Conformational Changes In Gp120 Research Articles

Novel Recombinant Engineered gp41 N-terminal Heptad Repeat Trimers and Their Potential as Anti-HIV-1 Therapeutics or Microbicides

Peptides derived from N-terminal heptad repeat (NHR) of the HIV-1 gp41 are generally poor inhibitors of HIV-1 entry, because they tend to aggregate and do not form a trimeric coiled-coil. In this study, we have fused portions of gp41 NHR, e.g. N36 or N28, to the T4 fibritin trimerization domain, Foldon (Fd), thus constructing novel NHR trimers, designated N36Fd or N28Fd, which could be expressed in Escherichia coli cells. The purified N36Fd and N28Fd exhibited SDS-resistant trimeric coiled-coil conformation with improved alpha-helicity compared with the corresponding N-peptides. They could interact with a C-peptide (e.g. C34) to form stable six-helix bundle and possessed potent anti-HIV-1 activity against a broad spectrum of HIV-1 strains. N28Fd was effective against T20-resistant HIV-1 variants and more resistant to proteinase K compared with T20 (enfuvirtide), a C-peptide-based HIV fusion inhibitor. Therefore, N28Fd trimer has great potentials for further development as an affordable therapeutic or microbicide for treatment and prevention of HIV-1 infection.

A Modeled Structure of an Aptamer−gp120 Complex Provides Insight into the Mechanism of HIV-1 Neutralization

The HIV-1 envelope glycoprotein, gp120, is a key target for a class of drugs called entry inhibitors. Here we used molecular modeling to construct a three-dimensional model of an anti-gp120 RNA aptamer, B40t77, alone and in complex with gp120. An initial model of B40t77 was built from the predicted secondary structure and then subjected to a combination of energy minimization and molecular dynamics. To model the B40t77-gp120 complex, we docked the B40t77 predicted structure onto the CD4-induced epitope of the gp120 crystal structure. A series of gp120 point mutations in the predicted B40t77-gp120 interface were measured for their binding affinity for B40t77 by surface plasmon resonance. According to the model, of the 10 gp120 amino acids that showed a reduction in the level of binding when mutated to alanine, all of them are modeled as making direct contact with B40t77 as part of a hydrogen bonding network. Comparison by electron microscopy of the B40t77-gp120 complex with gp120 alone revealed that only the longest dimension of the complex significantly increased in length, in a manner consistent with the predicted model. Binding assays revealed that B40t77 can weaken the binding of gp120 to the monoclonal antibodies B6, B12, and 2G12, none of which have binding sites that overlap with B40t77, as well as strengthen the binding to the antibody 19b. Thus, B40t77 may induce distant conformational changes in gp120 that disrupt its association with host cells and may suggest a mechanism for aptamer neutralization of HIV-1.

Role for the disulfide-bonded region of human immunodeficiency virus type 1 gp41 in receptor-triggered activation of membrane fusion function

The conserved disulfide-bonded region (DSR) of the human immunodeficiency virus type 1 (HIV-1) fusion glycoprotein, gp41, mediates association with the receptor-binding glycoprotein, gp120. Interactions between gp120, CD4 and chemokine receptors activate the fusion activity of gp41. The introduction of W596L and W610F mutations to the DSR of HIV-1 QH1549.13 blocked viral entry and hemifusion without affecting gp120–gp41 association. The fusion defect correlated with inhibition of CD4-triggered gp41 pre-hairpin formation, consistent with the DSR mutations having decoupled receptor-induced conformational changes in gp120 from gp41 activation. Our data implicate the DSR in sensing conformational changes in the gp120–gp41 complex that lead to fusion activation.

Topological Layers in the HIV-1 gp120 Inner Domain Regulate gp41 Interaction and CD4-Triggered Conformational Transitions

The entry of human immunodeficiency virus (HIV-1) into cells is initiated by binding of the gp120 exterior envelope glycoprotein to the receptor, CD4. How does CD4 binding trigger conformational changes in gp120 that allow the gp41 transmembrane envelope glycoprotein to mediate viral-cell membrane fusion? The transition from the unliganded to the CD4-bound state is regulated by two potentially flexible topological layers (layers 1 and 2) in the gp120 inner domain. Both layers apparently contribute to the noncovalent association of unliganded gp120 with gp41. After CD4 makes initial contact with the gp120 outer domain, layer 1-layer 2 interactions strengthen gp120-CD4 binding by reducing the off rate. Layer 1-layer 2 interactions also destabilize the activated state induced on HIV-1 by treatment with soluble CD4. Thus, despite lack of contact with CD4, the gp120 inner-domain layers govern CD4 triggering by participating in conformational transitions within gp120 and regulating the interaction with gp41.

