Binding of full-length HIV-1 gp120 to CD4 induces structural reorientation around the gp120 core

Small-angle x-ray scattering data on the unliganded full-length fully glycosylated HIV-1 gp120, the soluble CD4 (domains 1-2) receptor, and their complex in solution are presented. Ab initio structure restorations using these data provides the first look at the envelope shape for the unliganded and the complexed gp120 molecule. Fitting known crystal structures of the unliganded SIV and the complexed HIV gp120 core regions within our resultant shape constraints reveals movement of the V3 loop upon binding.     

Use of Human CD4 Transgenic Mice for Studying Immunogenicity of HIV-1 Envelope Protein gp120

HIV-1 envelope protein, gp120, is a major immunogenic protein of the AIDS virus. A specific feature of this protein is its interaction with the receptor protein, human CD4, an important component of the immune system. This interaction might affect the immunogenic properties of the gp120 and modulate the immune response towards HIV. To test this hypothesis we used human CD4-transgenic mice for immunization with gp120. The dynamics of the immune response towards gp120, CD4 and other proteins was followed. The results show that the primary immune response to gp120 (two weeks) developed somewhat faster in CD4-transgenic mice versus non-transgenic mice. Both animals, however, ultimately mounted the same level of response over time. The primary immune response to gp120 when complexed with soluble CD4 before the immunization, developed similarly in both groups. The secondary immune response was earlier and markedly stronger in non-transgenic mice compared with the transgenic mice where a less efficient memory response to gp120 was observed. The ability of gp120 to directly interact with CD4+ helper lymphocytes appears to affect the humoral response towards this antigen. Moreover, these effects illustrate how viral modulation of these cells may in turn lead to potentially different states of immunological equilibrium.     

Host-soluble galectin-1 promotes HIV-1 replication through a direct interaction with glycans of viral gp120 and host CD4

Sexual transmission of HIV-1 requires virus adsorption to a target cell, typically a CD4(+) T lymphocyte residing in the lamina propria, beneath the epithelium. To escape the mucosal clearance system and reach its target cells, HIV-1 has evolved strategies to circumvent deleterious host factors. Galectin-1, a soluble lectin found in the underlayers of the epithelium, increases HIV-1 infectivity by accelerating its binding to susceptible cells. By comparison, galectin-3, a family member expressed by epithelial cells and part of the mucosal clearance system, does not perform similarly. We show here that galectin-1 directly binds to HIV-1 in a β-galactoside-dependent fashion through recognition of clusters of N-linked glycans on the viral envelope gp120. Unexpectedly, this preferential binding of galectin-1 does not rely on the primary sequence of any particular glycans. Instead, glycan clustering arising from the tertiary structure of gp120 hinders its binding by galectin-3. Increased polyvalency of a specific ligand epitope is a common strategy for glycans to increase their avidity for lectins. In this peculiar occurrence, glycan clustering is instead exploited to prevent binding of gp120 by galectin-3, which would lead to a biological dead-end for the virus. Our data also suggest that galectin-1 binds preferentially to CD4, the host receptor for gp120. Together, these results suggest that HIV-1 exploits galectin-1 to enhance gp120-CD4 interactions, thereby promoting virus attachment and infection events. Since viral adhesion is a rate-limiting step for HIV-1 entry, modulation of the gp120 interaction with galectin-1 could thus represent a novel approach for the prevention of HIV-1 transmission.     

Atomic insight into the CD4 binding-induced conformational changes in HIV-1 gp120

The entry of HIV-1 into a target cell requires gp120 and receptor CD4 as well as coreceptor CCR5/CXCR4 recognition events associated with conformational changes of the involved proteins. The binding of CD4 to gp120 is the initiation step of the whole process involving structural rearrangements that are crucial for subsequent pathways. Despite the wealth of knowledge about the gp120/CD4 interactions, details of the conformational changes occurring at this stage remain elusive. We have performed molecular dynamics simulations in explicit solvent based on the gp120/CD4/CD4i crystal structure in conjunction with modeled V3 and V4 loops to gain insight into the dynamics of the binding process. Three differentiated interaction modes between CD4 and gp120 were found, which involve electrostatics, hydrogen bond and van der Waals networks. A "binding funnel" model is proposed based on the dynamical nature of the binding interface together with a CD4-attraction gradient centered in gp120 at the CD4-Phe43-binding cavity. Distinct dynamical behaviors of free and CD4-bound gp120 were monitored, which likely represent the ground and pre-fusogenic states, respectively. The transition between these states revealed concerted motions in gp120 leading to: i) loop contractions around the CD4-Phe43-insertion cavity; ii) stabilization of the four-stranded "bridging sheet" structure; and iii) translocation and clustering of the V3 loop and the bridging sheet leading to the formation of the coreceptor binding site. Our results provide new insight into the dynamic of the underlying molecular recognition mechanism that complements the biochemical and structural studies.     

