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.     

Conformation of the HIV-1 gp120 V3 loop. Structure functional analysis of the HIV-Haiti viral strain

The structural model describing the conformational preferences of the HIV-Haiti gp120 V3 loop in geometric space of dihedral angles was generated in terms of NMR spectroscopy data using the methods of computer modeling. The elements of secondary structure anti conformations of irregular stretches were deciphered for the fragment making the virus principal neutralizing determinant as well as the determinants of cell tropism and syncytium formation. The structurally conserved amino acids of the HIV-1 V3 loop, that may present the forward-looking targets for AIDS drug design, were identified based on the combined analysis of the results obtained with those derived previously. In particular, it was demonstrated that the register of these amino acids comprises Asn-25 critical for virus binding with primary cell receptor CD4 as well as Arg-3 critical for utilization of CCR5 coreceptor and heparan sulfate proteoglycan syndecans. The results obtained are discussed in conjunction with the literature data on the biological activity of individual amino acid residues of the HIV-1 gpl20 V3 loop.     

Molecular Recognition of CCR5 by an HIV-1 gp120 V3 Loop

The binding of protein HIV-1 gp120 to coreceptors CCR5 or CXCR4 is a key step of the HIV-1 entry to the host cell, and is predominantly mediated through the V3 loop fragment of HIV-1 gp120. In the present work, we delineate the molecular recognition of chemokine receptor CCR5 by a dual tropic HIV-1 gp120 V3 loop, using a comprehensive set of computational tools predominantly based on molecular dynamics simulations and free energy calculations. We report, what is to our knowledge, the first complete HIV-1 gp120 V3 loop : CCR5 complex structure, which includes the whole V3 loop and the N-terminus of CCR5, and exhibits exceptional agreement with previous experimental findings. The computationally derived structure sheds light into the functional role of HIV-1 gp120 V3 loop and CCR5 residues associated with the HIV-1 coreceptor activity, and provides insights into the HIV-1 coreceptor selectivity and the blocking mechanism of HIV-1 gp120 by maraviroc. By comparing the binding of the specific dual tropic HIV-1 gp120 V3 loop with CCR5 and CXCR4, we observe that the HIV-1 gp120 V3 loop residues 13–21, which include the tip, share nearly identical structural and energetic properties in complex with both coreceptors. This result paves the way for the design of dual CCR5/CXCR4 targeted peptides as novel potential anti-AIDS therapeutics.     

Disulfide bond that constrains the HIV-1 gp120 V3 domain is cleaved by thioredoxin

A functional disulfide bond in both the HIV envelope glycoprotein, gp120, and its immune cell receptor, CD4, is involved in viral entry, and compounds that block cleavage of the disulfide bond in these proteins inhibit HIV entry and infection. The disulfide bonds in both proteins are cleaved at the cell surface by the small redox protein, thioredoxin. The target gp120 disulfide and its mechanism of cleavage were determined using a thioredoxin kinetic trapping mutant and mass spectrometry. A single disulfide bond was cleaved in isolated and cell surface gp120, but not the gp160 precursor, and the extent of the reaction was enhanced when gp120 was bound to CD4. The Cys(32) sulfur ion of thioredoxin attacks the Cys(296) sulfur ion of the gp120 V3 domain Cys(296)-Cys(331) disulfide bond, cleaving the bond. Considering that V3 sequences largely determine the chemokine receptor preference of HIV, we propose that cleavage of the V3 domain disulfide, which is facilitated by CD4 binding, regulates chemokine receptor binding. There are 20 possible disulfide bond configurations, and, notably, the V3 domain disulfide has the same unusual -RHStaple configuration as the functional disulfide bond cleaved in CD4.     