Structure of HIV-1 gp120 with gp41-interactive region reveals layered envelope architecture and basis of conformational mobility

The viral spike of HIV-1 is composed of three gp120 envelope glycoproteins attached noncovalently to three gp41 transmembrane molecules. Viral entry is initiated by binding to the CD4 receptor on the cell surface, which induces large conformational changes in gp120. These changes not only provide a model for receptor-triggered entry, but affect spike sensitivity to drug- and antibody-mediated neutralization. Although some of the details of the CD4-induced conformational change have been visualized by crystal structures and cryoelectron tomograms, the critical gp41-interactive region of gp120 was missing from previous atomic-level characterizations. Here we determine the crystal structure of an HIV-1 gp120 core with intact gp41-interactive region in its CD4-bound state, compare this structure to unliganded and antibody-bound forms to identify structurally invariant and plastic components, and use ligand-oriented cryoelectron tomograms to define component mobility in the viral spike context. Newly defined gp120 elements proximal to the gp41 interface complete a 7-stranded beta-sandwich, which appeared invariant in conformation. Loop excursions emanating from the sandwich form three topologically separate--and structurally plastic--layers, topped off by the highly glycosylated gp120 outer domain. Crystal structures, cryoelectron tomograms, and interlayer chemistry were consistent with a mechanism in which the layers act as a shape-changing spacer, facilitating movement between outer domain and gp41-associated beta-sandwich and providing for conformational diversity used in immune evasion. A "layered" gp120 architecture thus allows movement among alternative glycoprotein conformations required for virus entry and immune evasion, whereas a beta-sandwich clamp maintains gp120-gp41 interaction and regulates gp41 transitions.

CD4 mimics targeting the mechanism of HIV entry

A structure–activity relationship study was conducted of several CD4 mimicking small molecules which block the interaction between HIV-1 gp120 and CD4. These CD4 mimics induce a conformational change in gp120, exposing its co-receptor-binding site. This induces a highly synergistic interaction in the use in combination with a co-receptor CXCR4 antagonist and reveals a pronounced effect on the dynamic supramolecular mechanism of HIV-1 entry.

Molecular docking studies of dithionitrobenzoic acid and its related compounds to protein disulfide isomerase: computational screening of inhibitors to HIV-1 entry

BackgroundEntry of HIV-1 into human lymphoid requires activities of viral envelope glycoproteins gp120 and gp41, and two host-cell proteins, the primary receptor CD4 and a chemokine co-receptor. In addition, a third cell-surface protein called protein disulfide isomerase (PDI) is found to play a major role in HIV-1 entry. PDI is capable of mediating thio-disulfide interchange reactions and could enable the reduction of gp120 disulfide bonds, which triggers the major conformational changes in gp120 and gp41 required for virus entry. In this scenario, inhibition of HIV-1 entry can be brought about by introducing agents that can block thiol-disulfide interchange reaction of cell surface PDI. There have been studies with agents that inhibit PDI activity, but the exact mode of binding remains to be elucidated; this might provide insights to develop new drugs to target PDI. This study attempts to perceive the mode of binding of dithionitrobenzoic acid (DTNB), and its structurally related compounds on PDI enzyme.ResultsWe performed molecular docking simulation with six different inhibitors (ligand), which includes DTNB, NSC695265, thionitrobenzoic acid, 2-nitro-5-thiocyanobenzoic acid, 2-nitro-5-sulfo-sulfonyl-benzoic acid and NSC517871 into the redox-active site [C37-G38-H39-C40] of the PDI enzyme and the activity was inferred by redox inhibitory models. All ligands showed favorable interactions and most of them seemed to bind to hydrophobic amino acids Ala34, Trp36, Cys37, Cys40, His39, Thr68 and Phe80. The redox inhibitory conformations were energetically and statistically favored and supported the evidence from wet laboratory experiments reported in the literature.ConclusionWe demonstrated that in silico docking experiment can be effectively carried out to recognize the redox inhibitory models of PDI with inhibitor molecules. Interestingly we found that number of docked clusters with each ligand varies in the range of five to eight and conveys that the binding specificity of each inhibitor varies for PDI. We also identified that Cys37 of the enzyme plays an important role in hydrogen bonding with inhibitors. This residue can be considered to being an active site for anti-HIV drug design. Therefore, by inhibiting PDI, one can, not only prevent the viral entry but also circumvent the problem of viral resistance