Broad-spectrum inhibition of HIV-1 by a monoclonal antibody directed against a gp120-induced epitope of CD4

To penetrate susceptible cells, HIV-1 sequentially interacts with two highly conserved cellular receptors, CD4 and a chemokine receptor like CCR5 or CXCR4. Monoclonal antibodies (MAbs) directed against such receptors are currently under clinical investigation as potential preventive or therapeutic agents. We immunized Balb/c mice with molecular complexes of the native, trimeric HIV-1 envelope (Env) bound to a soluble form of the human CD4 receptor. Sera from immunized mice were found to contain gp120-CD4 complex-enhanced antibodies and showed broad-spectrum HIV-1-inhibitory activity. A proportion of MAbs derived from these mice preferentially recognized complex-enhanced epitopes. In particular, a CD4-specific MAb designated DB81 (IgG1Κ) was found to preferentially bind to a complex-enhanced epitope on the D2 domain of human CD4. MAb DB81 also recognized chimpanzee CD4, but not baboon or macaque CD4, which exhibit sequence divergence in the D2 domain. Functionally, MAb DB81 displayed broad HIV-1-inhibitory activity, but it did not exert suppressive effects on T-cell activation in vitro. The variable regions of the heavy and light chains of MAb DB81 were sequenced. Due to its broad-spectrum anti-HIV-1 activity and lack of immunosuppressive effects, a humanized derivative of MAb DB81 could provide a useful complement to current preventive or therapeutic strategies against HIV-1.     

Thermodynamics of binding of a low-molecular-weight CD4 mimetic to HIV-1 gp120

NBD-556 and the chemically and structurally similar NBD-557 are two low-molecular weight compounds that reportedly block the interaction between the HIV-1 envelope glycoprotein gp120 and its receptor, CD4. NBD-556 binds to gp120 with a binding affinity of 2.7 x 10(5) M(-1) (K(d) = 3.7 muM) in a process characterized by a large favorable change in enthalpy partially compensated by a large unfavorable entropy change, a thermodynamic signature similar to that observed for binding of sCD4 to gp120. NBD-556 binding is associated with a large structuring of the gp120 molecule, as also demonstrated by CD spectroscopy. NBD-556, like CD4, activates the binding of gp120 to the HIV-1 coreceptor, CCR5, and to the 17b monoclonal antibody, which recognizes the coreceptor binding site of gp120. NBD-556 stimulates HIV-1 infection of CD4-negative, CCR5-expressing cells. The thermodynamic signature of the binding of NBD-556 to gp120 is very different from that of another viral entry inhibitor, BMS-378806. Whereas NBD-556 binds gp120 with a large favorable enthalpy and compensating unfavorable entropy changes, BMS-378806 does so with a small binding enthalpy change in a mostly entropy-driven process. NBD-556 is a competitive inhibitor of sCD4 and elicits a similar structuring of the coreceptor binding site, whereas BMS-378806 does not compete with sCD4 and does not induce coreceptor binding. These studies demonstrate that low-molecular-weight compounds can induce conformational changes in the HIV-1 gp120 glycoprotein similar to those observed upon CD4 binding, revealing distinct strategies for inhibiting the function of the HIV-1 gp120 envelope glycoprotein. Furthermore, competitive and noncompetitive compounds have characteristic thermodynamic signatures that can be used to guide the design of more potent and effective viral entry inhibitors.     

Molecular mechanism of HIV-1 gp120 mutations that reduce CD4 binding affinity

The interaction of the HIV-1 fusion protein gp120 with its cellular receptor CD4 represents a crucial step of the viral infection process, thus rendering gp120 a promising target for the intervention with anti-HIV drugs. Naturally occurring mutations of gp120, however, can decrease its affinity for anti-infective ligands like therapeutic antibodies or soluble CD4. To understand this phenomenon on a structural level, we performed molecular dynamics simulations of two gp120 variants (termed gp120_3-2 and gp120_2-1), which exhibit a significantly decreased binding of soluble CD4. In both variants, the exchange of a nonpolar residue byglutamate was identified as an important determinant for reduced binding. However, those glutamates are located at different sequence positions and affect different steps of the recognition process: E471 in gp120_3-2 predominantly affects the CD4-bound conformation, whereas E372 in gp120_2-1 mainly modulates the conformational sampling of free gp120. Despite these differences, there exists an interesting similarity between the two variants: both glutamates exert their function by modulating the conformation and interactions of glycine-rich motifs (G366–G367, G471–G473) resulting in an accumulation of binding incompetent gp120 conformations or a loss of intermolecular gp120–CD4 hydrogen bonds. Thus, the present data suggests that interference with the structure and dynamics of glycine-rich stretches might represent a more widespread mechanism, by which gp120 mutations reduce binding affinity. This knowledge should be helpful to predict the resistance of novel gp120 mutations or to design gp120–ligands with improved binding properties.