Structural analysis of the HIV-1 gp120 V3 loop: application to the HIV-Haiti isolates

The model describing the structure and conformational preferences of the HIV-Haiti V3 loop in the geometric spaces of Cartesian coordinates and dihedral angles was generated in terms of NMR spectroscopy data published in literature. To this end, the following successive steps were put into effect: (i) the NMR-based 3D structure for the HIV-Haiti V3 loop in water was built by computer modeling methods; (ii) the conformations of its irregular segments were analyzed and the secondary structure elements identified; and (iii) to reveal a common structural motifs in the HIV-Haiti V3 loop regardless of its environment variability, the simulated structure was collated with the one deciphered previously for the HIV-Haiti V3 loop in a water/trifluoroethanol (TFE) mixed solvent. As a result, the HIV-Haiti V3 loop was found to offer the highly variable fragment of gp120 sensitive to its environment whose changes trigger the large-scale structural rearrangements, bringing in substantial altering the secondary and tertiary structures of this functionally important site of the virus envelope. In spite of this fact, over half of amino acid residues that reside, for the most part, in the functionally important regions of the gp120 protein and may present promising targets for AIDS drug researches, were shown to preserve their conformational states in the structures under review. In particular, the register of these amino acids holds Asn-25 that is critical for the virus binding with primary cell receptor CD4 as well as Arg-3 that is critical for utilization of CCR5 co-receptor and heparan sulfate proteoglycans. The conservative structural motif embracing one of the potential sites of the gp120 N-linked glycosylation was detected, which seems to be a promising target for the HIV-1 drug design. The implications are discussed in conjunction with the literature data on the biological activity of the individual amino acids for the HIV-1 gp120 V3 loop.     

Structural Analysis of the HIV-1 gp120 V3 Loop: Application to the HIV-Haiti Isolates

Abstract

The model describing the structure and conformational preferences of the HIV-Haiti V3 loop in the geometric spaces of Cartesian coordinates and dihedral angles was generated in terms of NMR spectroscopy data published in literature. To this end, the following successive steps were put into effect: (i) the NMR-based 3D structure for the HIV-Haiti V3 loop in water was built by computer modeling methods; (ii) the conformations of its irregular segments were analyzed and the secondary structure elements identified; and (iii) to reveal a common structural motifs in the HIV-Haiti V3 loop regardless of its environment variability, the simulated structure was collated with the one deciphered previously for the HIV-Haiti V3 loop in a water/trifluoroethanol (TFE) mixed solvent.

As a result, the HIV-Haiti V3 loop was found to offer the highly variable fragment of gp120 sensitive to its environment whose changes trigger the large-scale structural rearrangements, bringing in substantial altering the secondary and tertiary structures of this functionally important site of the virus envelope. In spite of this fact, over half of amino acid residues that reside, for the most part, in the functionally important regions of the gp120 protein and may present promising targets for AIDS drug researches, were shown to preserve their conformational states in the structures under review. In particular, the register of these amino acids holds Asn-25 that is critical for the virus binding with primary cell receptor CD4 as well as Arg-3 that is critical for utilization of CCR5 co-receptor and heparan sulfate proteoglycans. The conservative structural motif embracing one of the potential sites of the gp120 N-linked glycosylation was detected, which seems to be a promising target for the HIV-1 drug design.

The implications are discussed in conjunction with the literature data on the biological activity of the individual amino acids for the HIV-1 gp120 V3 loop.     

Ion channel activation by SPC3, a peptide derived from the HIV-1 gp120 V3 loop.