Small-Molecule CD4 Mimics Interact with a Highly Conserved Pocket on HIV-1 gp120

Human immunodeficiency virus (HIV-1) interaction with the primary receptor, CD4, induces conformational changes in the viral envelope glycoproteins that allow binding to the CCR5 second receptor and virus entry into the host cell. The small molecule NBD-556 mimics CD4 by binding the gp120 exterior envelope glycoprotein, moderately inhibiting virus entry into CD4-expressing target cells and enhancing CCR5 binding and virus entry into CCR5-expressing cells lacking CD4. Studies of NBD-556 analogs and gp120 mutants suggest that (1) NBD-556 binds within the Phe 43 cavity, a highly conserved, functionally important pocket formed as gp120 assumes the CD4-bound conformation; (2) the NBD-556 phenyl ring projects into the Phe 43 cavity; (3) enhancement of CD4-independent infection by NBD-556 requires the induction of conformational changes in gp120; and (4) increased affinity of NBD-556 analogs for gp120 improves antiviral potency during infection of CD4-expressing cells.

Peptide Mimic of the HIV Envelope gp120–gp41 Interface

The human immunodeficiency virus envelope glycoprotein (Env) is composed of surface (gp120) and transmembrane (gp41) subunits, which are noncovalently associated on the viral surface. Human immunodeficiency virus Env mediates viral entry after undergoing a complex series of conformational changes induced by interaction with cellular CD4 and a chemokine coreceptor. These changes propagate from gp120 to gp41 via the gp120–gp41 interface, ultimately exposing gp41 and allowing it to form the trimer-of-hairpins structure that provides the driving force for membrane fusion. Key unresolved questions about the gp120–gp41 interface include the specific regions of gp41 and gp120 involved, the mechanism by which receptor and coreceptor-binding-induced conformational changes in gp120 are communicated to gp41, how trimer-of-hairpins formation is prevented in the prefusogenic gp120–gp41 complex, and, ultimately, the structure of the prefusion gp120–gp41 complex. Here, we develop a biochemical model system that mimics a key portion of the gp120–gp41 interface in the prefusogenic state. We find that a gp41 fragment containing the disulfide bond loop and C-peptide region binds primarily to the gp120 C5 region and that this interaction is incompatible with trimer-of-hairpins formation. Based on these data, we propose that in prefusogenic Env, gp120 sequesters the gp41 C-peptide region away from the N-trimer region, preventing trimer-of-hairpins formation until coreceptor binding disrupts this interface. This model system is a valuable tool for studying the gp120–gp41 complex, conformational changes induced by CD4 and coreceptor binding, and the mechanism of membrane fusion.

CD4 Interacts Constitutively with Multiple CCR5 at the Plasma Membrane of Living Cells

The entry of human immunodeficiency virus into target cells requires successive interactions of the viral envelope glycoprotein gp120 with CD4 and the chemokine receptors CCR5 or CXCR4. We previously demonstrated, by Förster resonance energy transfer experiments, the constitutive association of CD4 and CCR5 at the surface of living cells. We therefore speculated that this interaction may correlate with compartmentalization of CD4 and CCR5 within the plasma membrane. Here, we characterize the lateral distribution, the dynamics, and the stoichiometry of these receptors in living cells stably expressing CD4 and/or CCR5 by means of fluorescence recovery after photobleaching at variable radii experiments. We found that (i) these receptors expressed alone are confined into 1-microm-sized domains, (ii) CD4-CCR5 associations occur outside and inside smaller domains, and (iii) these interactions involve multiple CCR5 molecules per CD4.

