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.     

HIV-1 Accessory Protein Vpu Internalizes Cell-surface BST-2/Tetherin through Transmembrane Interactions Leading to Lysosomes

Bone marrow stromal antigen 2 (BST-2, also known as tetherin) is a recently identified interferon-inducible host restriction factor that can block the production of enveloped viruses by trapping virus particles at the cell surface. This antiviral effect is counteracted by the human immunodeficiency virus type 1 (HIV-1) accessory protein viral protein U (Vpu). Here we show that HIV-1 Vpu physically interacts with BST-2 through their mutual transmembrane domains and leads to the degradation of this host factor via a lysosomal, not proteasomal, pathway. The degradation is partially controlled by a cellular protein, β-transducin repeat-containing protein (βTrCP), which is known to be required for the Vpu-induced degradation of CD4. Importantly, targeting of BST-2 by Vpu occurs at the plasma membrane followed by the active internalization of this host protein by Vpu independently of constitutive endocytosis. Thus, the primary site of action of Vpu is the plasma membrane, where Vpu targets and internalizes cell-surface BST-2 through transmembrane interactions, leading to lysosomal degradation, partially in a βTrCP-dependent manner. Also, we propose the following configuration of BST-2 in tethering virions to the cell surface; each of the dimerized BST-2 molecules acts as a bridge between viral and cell membranes.     

Transmembrane domain determinants of CD4 Downregulation by HIV-1 Vpu

The transmembrane domains (TMDs) of integral membrane proteins do not merely function as membrane anchors but play active roles in many important biological processes. The downregulation of the CD4 coreceptor by the Vpu protein of HIV-1 is a prime example of a process that is dependent on specific properties of TMDs. Here we report the identification of Trp22 in the Vpu TMD and Gly415 in the CD4 TMD as critical determinants of Vpu-induced targeting of CD4 to endoplasmic reticulum (ER)-associated degradation (ERAD). The two residues participate in different aspects of ERAD targeting. Vpu Trp22 is required to prevent assembly of Vpu into an inactive, oligomeric form and to promote CD4 polyubiquitination and subsequent recruitment of the VCP-UFD1L-NPL4 dislocase complex. In the presence of a Vpu Trp22 mutant, CD4 remains integrally associated with the ER membrane, suggesting that dislocation from the ER into the cytosol is impaired. CD4 Gly415, on the other hand, contributes to CD4-Vpu interactions. We also identify two residues, Val20 and Ser23, in the Vpu TMD that mediate retention of Vpu and, by extension, CD4 in the ER. These findings highlight the exploitation of several TMD-mediated mechanisms by HIV-1 Vpu in order to downregulate CD4 and thus promote viral pathogenesis.     

Synaptobrevin Transmembrane Domain Dimerization Studied by Multiscale Molecular Dynamics Simulations

Synaptic vesicle fusion requires assembly of the SNARE complex composed of SNAP-25, syntaxin-1, and synaptobrevin-2 (sybII) proteins. The SNARE proteins found in vesicle membranes have previously been shown to dimerize via transmembrane (TM) domain interactions. While syntaxin homodimerization is supposed to promote the transition from hemifusion to complete fusion, the role of synaptobrevin’s TM domain association in the fusion process remains poorly understood. Here, we combined coarse-grained and atomistic simulations to model the homodimerization of the sybII transmembrane domain and of selected TM mutants. The wild-type helix is shown to form a stable, right-handed dimer with the most populated helix-helix interface, including key residues predicted in a previous mutagenesis study. In addition, two alternative binding interfaces were discovered, which are essential to explain the experimentally observed higher-order oligomerization of sybII. In contrast, only one dimerization interface was found for a fusion-inactive poly-Leu mutant. Moreover, the association kinetics found for this mutant is lower as compared to the wild-type. These differences in dimerization between the wild-type and the poly-Leu mutant are suggested to be responsible for the reported differences in fusogenic activity between these peptides. This study provides molecular insight into the role of TM sequence specificity for peptide aggregation in membranes.     