An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:41     

Thioredoxin-1 and protein disulfide isomerase catalyze the reduction of similar disulfides in HIV gp120

HIV-1 enters cells via interaction of the viral glycoprotein gp120, the host cell surface receptor CD4 and the co-receptors CCR5 or CXCR4. For entry, gp120 undergoes conformational changes that depend on the reduction of one or more disulfides. Previous studies indicate that protein disulfide isomerase (PDI), thioredoxin-1 (Trx1), and glutaredoxin-1 (Grx1) catalyze gp120 reduction, but their specific disulfide targets are not known. Here, it was demonstrated that PDI and Trx1 have similar gp120 disulfide targets as determined by labeling after reduction, but with some pattern differences, including overall stronger labeling with Trx1 than with PDI. Furthermore, uneven labeling of the residues of a disulfide may reflect altered accessibility by conformational changes upon the reduction process. Since both PDI and Trx1 may be involved in viral entry, compounds that target the host redox system or the viral gp120 were tested in vitro to investigate whether redox regulation is a target for anti-HIV therapy. Carbohydrate binding agents (CBAs), previously shown to bind gp120 and inhibit HIV entry, were now demonstrated to inhibit gp120 disulfide reduction. Auranofin, an inhibitor of thioredoxin reductase 1 (TrxR1), also showed inhibitory activity towards HIV infection, although close to its cytotoxic concentration. Our results demonstrate that both the host redox system and the viral surface glycoproteins are of interest for the development of new generations of anti-HIV therapeutics.     

Structure of an HIV-2 gp120 in Complex with CD4

HIV-2 is a nonpandemic form of the virus causing AIDS, and the majority of HIV-2-infected patients exhibit long-term nonprogression. The HIV-1 and HIV-2 envelope glycoproteins, the sole targets of neutralizing antibodies, share 30 to 40% identity. As a first step in understanding the reduced pathogenicity of HIV-2, we solved a 3.0-Å structure of an HIV-2 gp120 bound to the host receptor CD4, which reveals structural similarity to HIV-1 gp120 despite divergence in amino acid sequence.     

Role of Curdlan Sulfate in the Binding of HIV-1 gp120 to CD4 Molecules and the Production of gp120-Mediated TNF-α

To clarify the mechanism by which curdlan sulfate (CRDS) inhibits human immunodeficiency virus (HIV)-1 infection, we examined its influence on the binding of gp120 to CD4 molecules on T cells and macrophages, as well as on the production of TNF-α by gp120-stimulated macrophages (which promotes HIV-1 replication). CRDS treatment of cells not only inhibited the binding of HIV-1 gp120 to CD4⁺ cells, but also inhibited TNF-α production induced by gp120. Inhibition of HIV-1 infection by CRDS may be related to these two actions.     

A rationally designed synthetic mimic of the discontinuous CD4-binding site of HIV-1 gp120

Synthetic mimetics of the CD4-binding site of HIV-1 gp120 are promising candidates for HIV-1 entry inhibition, as well as immunogen candidates for the elicitation of virus-neutralizing antibodies. On the basis of the crystal structure of gp120 in complex with CD4, we have used a recently introduced strategy for the generation of structurally diverse scaffolds to design and synthesize a scaffolded peptide, in which three fragments, making up the sequentially discontinuous binding site of gp120 for CD4, are presented in a nonlinear and discontinuous fashion through a molecular scoffold, which restrains conformational flexibility. The affinities of this molecule to CD4, as well as to the broadly neutralizing antibody mAb b12, whose epitope overlaps the CD4-binding site of gp120, were determined in competitive binding assays.     

A Rationally Designed Synthetic Mimic of the Discontinuous CD4-Binding Site of HIV-1 gp120

Synthetic mimetics of the CD4-binding site of HIV-1 gp120 are promising candidates for HIV-1 entry inhibition, as well as immunogen candidates for the elicitation of virus-neutralizing antibodies. On the basis of the crystal structure of gp120 in complex with CD4, we have used a recently introduced strategy for the generation of structurally diverse scaffolds to design and synthesize a scaffolded peptide, in which three fragments, making up the sequentially discontinuous binding site of gp120 for CD4, are presented in a nonlinear and discontinuous fashion through a molecular scoffold, which restrains conformational flexibility. The affinities of this molecule to CD4, as well as to the broadly neutralizing antibody mAb b12, whose epitope overlaps the CD4-binding site of gp120, were determined in competitive binding assays.     