Critical role of Arg 59 in the high-affinity gp120-binding region of CD4 for human immunodeficiency virus type 1 infection

Human immunodeficiency virus type 1 (HIV-1) infection is initiated by the binding of the viral envelope protein gp120 to the host cell CD4 receptor through a high-affinity interaction involving amino acids 39–60 within the CD4. We obtained evidence demonstrating functional importance of this region in CD4 for viral infectivity by showing that a synthetic peptide corresponding to this CD4 sequence exhibited competitive binding to gp120 and significantly reduced infection by diverse HIV-1 strains, including primary isolates. Treatment of HIV-1-infected cells with this CD4 peptide induced shedding of gp120 and exposure of the transmembrane protein gp41. Furthermore, we observed that deletion or substitution of arginine at position 59 (Arg 59) within the CD4 peptide sequence abrogated its gp120-shedding property. These results indicate a critical role for Arg 59 in the CD4 for conformational changes in gp120 during the sequential process of entry and infection by HIV-1.

Induction of a Nonproductive Conformational Change in gp120 by a Small Molecule HIV Type 1 Entry Inhibitor

Conformational changes in HIV-1 envelope glycoproteins, gp120 and gp41, is a dynamic process essential for HIV-1 entry. Here we show that a small molecule HIV-1 entry inhibitor, IC9564, induces a conformational change in gp120. The conformational change in gp120 is evidenced by a significant increase in the binding of a conformational monoclonal antibody 17b. As a result of the conformational effect, IC9564 significantly enhances the neutralizing activity of 17b. Unlike CD4, IC9564 does not trigger conformational changes in gp41. In fact, IC9564 inhibits CD4-induced conformational changes in gp41. Thus, IC9564 exploits the dynamic nature of gp120 by inducing a nonproductive gp120 conformation that is not able to trigger a conformational change in gp41 for membrane fusion.

HIV-1 infection: Is it time to reconsider our concepts?

The long asymptomatic phase of HIV infection is critical in the progression to AIDS. It probably reflects an ancestral relationship with lentiviruses stemming from the primate-simian immunodeficiency virus evolutionary pathway leading to an idiosyncratic immune tolerance, which needs to be understood if effective vaccines are to be rationally designed. The majority of CD4+ T cells that die due to HIV-1 in the asymptomatic phase are not infected with the virus. Transmission of the predominant HIV-1 R5 variants to T cells is mediated by infected monocyte-derived macrophages. The two cell populations come into intimate contact mainly in the lymph nodes during antigen presentation where there is also active viral replication. We propose that HIV exploits antigen presentation to access target T cells and evade immune surveillance. This is achieved at the assembly point of an immunological synapse between an antigen presenting, HIV-1-infected macrophage and a responding effector/memory CD4+ T cell. Viral envelope gp120 glycoproteins proximal to MHC II molecules cross-link with T cell CD4 molecules, thus establishing a supra molecular immuno-viral synapse. The interaction results in conformational changes of gp120 exposing its V3 domain. Ionic interaction of this domain with the synapse-recruited chemokine receptor CCR5 dimerizes the receptor triggering intracellular signals that contribute to T cell receptor transactivation pathways and subsequent enhancement of T cell activation. HIV-downregulated MHC II gives weak immune complexes. Disruption of the immuno-viral synapse before completion of cell entry is a frequent outcome condemning the responding T cell to a premature activation-induced T cell death. Information on the assembly, mechanistic and functional interactions at the immuno-viral synapses may well assist in elucidating new strategies to combat HIV infection.

Inhibition of HIV-1 Entry by Inducing a Nonproductive Conformational Change in gp120

Conformational change in HIV-1 gp120 is a dynamic process essential for HIV-1 entry. It is not clear whether the dynamic nature of gp120 could be exploited to abort the viral entry machinery. Here we show that a small molecule entry inhibitor, IC9564, induces a conformational change in gp120 and locks the envelope into fusion incompetent conformation. Binding of IC9564 to HIV-1 envelope results in the exposure of CD4i epitopes that are ally concealed in gp120. As a result of the conformational effect, IC9564 significantly enhances the neutralizing activity of 17b that binds to an epitope overlapping chemokine receptor binding site. Unlike CD4, IC9564-induced conformational change in gp120 does not trigger a conformational change in gp41. In fact, IC9564 inhibits CD4 induced conformational changes in gp41. The binding site of IC9564 remains elusive due to the fact that mutations in both gp120 and gp41 could change IC9564 sensitivity. Nevertheless, a common effect of these mutations is that conformation of gp120 is changed to conceal conserved epitopes such as CD4i. In summary, IC9564 exploits the dynamic nature of HIV-1 gp120 by inducing a nonproductive conformational change in gp120 and prevents HIV-1 from entering the cells. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