HIV-1辅助调节蛋白Vpu的结构与功能

Vpu蛋白是HIV病毒的辅助调节蛋白之一,仅存在HIV一1型病毒中。在病毒的复制过程中Vpu蛋白下调CD4受体的表达,调节Pr55^gag蛋白的核定位影响病毒的装配,细胞膜上类似离子通道作用促进子代病毒颗粒的释放。该文介绍Vpu蛋白的结构与功能。     

HIV-1 Vpu蛋白的原核表达、鉴定和纯化

目的：克隆、表达、纯化人免疫缺陷病毒Ⅰ型（HIV-1）Vpu蛋白,为其功能及免疫学研究奠定基础。方法：PCR扩增Vpu基因,纯化、酶切后克隆到原核表达载体pET32a中,转化大肠杆菌BL21（DE3）菌株获得表达工程菌株,IPTG诱导蛋白表达,免疫印迹鉴定目的蛋白,亲和层析纯化蛋白。结果：构建了HIV-1Vpu蛋白的原核表达载体Vpu-pET32a,并在大肠杆菌中高效表达,目的蛋白呈可溶性形式存在,免疫印迹检测显示为目的蛋白,经Ni—NTAAgarose纯化获得了高纯度的目的蛋白。结论：在原核表达系统中表达了可溶性HIV-1Vpu蛋白,为进一步进行HIV-1Vpu蛋白的免疫原性和功能研究奠定了基础。     

Molecular Dynamics Simulations of Micelle Formation around Dimeric Glycophorin A Transmembrane Helices

Insertion and formation of membrane proteins involves the interaction of protein helices with one another in lipid environments. Researchers have studied glycophorin A (GpA) transmembrane helices embedded in sodium dodecyl sulfate (SDS) micelles to identify contacts significant for helix dimerization. However, a detailed picture of the conformation and dynamics of the GpA-SDS system cannot be obtained solely through experiment. Molecular dynamics simulations of SDS and a GpA dimer can provide an atomic-level picture of SDS aggregation and helix association. We report 2.5-ns simulations of GpA wild-type and mutants in a preformed micelle as well as a 32-ns simulation showing the formation of a complete micelle around wild-type GpA from an initially random placement of SDS molecules in an aqueous environment. In the latter case, an initial instability of GpA helices in water is reversed after the helices become surrounded by SDS. The properties of the spontaneously formed micelle surrounding the GpA are indistinguishable from those of the preformed micelle surrounding the GpA dimer.     

Species-Specific Activity of HIV-1 Vpu and Positive Selection of Tetherin Transmembrane Domain Variants

Tetherin/BST-2/CD317 is a recently identified antiviral protein that blocks the release of nascent retrovirus, and other virus, particles from infected cells. An HIV-1 accessory protein, Vpu, acts as an antagonist of tetherin. Here, we show that positive selection is evident in primate tetherin sequences and that HIV-1 Vpu appears to have specifically adapted to antagonize variants of tetherin found in humans and chimpanzees. Tetherin variants found in rhesus macaques (rh), African green monkeys (agm) and mice were able to inhibit HIV-1 particle release, but were resistant to antagonism by HIV-1 Vpu. Notably, reciprocal exchange of transmembrane domains between human and monkey tetherins conferred sensitivity and resistance to Vpu, identifying this protein domain as a critical determinant of Vpu function. Indeed, differences between hu-tetherin and rh-tetherin at several positions in the transmembrane domain affected sensitivity to antagonism by Vpu. Two alterations in the hu-tetherin transmembrane domain, that correspond to differences found in rh- and agm-tetherin proteins, were sufficient to render hu-tetherin completely resistant to HIV-1 Vpu. Interestingly, transmembrane and cytoplasmic domain sequences in primate tetherins exhibit variation at numerous codons that is likely the result of positive selection, and some of these changes coincide with determinants of HIV-1 Vpu sensitivity. Overall, these data indicate that tetherin could impose a barrier to viral zoonosis as a consequence of positive selection that has been driven by ancient viral antagonists, and that the HIV-1 Vpu protein has specialized to target the transmembrane domains found in human/chimpanzee tetherin proteins.     

Molecular Dynamics Simulations of the Periplasmic Ferric-hydroxamate Binding Protein FhuD

FhuD is a periplasmic binding protein (PBP) that, under iron-limiting conditions, transports various hydroxamate-type siderophores from the outer membrane receptor (FhuA) to the inner membrane ATP-binding cassette transporter (FhuBC). Unlike many other PBPs, FhuD possesses two independently folded domains that are connected by an α-helix rather than two or three central β-strands. Crystal structures of FhuD with and without bound gallichrome have provided some insight into the mechanism of siderophore binding as well as suggested a potential mechanism for FhuD binding to FhuB. Since the α-helix connecting the two domains imposes greater rigidity on the structure relative to the β-strands in other ‘classical’ PBPs, these structures reveal no large conformational change upon binding a hydroxamate-type siderophore. Therefore, it is difficult to explain how the inner membrane transporter FhuB can distinguish between ferrichrome-bound and ferrichrome-free FhuD. In the current study, we have employed a 30 ns molecular dynamics simulation of FhuD with its bound siderophore removed to explore the dynamic behavior of FhuD in the substrate-free state. The MD simulation suggests that FhuD is somewhat dynamic with a C-terminal domain closure of 6° upon release of its siderophore. This relatively large motion suggests differences that would allow FhuB to distinguish between ferrichrome-bound and ferrichrome-free FhuD.     