SPC3 is a multibranched peptide containing eight identical GPGRAF motifs which are derived from the human immunodeficiency virus (HIV)-1 gp120 V3 loop consensus sequence. This molecule was reported to prevent the infection of CD4+ cells by various HIV-1 and HIV-2 strains. However, the molecular mode of action of SPC3 remains unclear. Here, we investigated the possibility that SPC3 could interact with alpha/beta-chemokine receptors following observations that, first, the V3 loop is likely to be involved in alpha/beta-chemokine receptor-dependent HIV entry and, second, natural ligands of these receptors are potent inhibitors of cell infection. To address this point, we examined the effects of SPC3 on Xenopus oocytes either uninjected or expressing exogenous human CXCR4 alpha-chemokine receptors. Extracellular applications of micromolar concentrations of SPC3 onto Xenopus oocytes trigger potent inward chloride currents which can be inhibited by increasing extracellular Ca2+ concentration. This effect can be blocked by chloride channel antagonists and is highly specific to SPC3 as it is not triggered by structural analogs of SPC3. The SPC3-induced chloride conductance in oocytes is alpha/beta-chemokine receptor dependent because: (i) SPC3 alters the sensitivity of this channel to external applications of human recombinant MIP-1alpha, a natural ligand of human CCR5 receptor, and (ii) the amplitude of the inward current could be increased by the expression of exogenous human CXCR4 chemokine receptor. The effect of SPC3 appears to rely on the activation of a phospholipase A2 signaling pathway, but is not affected by changes in cytosolic Ca2+ concentration, or by alterations in Gi/Go protein, adenylate cyclase, phospholipase C or protein kinase C activity. Altogether, the data indicate that SPC3 is capable of activating a surface alpha/beta-chemokine-like receptor-mediated signaling pathway in competent cells, thereby triggering, either directly or indirectly, a Ca2+-inactivated chloride conductance.     

Structure and Dynamics of the gp120 V3 Loop That Confers Noncompetitive Resistance in R5 HIV-1JR-FL to Maraviroc

Maraviroc, an (HIV-1) entry inhibitor, binds to CCR5 and efficiently prevents R5 human immunodeficiency virus type 1 (HIV-1) from using CCR5 as a coreceptor for entry into CD4⁺ cells. However, HIV-1 can elude maraviroc by using the drug-bound form of CCR5 as a coreceptor. This property is known as noncompetitive resistance. HIV-1_V3-M5 derived from HIV-1_JR-FLan is a noncompetitive-resistant virus that contains five mutations (I304V/F312W/T314A/E317D/I318V) in the gp120 V3 loop alone. To obtain genetic and structural insights into maraviroc resistance in HIV-1, we performed here mutagenesis and computer-assisted structural study. A series of site-directed mutagenesis experiments demonstrated that combinations of V3 mutations are required for HIV-1_JR-FLan to replicate in the presence of 1 µM maraviroc, and that a T199K mutation in the C2 region increases viral fitness in combination with V3 mutations. Molecular dynamic (MD) simulations of the gp120 outer domain V3 loop with or without the five mutations showed that the V3 mutations induced (i) changes in V3 configuration on the gp120 outer domain, (ii) reduction of an anti-parallel β-sheet in the V3 stem region, (iii) reduction in fluctuations of the V3 tip and stem regions, and (iv) a shift of the fluctuation site at the V3 base region. These results suggest that the HIV-1 gp120 V3 mutations that confer maraviroc resistance alter structure and dynamics of the V3 loop on the gp120 outer domain, and enable interactions between gp120 and the drug-bound form of CCR5.     

Solid-state NMR evidence for an antibody-dependent conformation of the V3 loop of HIV-1 gp120

Solid-state NMR measurements have been carried out on frozen solutions of the complex of a 24-residue peptide derived from the third variable (V3) loop of the HIV-1 envelope glycoprotein gp120 bound to the Fab fragment of an anti-gp120 antibody. The measurements place strong constraints on the conformation of the conserved central GPGR motif of the V3 loop in the antibody-bound state. In combination with earlier crystal structures of V3 peptide-antibody complexes and existing data on the cross-reactivity of the antibodies, the solid-state NMR measurements suggest that the Gly-Pro-Gly-Arg (GPGR) motif adopts an antibody-dependent conformation in the bound state and may be conformationally heterogeneous in unbound, full-length gp120. These measurements are the first application of solid-state NMR methods in a structural study of a peptide-protein complex.     