The Synthetic Peptide Derived from the NH2-terminal Extracellular Region of an Orphan G Protein-coupled Receptor, GPR1, Preferentially Inhibits Infection of X4 HIV-1

Several G protein-coupled receptors (GPCRs) serve as co-receptors for entry of human immunodeficiency virus type 1 (HIV-1) into target cells. Here we report that a synthetic peptide derived from the NH2-terminal extracellular region of an orphan GPCR, GPR1 (GPR1ntP-(1-27); MEDLEETLFEEFENYSYDLDYYSLESC), inhibited infection of not only an HIV-1 variant that uses GPR1 as a co-receptor, but also X4, R5, and R5X4 viruses. Among these HIV-1 strains tested, viruses that can utilize CXCR4 as their co-receptors were preferentially inhibited. Inhibition of early steps in X4 virus replication was also detected in the primary human peripheral blood lymphocytes. GPR1ntP-(1-27) directly interacted with recombinant X4 envelope glycoprotein (rgp120). This interaction was neither inhibited nor enhanced by the soluble CD4 (sCD4) but inhibited by the anti-third variable (V3) loop-specific monoclonal antibody and heparin known to bind to the V3 loop. Although the conformational changes in gp120, including the V3 loop, have been reported to be required for its interaction with a co-receptor after binding of gp120 to CD4, it has also been reported that the V3 loop is already exposed on the surface of virions before interaction with CD4. We found that GPR1ntP-(1-27) blocked binding of virus to the cells, and this peptide equally bound to rgp120 in the presence or absence of sCD4. Because we detected the binding of GPR1ntP-(1-27) to the highly purified virions even in the absence of sCD4, GPR1ntP-(1-27) probably recognized the V3 loop exposed on the virions, and this interaction was responsible for the anti-HIV-1 activity of GPR1ntP-(1-27).

Potency of HIV-1 envelope glycoprotein gp120 antibodies to inhibit the interaction of DC-SIGN with HIV-1 gp120

The interaction of DC-SIGN with gp120 provides an attractive target for intervention of HIV-1 transmission. Here, we have investigated the potency of gp120 antibodies to inhibit the DC-SIGN–gp120 interaction. We demonstrate that although the V3 loop is not essential for DC-SIGN binding, antibodies against the V3 loop partially inhibit DC-SIGN binding, suggesting that these antibodies sterically hinder DC-SIGN binding to gp120. Polyclonal antibodies raised against non-glycosylated gp120 inhibited both low and high avidity DC-SIGN–gp120 interactions in contrast to polyclonal antibodies raised against glycosylated gp120. Thus, glycans present on gp120 may prevent the generation of antibodies that block the DC-SIGN–gp120 interactions. Moreover, the polyclonal antibodies against non-glycosylated gp120 efficiently inhibited HIV-1 capture by both DC-SIGN transfectants and immature dendritic cells. Therefore, non-glycosylated gp120 may be an attractive immunogen to elicit gp120 antibodies that block the binding to DC-SIGN. Furthermore, we demonstrate that DC-SIGN binding to gp120 enhanced CD4 binding, suggesting that DC-SIGN induces conformational changes in gp120, which may provide new targets for neutralizing antibodies.

Recent advances in understanding the molecular mechanisms of HIV-1 entry and fusion: revisiting current targets and considering new options for therapeutic intervention.

