats had been designed previously using a sequence-consensus method. Heteronuclear NMR revealed unfavorable interactions present at neutral but absent at high pH. Two lysines per repeat were involved in repulsive interactions, and stability was increased by mutating both to glutamine. Five point mutations in the capping repeats were suggested by the analysis of positional fluctuations and configurational entropy along multiple MD simulations. The most stabilizing single C-cap mutation Q240L was inferred from explicit solvent MD simulations, in which water penetrated the ArmRP. All mutants were characterized by temperature- and denaturant-unfolding studies and the improved mutants were established as monomeric species with cooperative folding and increased stability against heat and denaturant. Importantly, the mutations tested resulted in a cumulative decrease of flexibility of the folded state in silico and a cumulative increase of thermodynamic stability in vitro. The final construct has a melting temperature of about 85°C, 14.5° higher than the starting sequence. This work indicates that in silico studies in combination with heteronuclear NMR and other biophysical tools may provide a basis for successfully selecting mutations that rapidly improve biophysical properties of the target proteins.     

Bundles consisting of extended transmembrane segments of Vpu from HIV-1: computer simulations and conductance measurements

Part of the genome of the human immunodeficiency virus type 1 (HIV-1) encodes for a short membrane protein Vpu, which has a length of 81 amino acids. It has two functional roles: (i) to downregulate CD4 and (ii) to support particle release. These roles are attributed to two distinct domains of the peptide, the cytoplasmic and transmembrane (TM) domains, respectively. It has been suggested that the enhanced particle release function is linked to the ion channel activity of Vpu, with a slight preference for cations over anions. To allow ion flux across the membrane Vpu would be required to assemble in homooligomers to form functional water-filled pores. In this study molecular dynamics simulations are used to address the role of particular amino acids in 4, 5, and 6 TM helix bundle structures. The helices (Vpu(6-33)) are extended to include hydrophilic residues such as Glu, Tyr, and Arg (EYR motif). Our simulations indicate that this motif destabilizes the bundles at their C-terminal ends. The arginines point into the pore to form a positive charged ring that could act as a putative selectivity filter. The helices of the bundles adopt slightly higher average tilt angles with decreasing number of helices. We also suggest that the helices are kinked. Conductance measurements on a peptide (Vpu(1-32)) reconstituted into lipid membranes show that the peptide forms ion channels with several conductance levels.     

Denaturation of HIV-1 protease (PR) monomer by acetic acid: mechanistic and trajectory insights from molecular dynamics simulations and NMR

Inside a living cell there can be a variety of interactions for any given protein, which serve to regulate denaturation and renaturation processes. Insights into some of them can be obtained by in vitro studies using various denaturing agents. In this study, all-atom MD simulations in explicit solvent and NMR relaxation studies were performed on HIV-1 Protease (PR) in 9 M acetic acid (AcOH) (the commonly used denaturant during PR preparation). Following previous reports that denaturation proceeds via dissociation of the dimer into monomers, unfolding of the monomer by acetic acid has been explicitly investigated here. Direct visualization of the denaturation process and evidence for the mechanism of denaturation have been presented. Our simulations reveal that the denaturation of the PR monomer is caused due to direct interaction between acetic acid molecules and PR. Autocorrelation of N-H vectors calculated from the simulations have revealed that the α-helix and the surrounding β-strands represent the sensitive regions of the PR that respond maximally to the change in the solvent environment around the PR and are prone to disruption by acetic acid. This disruption is caused due to increased penetration of the acetic acid molecules into the PR structure by formation of preferred tertiary contacts and hydrogen bonds between the PR and acetic acid molecules. Following the loss of these critical interactions, the PR follows a random and non-equilibrating path on the conformation landscape and cycles between different denatured extended and compact states.     

Analysis of correlated motion in antibody combining sites from molecular dynamics simulations

The crystal structures of the NC6.8-antisweet taste ligand complex and the uncomplexed antibody structures display significant differences in the conformations of residues in the combining site. A molecular dynamics method was employed to understand the flexibility and correlated motion of key combining site residues in the uncomplexed antibody. The simulations reveal that residues that show conformational differences between the complex and uncomplexed structures display strong dynamical correlations. Extensive analysis of the dynamics trajectory using time correlation methods is presented.     