Enhancing Protein Adsorption Simulations by Using Accelerated Molecular Dynamics

The atomistic modeling of protein adsorption on surfaces is hampered by the different time scales of the simulation (s) and experiment (up to hours), and the accordingly different ‘final’ adsorption conformations. We provide evidence that the method of accelerated molecular dynamics is an efficient tool to obtain equilibrated adsorption states. As a model system we study the adsorption of the protein BMP-2 on graphite in an explicit salt water environment. We demonstrate that due to the considerably improved sampling of conformational space, accelerated molecular dynamics allows to observe the complete unfolding and spreading of the protein on the hydrophobic graphite surface. This result is in agreement with the general finding of protein denaturation upon contact with hydrophobic surfaces.     

Structural and Dynamical Properties of a Full-length HIV-1 Integrase: Molecular Dynamics Simulations

Abstract

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 Glul52; (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.     

Influence of a Mutation in the Transmembrane Domain of the pl85c-erbB2 Oncogene-Encoded Protein Studied by Molecular Dynamics Simulations

Abstract

The c-erbB2 proto-oncogene encodes for a protein of 185kDa (p 185) which becomes transforming upon the Val→-Glu transmembrane amino acid substitution. The transforming ability seems to be due to a substitution-resulting constitutive activation of the tyrosine kinase cytosolic domain of the protein. These observations prompted us to evaluate the structural and dynamical behavior of the transmembrane region of the wild and transforming p 185 protein in order to understand the role of this region in the transduction mechanism. 160 ps molecular dynamics simulations in vacuo have been performed on two peptides corresponding to the sequence [651-679] of p 185^c-erbB2 protein and its transforming mutant Val⁶⁵⁹→Glu⁶⁵⁹. These two sequences include the transmembrane domain and are initially postulated to be in an α- helix conformation. Noticeable differences in the flexibility of the two peptides are shown. The nontransforming sequence seems rather flexible and several conformational changes are detected at the junction of the mutation point [658-659] and at position Val⁶⁶⁵-Val⁶⁶⁶ during the 160 ps simulations. On the contrary, no transitions were observed for the mutated sequence which adopts a stable α-helix conformation. This difference in flexibility could be hypothesized as a factor involved in the regulation of the tyrosine kinase activity of 185^c-erbB2     

Revealing Atomic-Level Mechanisms of Protein Allostery with Molecular Dynamics Simulations

Molecular dynamics (MD) simulations have become a powerful and popular method for the study of protein allostery, the widespread phenomenon in which a stimulus at one site on a protein influences the properties of another site on the protein. By capturing the motions of a protein’s constituent atoms, simulations can enable the discovery of allosteric binding sites and the determination of the mechanistic basis for allostery. These results can provide a foundation for applications including rational drug design and protein engineering. Here, we provide an introduction to the investigation of protein allostery using molecular dynamics simulation. We emphasize the importance of designing simulations that include appropriate perturbations to the molecular system, such as the addition or removal of ligands or the application of mechanical force. We also demonstrate how the bidirectional nature of allostery—the fact that the two sites involved influence one another in a symmetrical manner—can facilitate such investigations. Through a series of case studies, we illustrate how these concepts have been used to reveal the structural basis for allostery in several proteins and protein complexes of biological and pharmaceutical interest.     

Molecular Dynamics Simulations of a Membrane Protein/Amphipol Complex

Amphipathic polymers known as “amphipols” provide a highly stabilizing environment for handling membrane proteins in aqueous solutions. A8-35, an amphipol with a polyacrylate backbone and hydrophobic grafts, has been extensively characterized and widely employed for structural and functional studies of membrane proteins using biochemical and biophysical approaches. Given the sensitivity of membrane proteins to their environment, it is important to examine what effects amphipols may have on the structure and dynamics of the proteins they complex. Here we present the first molecular dynamics study of an amphipol-stabilized membrane protein, using Escherichia coli OmpX as a model. We begin by describing the structure of the complexes formed by supplementing OmpX with increasing amounts of A8-35, in order to determine how the amphipol interacts with the transmembrane and extramembrane surfaces of the protein. We then compare the dynamics of the protein in either A8-35, a detergent, or a lipid bilayer. We find that protein dynamics on all accessible length scales is restrained by A8-35, which provides a basis to understanding some of the stabilizing and functional effects of amphipols that have been experimentally observed.     