Recent advances in our understanding of the cellular and molecular mechanisms of HIV-1 entry provide the basis for novel therapeutic strategies that prevent viral penetration of the target cell-membrane, while reducing detrimental virus and treatment effects on cells and prolonging virion exposure to immune defenses. A number of potential sites for therapeutic intervention become accessible during the narrow window between virus attachment and the subsequent fusion of viral envelope with the cell membrane. Initial approaches considered for prevention of HIV-1 entry included the use of natural ligands, small-molecule inhibitors and/or monoclonal antibodies, which could interfere with gp120-CD4 and/or gp120-CXCR4/CCR5 interaction. Others avenues pursued were the use of agents that interfere with the conformational changes of gp120 or gp41 leading to subsequent fusion of viral and cellular membranes. More recently, strategies have emerged that involve inhibition of thiol/disulfide oxidoreductases (factors which facilitate Env transition from an inactive to a fusion-competent conformation) to block redox exchange, thereby impeding the entry process. This review provides a summary of the cellular and viral factors mediating the HIV-1 entry process, with an emphasis on novel therapeutics targeting Env-receptor/coreceptor interaction, Env conformational change and the membrane fusion process.

Role of hydrophobic residues in the central ectodomain of gp41 in maintaining the association between human immunodeficiency virus type 1 envelope glycoprotein subunits gp120 and gp41.

The interaction between the gp120 and gp41 subunits of the human immunodeficiency virus envelope glycoprotein serves to stabilize the virion form of the complex and to transmit receptor-induced conformational changes in gp120 to trigger the membrane fusion activity of gp41. In this study, we used site-directed mutagenesis to identify amino acid residues in the central ectodomain of gp41 that contribute to the stability of the gp120-gp41 association. We identified alanine mutations at six positions, including four tryptophan residues, which result in mutant envelope glycoprotein complexes that fail to retain gp120 on the cell surface. These envelope glycoproteins readily shed their gp120 and are unable to mediate cell-cell fusion. These findings suggest an important role for the conserved bulky hydrophobic residues in stabilizing the gp120-gp41 complex.

Mutations in gp41 and gp120 of HIV-1 isolates resistant to hexa-arginine neomycin B conjugate

Aminoglycoside–arginine conjugates (AACs) inhibit HIV-1 replication and act as Tat antagonists. AACs compete with monoclonal antibody binding to CXCR4, compete with SDF-1α and HIV-1 gp120 cellular uptake, indicating that they interfere with initial steps of HIV-1 infection. We here present the selection of HIV-1 isolates resistant to hexa-arginine neomycin B conjugate (NeoR6), the most potent anti-HIV-1 AAC. We found in the NeoR6-resistant isolates the following mutations in gp120: I339T in the C3 region, S372L in the V4 region, and Q395K in the C4 region; and in gp41: S668R and F672Y in the ‘heptad repeat’ 2 (HR2) region. These findings strongly suggest that NeoR6 obstructs HIV-1 replication by interfering with the fusion step, dependent on both conformational changes in gp120 following CD4 and CXCR4 interaction, as well as by conformational changes in gp41 induced by HR1 and HR2 interaction. The AACs may thus represent a novel family of fusion inhibitors.

Structural and functional characterization of the human CCR5 receptor in complex with HIV gp120 envelope glycoprotein and CD4 receptor by molecular modeling studies.

The entry of human immunodeficiency virus (HIV) into cells depends on a sequential interaction of the gp120 envelope glycoprotein with the cellular receptors CD4 and members of the chemokine receptor family. The CC chemokine receptor CCR5 is such a receptor for several chemokines and a major coreceptor for the entry of R5 HIV type-1 (HIV-1) into cells. Although many studies focus on the interaction of CCR5 with HIV-1, the corresponding interaction sites in CCR5 and gp120 have not been matched. Here we used an approach combining protein structure modeling, docking and molecular dynamics simulation to build a series of structural models of the CCR5 in complexes with gp120 and CD4. Interactions such as hydrogen bonds, salt bridges and van der Waals contacts between CCR5 and gp120 were investigated. Three snapshots of CCR5-gp120-CD4 models revealed that the initial interactions of CCR5 with gp120 are involved in the negatively charged N-terminus (Nt) region of CCR5 and positively charged bridging sheet region of gp120. Further interactions occurred between extracellular loop2 (ECL2) of CCR5 and the base of V3 loop regions of gp120. These interactions may induce the conformational changes in gp120 and lead to the final entry of HIV into the cell. These results not only strongly support the two-step gp120-CCR5 binding mechanism, but also rationalize extensive biological data about the role of CCR5 in HIV-1 gp120 binding and entry, and may guide efforts to design novel inhibitors.