All-atom molecular dynamics simulations using orientational constraints from anisotropic NMR samples

Orientational constraints obtained from solid state NMR experiments on anisotropic samples are used here in molecular dynamics (MD) simulations for determining the structure and dynamics of several different membrane-bound molecules. The new MD technique is based on the inclusion of orientation dependent pseudo-forces in the COSMOS-NMR force field. These forces drive molecular rotations and re-orientations in the simulation, such that the motional time-averages of the tensorial NMR properties approach the experimentally measured parameters. The orientational-constraint-driven MD simulations are universally applicable to all NMR interaction tensors, such as chemical shifts, dipolar couplings and quadrupolar interactions. The strategy does not depend on the initial choice of coordinates, and is in principle suitable for any flexible molecule. To test the method on three systems of increasing complexity, we used as constraints some deuterium quadrupolar couplings from the literature on pyrene, cholesterol and an antimicrobial peptide embedded in oriented lipid bilayers. The MD simulations were able to reproduce the NMR parameters within experimental error. The alignment of the three membrane-bound molecules and some aspects of their conformation were thus derived from the NMR data, in good agreement with previous analyses. Furthermore, the new approach yielded for the first time the distribution of segmental orientations with respect to the membrane and the order parameter tensors of all three systems.     

Structural and dynamical properties of a full-length HIV-1 integrase: molecular dynamics simulations

The structural and dynamical properties of the complete full-length structure of HIV-1 integrase were investigated using Molecular Dynamics approach. Simulations were carried out for the three systems, core domain only (CORE), full-length structure without (FULL) and with a Mg2+ (FULL+ION) in its active site, aimed to investigate the difference in the molecular properties of the full-length models due to their different construction procedures as well as the effects of the two ends, C- and N-terminal, on those properties in the core domain. The full-length structure was prepared from the two experimental structures of two-domain fragment. The following properties were observed to differ significantly from the previous reports: (i) relative topology formed by an angle between the three domains; (ii) the cavity size defined by the catalytic triad, Asp64, Asp116, and Glu152; (iii) distances and solvation of the Mg2+; and (iv) conformation of the catalytic residues. In addition, the presence of the two terminal domains decreases the mobility of the central core domain significantly.     

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

Background  

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

Studies on somatostatin with time-resolved spectroscopy and molecular dynamics simulations

Somatostatin was studied by time-resolved fluorescence spectroscopy and by molecular dynamics simulations. The results obtained indicate the existence of mainly one conformation in DMSO, and the existence of several conformations in other solvents. Molecular dynamics simulations showed that the most important region for activity (residue 7-9) is also the most flexible region in the peptide.     

Conformational studies on a unique bis-sulfated glycolipid using NMR spectroscopy and molecular dynamics simulations.

The time-averaged solution conformation of a unique bis-sulfated glycolipid (HSO3)2-2,6Manalpha-2Glcalpha-1-sn-2,3-O-alkylglycerol , was studied in terms of the torsional angles of two glycosidic linkages, phi (H1-C1-O-Cx) and psi (C1-O-Cx-Hx), derived from heteronuclear three-bond coupling constants (3JC,H), and inter-residual proton-proton distances from J-HMBC 2D and ROESY experiments, respectively. The dihedral angles of Glcalpha1Gro in glycolipids were determined for the first time. The C1-C4 diagonal line of the alpha-glucose ring makes an angle of approximately 120 degrees with the glycerol backbone, suggesting that the alpha-glucose ring is almost parallel to the membrane surface in contrast with the perpendicular orientation of the beta-isomer. Furthermore, minimum-energy states around the conformation were estimated by Monte Carlo/stochastic dynamics (MCSD) mixed-mode simulations and the energy minimization with assisted model building and energy refinement (AMBER) force field. The Glcalpha1Gro linkage has a single minimum-energy structure. On the other hand, three conformers were observed for the Manalpha2Glc linkage. The flexibility of Manalpha2Glc was further confirmed by the absence of inter-residual hydrogen bonds which were judged from the temperature coefficients of the chemical shifts, ddelta/dT (-10-3 p.p.m. degrees C-1), of hydroxy protons. The conformational flexibility may facilitate interaction of extracellular substances with both sulfate groups.     

Conformational investigation of a cyclic enterobacterial common antigen employing NMR spectroscopy and molecular dynamics simulations

The three-dimensional structure of a cyclic enterobacterial common antigen (ECA) having four trisaccharide repeating units has been investigated by NMR spectroscopy and molecular dynamics simulations. Three different NMR parameters were determined: (a) (1)H,(1)H cross-relaxation rates from NOE experiments were used for determination of proton-proton distances; (b) trans-glycosidic (3)J(C,H) scalar coupling constants analyzed via a Karplus-type relationship provided information on torsion angles; and (c) (1)H,(13)C one-bond dipolar couplings obtained in a dilute liquid-crystalline medium were interpreted in terms of the orientational order and molecular conformations. The molecular dynamics simulations of the dodecasaccharide were performed with explicit water and counterions, which are important factors that strongly influence molecular conformation. Subsequently, the results from computer simulation were used to generate a three-dimensional structure of the cyclic ECA which is consistent with the experimental NMR parameters.     

Molecular dynamics simulations on HIV-1 Tat

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