Insight Derived from Molecular Dynamics Simulations into Molecular Motions,Thermodynamics and Kinetics of HIV-1 gp120

Although the crystal structures of the HIV-1 gp120 core bound and pre-bound by CD4 are known, the details of dynamics involved in conformational equilibrium and transition in relation to gp120 function have remained elusive. The homology models of gp120 comprising the N- and C-termini and loops V3 and V4 in the CD4-bound and CD4-unbound states were built and subjected to molecular dynamics (MD) simulations to investigate the differences in dynamic properties and molecular motions between them. The results indicate that the CD4-bound gp120 adopted a more compact and stable conformation than the unbound form during simulations. For both the unbound and bound gp120, the large concerted motions derived from essential dynamics (ED) analyses can influence the size/shape of the ligand-binding channel/cavity of gp120 and, therefore, were related to its functional properties. The differences in motion direction between certain structural components of these two forms of gp120 were related to the conformational interconversion between them. The free energy calculations based on the metadynamics simulations reveal a more rugged and complex free energy landscape (FEL) for the unbound than for the bound gp120, implying that gp120 has a richer conformational diversity in the unbound form. The estimated free energy difference of ∼−6.0 kJ/mol between the global minimum free energy states of the unbound and bound gp120 indicates that gp120 can transform spontaneously from the unbound to bound states, revealing that the bound state represents a high-probability “ground state” for gp120 and explaining why the unbound state resists crystallization. Our results provide insight into the dynamics-and-function relationship of gp120, and facilitate understandings of the thermodynamics, kinetics and conformational control mechanism of HIV-1 gp120.     

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.     

Insight into Signal Transduction: Structural Alterations in Transmembrane Helices Probed by Multi-1 ns Molecular Dynamics Simulations

Abstract

The hypothesis of structural alteration in transmembrane helices for signal transduction process is viewed by molecular dynamics simulation techniques. For the c-erbB-2 transmembrane domain involved in oncogenicity, the occurrence of conformational changes has been previously described as transition from the α to π helix. This dynamical feature is thoroughly analyzed for the wild phenotype and oncogenic sequences from a series of 18 simulations carried out on one nanosecond time scale. We show that these structural events do not depend upon the conditions of simulations like force field or starting helix coordinates. We demonstrate that the oncogenic mutations Val659 Glu, Gin and Asp do not prevent the transition. Furthermore, we show that β branched residues, in conjunction with Gly residues in the c-erbB-2 sequence, act as destabilizers for the α helix structure, π deformations are tightly related to other local structural motifs found in soluble and membrane proteins. These structural alterations are discussed in term of structure-activity relationships for the c-erbB-2 activating mechanism mediated by transmembrane domain dimerization.     

HIV-1 Vpu: putting a channel to the TASK

Vpu is an HIV-encoded protein that enhances virus release. Previously, this activity was correlated with an intrinsic ion channel activity of Vpu. In this issue of Molecular Cell, Hsu at all. propose an alternative mechanism: they suggest that Vpu functions by inhibiting another ion channel, TASK-1.     

Human Tetherin Exerts Strong Selection Pressure on the HIV-1 Group N Vpu Protein

HIV-1 groups M and N emerged within the last century following two independent cross-species transmissions of SIVcpz from chimpanzees to humans. In contrast to pandemic group M strains, HIV-1 group N viruses are exceedingly rare, with only about a dozen infections identified, all but one in individuals from Cameroon. Poor adaptation to the human host may be responsible for this limited spread of HIV-1 group N in the human population. Here, we analyzed the function of Vpu proteins from seven group N strains from Cameroon, the place where this zoonosis originally emerged. We found that these N-Vpus acquired four amino acid substitutions (E15A, V19A and IV25/26LL) in their transmembrane domain (TMD) that allow efficient interaction with human tetherin. However, despite these adaptive changes, most N-Vpus still antagonize human tetherin only poorly and fail to down-modulate CD4, the natural killer (NK) cell ligand NTB-A as well as the lipid-antigen presenting protein CD1d. These functional deficiencies were mapped to amino acid changes in the cytoplasmic domain that disrupt putative adaptor protein binding sites and an otherwise highly conserved ßTrCP-binding DSGxxS motif. As a consequence, N-Vpus exhibited aberrant intracellular localization and/or failed to recruit the ubiquitin-ligase complex to induce tetherin degradation. The only exception was the Vpu of a group N strain recently discovered in France, but originally acquired in Togo, which contained intact cytoplasmic motifs and counteracted tetherin as effectively as the Vpus of pandemic HIV-1 M strains. These results indicate that HIV-1 group N Vpu is under strong host-specific selection pressure and that the acquisition of effective tetherin antagonism may lead to the emergence of viral variants with increased transmission fitness.