Probing of CD4 binding pocket of HIV-1 gp120 glycoprotein using unnatural phenylalanine analogues

CD4-gp120 interaction is the first step for HIV-1 entry into host cells. A highly conserved pocket in gp120 protein is an attractive target for developing gp120 inhibitors or novel HIV detection tools. Here we incorporate seven phenylalanine derivatives having different sizes and steric conformations into position 43 of domain 1 of CD4 (mD1.2) to explore the architecture of the ‘Phe43 cavity’ of HIV-1 gp120. The results show that the conserved hydrophobic pocket in gp120 tolerates a hydrophobic side chain of residue 43 of CD protein, which is 12.2 Å in length and 8.0 Å in width. This result provides useful information for developing novel gp120 inhibitors or new HIV detection tools.     

AFM force measurements of the gp120-sCD4 and gp120 or CD4 antigen-antibody interactions

Soluble CD4 (sCD4), anti-CD4 antibody, and anti-gp120 antibody have long been regarded as entry inhibitors in human immunodeficiency virus (HIV) therapy. However, the interactions between these HIV entry inhibitors and corresponding target molecules are still poorly understood. In this study, atomic force microscopy (AFM) was utilized to investigate the interaction forces among them. We found that the unbinding forces of sCD4–gp120 interaction, CD4 antigen–antibody interaction, and gp120 antigen–antibody interaction were 25.45 ± 20.46, 51.22 ± 34.64, and 89.87 ± 44.63 pN, respectively, which may provide important mechanical information for understanding the effects of viral entry inhibitors on HIV infection. Moreover, we found that the functionalization of an interaction pair on AFM tip or substrate significantly influenced the results, implying that we must perform AFM force measurement and analyze the data with more caution.     

Computational identification of novel entry inhibitor scaffolds mimicking primary receptor CD4 of HIV-1 gp120

Virtual screening of novel entry inhibitor scaffolds mimicking primary receptor CD4 of HIV-1 gp120 was carried out in conjunction with evaluation of their potential inhibitory activity by molecular modeling. To do this, pharmacophore models presenting different sets of the hotspots of cellular receptor CD4 for its interaction with gp120 were generated. These models were used as the templates for identification of CD4-mimetic candidates by the pepMMsMIMIC screening platform. Complexes of these candidates with gp120 were built by high-throughput ligand docking and their stability was estimated by molecular dynamics simulations and binding free energy calculations. As a result, five top hits that exhibited strong attachment to the two well-conserved hotspots of the gp120 CD4-binding site were selected for the final analysis. In analogy to CD4, the identified compounds make hydrogen bonds with Asp-368_gp120 and multiple van der Waals contacts with the gp120 residues that bind to Phe-43_CD4, resulting in destruction of the critical interactions of gp120 with Phe-43_CD4 and Arg-59_CD4. The complexes of the CD4-mimetic candidates with gp120 show relative conformational stability within the molecular dynamics simulations and expose the high percentage occupancies of intermolecular hydrogen bonds, in line with the data on the binding free energy calculations. In light of these findings, the identified compounds are considered as good scaffolds for the development of new functional antagonists of viral entry with broad HIV-1 neutralization.     

Combinatorial optimization of a CD4-mimetic miniprotein and cocrystal structures with HIV-1 gp120 envelope glycoprotein

Miniproteins provide a bridge between proteins and small molecules. Here we adapt methods from combinatorial chemistry to optimize CD4M33, a synthetic miniprotein into which we had previously transplanted the HIV-1 gp120 binding surface of the CD4 receptor. Iterative deconvolution of generated libraries produced CD4M47, a derivative of CD4M33 that had been optimized at four positions. Surface plasmon resonance demonstrated fourfold to sixfold improvement in CD4M47 affinity for gp120 to a level about threefold tighter than that of CD4 itself. Assessment of the neutralization properties of CD4M47 against a diverse range of isolates spanning from HIV-1 to SIVcpz showed that CD4M47 retained the extraordinary breadth of the parent CD4M33, but yielded only limited improvements in neutralization potencies. Crystal structures of CD4M47 and a phenylalanine variant ([Phe23]M47) were determined at resolutions of 2.4 and 2.6 Å, in ternary complexes with HIV-1 gp120 and the 17b antibody. Analysis of these structures revealed a correlation between mimetic affinity for gp120 and overall mimetic-gp120 interactive surface. A correlation was also observed between CD4- and mimetic-induced gp120 structural similarity and CD4- and mimetic-induced gp120 affinity for the CCR5 coreceptor. Despite mimetic substitutions, including a glycine-to-(d)-proline change, the gp120 conformation induced by CD4M47 was as close or closer to the conformation induced by CD4 as the one induced by the parent CD4M33. Our results demonstrate the ability of combinatorial chemistry to optimize a disulfide-containing miniprotein, and of structural biology to decipher the resultant interplay between binding affinity, neutralization breadth, molecular mimicry, and induced affinity for CCR5.