Structural and immunological reactivity of the principal neutralizing determinant V3 of glycoprotein gp 120 of HIV-1

The variable domain V3 in the outer glycoprotein gp 120 of HIV-1 is a highly important region with respect to immune response during the course of viral infection. Neutralizing antibodies are produced against this domain; in addition, it has been shown to be a functionally active epitope for T helper and cytotoxic T cells. The high degree of amino acid variability in individual HIV-isolates, however, limits the use of the V3-domain in approaches to vaccine development. In order to characterize the residues important for antibody interaction and binding to MHC class I proteins, we constructed a consensus sequence of the V3-domain with broad reactivity [1] and used synthetic peptides derived from this consensus with individual residues altered to alanine. These peptides were used as antigens in ELISA tests to define the amino acids which are important for binding to human and rabbit/anti-peptide immunoglobulins. In addition, we used these alanine-derived peptides in interaction studies with human HLA-A2.1 and mouse H-2D^d by testing their capacity to stabilize the respective MHC class I protein complexes on the surface of mutant cell lines T2 and RMA-S transfected with D^d gene. The experimental tests allowed us to define individual residues involved in antibody and MHC-protein interaction, respectively. In a further approach, we used those results to design interaction models with HLA-A2.1 and H-2D^d. Therefore, a structural model for H-2D^d was built that exhibits an overall similar conformation to the parental crystal structure of HLA-A2.1. The resulting interaction models show V3-peptide bound in an extended β-conformation with a bulge in its centre for both H-2D^d and HLA-A2.1 complexes. The N- and C-termini of V3 peptide reside in conserved pockets within both MHC-proteins. Anchoring residues could be determined that are crucial for the binding of the respective MHC class I haplotype. The cross-reactivity of V3-peptide in enhancing the expression of two different MHC class I molecules (H-2D^d and HLA-A2.1) is shown to be based on similar peptide binding that induces an almost identical peptide conformation.     

Molecular Recognition of CXCR4 by a Dual Tropic HIV-1 gp120 V3 Loop

HIV-1 cell entry is initiated by the interaction of the viral envelope glycoprotein gp120 with CD4, and chemokine coreceptors CXCR4 and CCR5. The molecular recognition of CXCR4 or CCR5 by the HIV-1 gp120 is mediated through the V3 loop, a fragment of gp120. The binding of the V3 loop to CXCR4 or CCR5 determines the cell tropism of HIV-1 and constitutes a key step before HIV-1 cell entry. Thus, elucidating the molecular recognition of CXCR4 by the V3 loop is important for understanding HIV-1 viral infectivity and tropism, and for the design of HIV-1 inhibitors. We employed a comprehensive set of computational tools, predominantly based on free energy calculations and molecular-dynamics simulations, to investigate the molecular recognition of CXCR4 by a dual tropic V3 loop. We report what is, to our knowledge, the first HIV-1 gp120 V3 loop:CXCR4 complex structure. The computationally derived structure reveals an abundance of polar and nonpolar intermolecular interactions contributing to the HIV-1 gp120:CXCR4 binding. Our results are in remarkable agreement with previous experimental findings. Therefore, this work sheds light on the functional role of HIV-1 gp120 V3 loop and CXCR4 residues associated with HIV-1 coreceptor activity.     

Molecular Recognition of CXCR4 by a Dual Tropic HIV-1 gp120 V3 Loop

HIV-1 cell entry is initiated by the interaction of the viral envelope glycoprotein gp120 with CD4, and chemokine coreceptors CXCR4 and CCR5. The molecular recognition of CXCR4 or CCR5 by the HIV-1 gp120 is mediated through the V3 loop, a fragment of gp120. The binding of the V3 loop to CXCR4 or CCR5 determines the cell tropism of HIV-1 and constitutes a key step before HIV-1 cell entry. Thus, elucidating the molecular recognition of CXCR4 by the V3 loop is important for understanding HIV-1 viral infectivity and tropism, and for the design of HIV-1 inhibitors. We employed a comprehensive set of computational tools, predominantly based on free energy calculations and molecular-dynamics simulations, to investigate the molecular recognition of CXCR4 by a dual tropic V3 loop. We report what is, to our knowledge, the first HIV-1 gp120 V3 loop:CXCR4 complex structure. The computationally derived structure reveals an abundance of polar and nonpolar intermolecular interactions contributing to the HIV-1 gp120:CXCR4 binding. Our results are in remarkable agreement with previous experimental findings. Therefore, this work sheds light on the functional role of HIV-1 gp120 V3 loop and CXCR4 residues associated with HIV-1 coreceptor activity.     

Identification of the potential regions of Epap-1 that interacts with V3 loop of HIV-1 gp120

Early pregnancy associated protein-1 (Epap-1), a 90 kDa glycoprotein present in first trimester placental tissue, inhibits HIV-1 entry through interaction with HIV-1 gp120 at V3 and C5 regions. In the present study, we have identified the specific 32 mer region of Epap-1 that can interact with V3 loop. This was achieved by docking between Epap-1 molecular model and gp120 and studying the interaction of peptides with gp120 in vitro. Out of four peptides analyzed, two peptides (P-2 and P-3) showed significant interaction with V3 domain (N = 8; N = 7) of gp120. In the studies conducted using soluble gp120 and virus, peptide P-2 has shown conserved interaction at V3 loop regions recognized by 257D and F425 antibodies and higher anti-viral activity. Also, P-2 inhibited cell fusion mediated dye transfer between gp120 expressing HL2/3 and CD4 expressing Sup T1 cells suggesting its inhibition of viral entry, which is further confirmed by its action on HIV infection mediated by Tat activated beta gal expression in TZM-bl cells. Further optimization of P-2 peptide showed that the anti-viral activity and gp120 interaction residues lie in the N-terminal region of the peptide. These results together suggest that P-2 inhibits viral entry through specific interaction at V3 loop region.     

The Influence of N-Linked Glycans on the Molecular Dynamics of the HIV-1 gp120 V3 Loop

N-linked glycans attached to specific amino acids of the gp120 envelope trimer of a HIV virion can modulate the binding affinity of gp120 to CD4, influence coreceptor tropism, and play an important role in neutralising antibody responses. Because of the challenges associated with crystallising fully glycosylated proteins, most structural investigations have focused on describing the features of a non-glycosylated HIV-1 gp120 protein. Here, we use a computational approach to determine the influence of N-linked glycans on the dynamics of the HIV-1 gp120 protein and, in particular, the V3 loop. We compare the conformational dynamics of a non-glycosylated gp120 structure to that of two glycosylated gp120 structures, one with a single, and a second with five, covalently linked high-mannose glycans. Our findings provide a clear illustration of the significant effect that N-linked glycosylation has on the temporal and spatial properties of the underlying protein structure. We find that glycans surrounding the V3 loop modulate its dynamics, conferring to the loop a marked propensity towards a more narrow conformation relative to its non-glycosylated counterpart. The conformational effect on the V3 loop provides further support for the suggestion that N-linked glycosylation plays a role in determining HIV-1 coreceptor tropism.     

HIV-1 gp120 V3 cholera toxin B subunit fusion gene expression in transgenic potato

A cDNA fragment encoding the V3 loop of human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein gp120 was fused to the cholera toxin B subunit gene (CTB-gp120) and transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation. The CTB-gp120 fusion gene was detected in genomic DNA from transformed potato leaves by PCR DNA amplification. Synthesis and assembly of the CTB-gp120 fusion protein into oligomeric structures of pentamer size was detected in transformed tuber extracts by immunoblot analysis. The binding of CTB-gp120 fusion protein pentamers to intestinal epithelial cell membrane glycolipid receptors was quantified by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). The ELISA results indicated that CTB-gp120 fusion protein made up 0.002-0.004% of the total soluble tuber protein. Synthesis of CTB-gp120 monomers and their assembly into biologically active oligomers in transformed potato tuber tissues demonstrates for the first time the expression of HIV-1 gp120 in plants and emphasizes the feasibility of using edible plant-based vaccination for protection against HIV-1 infection.     

Accurate and efficient gp120 V3 loop structure based models for the determination of HIV-1 co-receptor usage

Background  

HIV-1 targets human cells expressing both the CD4 receptor, which binds the viral envelope glycoprotein gp120, as well as either the CCR5 (R5) or CXCR4 (X4) co-receptors, which interact primarily with the third hypervariable loop (V3 loop) of gp120. Determination of HIV-1 affinity for either the R5 or X4 co-receptor on host cells facilitates the inclusion of co-receptor antagonists as a part of patient treatment strategies. A dataset of 1193 distinct gp120 V3 loop peptide sequences (989 R5-utilizing, 204 X4-capable) is utilized to train predictive classifiers based on implementations of random forest, support vector machine, boosted decision tree, and neural network machine learning algorithms. An in silico mutagenesis procedure employing multibody statistical potentials, computational geometry, and threading of variant V3 sequences onto an experimental structure, is used to generate a feature vector representation for each variant whose components measure environmental perturbations at corresponding structural positions.     

Dual conformations for the HIV-1 gp120 V3 loop in complexes with different neutralizing fabs.

BACKGROUND: The third hypervariable (V3) loop of HIV-1 gp120 has been termed the principal neutralizing determinant (PND) of the virus and is involved in many aspects of virus infectivity. The V3 loop is required for viral entry into the cell via membrane fusion and is believed to interact with cell surface chemokine receptors on T cells and macrophages. Sequence changes in V3 can affect chemokine receptor usage, and can, therefore, modulate which types of cells are infected. Antibodies raised against peptides with V3 sequences can neutralize laboratory-adapted strains of the virus and inhibit syncytia formation. Fab fragments of these neutralizing antibodies in complex with V3 loop peptides have been studied by X-ray crystallography to determine the conformation of the V3 loop. RESULTS: We have determined three crystal structures of Fab 58.2, a broadly neutralizing antibody, in complex with one linear and two cyclic peptides the amino acid sequence of which comes from the MN isolate of the gp120 V3 loop. Although the peptide conformations are very similar for the linear and cyclic forms, they differ from that seen for the identical peptide bound to a different broadly neutralizing antibody, Fab 59.1, and for a similar peptide bound to the MN-specific Fab 50.1. The conformational difference in the peptide is localized around residues Gly-Pro-Gly-Arg, which are highly conserved in different HIV-1 isolates and are predicted to adopt a type II beta turn. CONCLUSIONS: The V3 loop can adopt at least two different conformations for the highly conserved Gly-Pro-Gly-Arg sequence at the tip of the loop. Thus, the HIV-1 V3 loop has some inherent conformational flexibility that may relate to its biological function.     

Recognition properties of V3-specific antibodies to V3 loop peptides derived from HIV-1 gp120 presented in multiple conformations

To identify structural constraints and amino acid sequences important for antibody recognition of the third variable domain (V3) of HIV-1 gp120, we have studied the solution conformation of three 35-mer circular V3 loop peptides derived from HIV-1 strains which differ in syncytium- (SI) and non-syncytium-inducing (NSI) capacity. In addition to 2D NMR and CD analyses, fluid- and solid-phase immunoassays were performed using V3-specific antibodies to V3 peptides and gp120 derived from different strains of HIV-1. NMR and CD spectroscopy indicated that circular and linear V3 loops exist in water as a dynamic ensemble of multiple conformations. Amino acid substitutions and biochemical modifications of the V3 loop were found to affect antibody binding depending on the presentation of the antigens. From NMR observations and immunological experiments, we provide evidence for a V3 loop specific monoclonal antibody interaction which is directed predominantly against linear epitopes rather than against discontinuous epitopes. The absence of a single defined solution conformation of 35-mer circular V3 peptides should be taken into account when using V3-related peptides to investigate structural elements in the V3 domain of the gp120 envelope protein of HIV-1 involved in biological processes of the virus.