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1.
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.  相似文献   

2.
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.  相似文献   

3.
Development of successful AIDS vaccine immunogens continues to be a major challenge. One of the mechanisms by which HIV-1 evades antibody-mediated neutralizing responses is the remarkable conformational flexibility of its envelope glycoprotein (Env) gp120. Some recombinant gp120s do not preserve their conformations on gp140s and functional viral spikes, and exhibit decreased recognition by CD4 and neutralizing antibodies. CD4 binding induces conformational changes in gp120 leading to exposure of the coreceptor-binding site (CoRbs). In this study, we test our hypothesis that CD4-induced (CD4i) antibodies, which target the CoRbs, could also induce conformational changes in gp120 leading to better exposed conserved neutralizing antibody epitopes including the CD4-binding site (CD4bs). We found that a mixture of CD4i antibodies with gp120 only weakly enhanced CD4 binding. However, such interactions in single-chain fusion proteins resulted in gp120 conformations which bound to CD4 and CD4bs antibodies better than the original or mutagenically stabilized gp120s. Moreover, the two molecules in the fusion proteins synergized with each other in neutralizing HIV-1. Therefore, fusion proteins of gp120 with CD4i antibodies could have potential as components of HIV-1 vaccines and inhibitors of HIV-1 entry, and could be used as reagents to explore the conformational flexibility of gp120 and mechanisms of entry and immune evasion.  相似文献   

4.
CD4 is a co-receptor for binding of T cells to antigen-presenting cells and the primary receptor for the human immunodeficiency virus type 1 (HIV). CD4 exists in three different forms on the cell surface defined by the state of the domain 2 cysteine residues: an oxidized monomer, a reduced monomer, and a covalent dimer linked through the domain 2 cysteines. The disulfide-linked dimer is the preferred immune co-receptor. The form of CD4 that is preferred by HIV was examined in this study. HIV entry and envelope-mediated cell-cell fusion were tested using cells expressing comparable levels of wild-type or disulfide bond mutant CD4 in which the domain 2 cysteines were mutated to alanine. Eliminating the domain 2 disulfide bond increased entry of HIV reporter viruses and enhanced HIV envelope-mediated cell-cell fusion 2-4-fold. These observations suggest that HIV enters susceptible cells preferably through monomeric reduced CD4, whereas dimeric CD4 is the preferred receptor for binding to antigen-presenting cells. Cleavage of the domain 2 disulfide bond is possibly involved in the conformational change in CD4 associated with fusion of the HIV and cell membranes.  相似文献   

5.
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.  相似文献   

6.
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.  相似文献   

7.
The HIV-1 gp120 exterior envelope glycoprotein undergoes a series of conformational rearrangements while sequentially interacting with the receptor CD4 and coreceptor CCR5 or CXCR4 on the surface of host cells to initiate virus entry. Both the crystal structures of the HIV-1 gp120 core bound by the CD4 and antigen 17b, and the SIV gp120 core pre-bound by the CD4 are known. We have performed dynamic domain studies on the homology models of the CD4-bound and unliganded HIV-1 gp120 with modeled V3 and V4 loops to explore details of conformational changes, hinge axes, and hinge bending regions in the gp120 structures upon CD4 binding. Four dynamic domains were clustered and intricately motional modes for domain pairs were discovered. Together with the detailed comparative analyses of geometrical properties between the unliganded and liganded gp120 models, an induced fit model was proposed to explain events accompanying the CD4 engagement to the gp120, which provided new insight into the dynamics of the molecular induced binding mechanism that complements the molecular dynamics and crystallographic studies.  相似文献   

8.
Human immunodeficiency virus-1 (HIV-1) infects both humans and chimpanzees, but in the chimpanzee, HIV-1 infection leads only very rarely to loss of CD4 T cells or to AIDS-like disease. The pathogenetic basis for this difference in host range is not understood. In previous studies, using CD4 T cells from HIV-1 seronegative human donors, we demonstrated that crosslinking of CD4-bound gp120, followed by signaling through the T cell receptor for antigen (TCR), resulted in cell death by apoptosis. To determine whether activation-induced apoptosis correlates with progression to AIDS, we studied the chimpanzee. Our data suggest that, although human CD4 T cells respond to CD4 ligation with anergy and apoptosis upon activation, chimpanzee CD4 T cells do not undergo apoptosis after cross-linking of CD4-bound gp120, followed by signaling through the TCR. In addition, proliferation assays show that chimpanzee CD4 T cells do not become anergic after CD4 ligation. Thus, it is possible that, in the chimpanzee, the absence of cellular anergy and apoptotic cell death after CD4 ligation by HIV-1 gp120 protect this primate species from progression to AIDS-like disease.This investigation was supported by National Institute of Health grants AI-30575, AI-29903, AI-35513, and RR00015 (TH Finkel), AI-05060 (WC Satterfield), American Foundation for AIDS Research grants 02270-16-RG (TH Finkel) and 770188-11-PF (NK Banda), the Concerned Parents for AIDS Research, the UCHSC Cancer Center, the Eleanore and Michael Stobin Trust, and the Bender Foundation.  相似文献   

9.
10.
The low-molecular-weight compound JRC-II-191 inhibits infection of HIV-1 by blocking the binding of the HIV-1 envelope glycoprotein gp120 to the CD4 receptor and is therefore an important lead in the development of a potent viral entry inhibitor. Reported here is the use of two orthogonal screening methods, gold docking and ROCS shape-based similarity searching, to identify amine-building blocks that, when conjugated to the core scaffold, yield novel analogs that maintain similar affinity for gp120. Use of this computational approach to expand SAR produced analogs of equal inhibitory activity but with diverse capacity to enhance viral infection. The novel analogs provide additional lead scaffolds for the development of HIV-1 entry inhibitors that employ protein-ligand interactions in the vestibule of gp120 Phe 43 cavity.  相似文献   

11.
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.  相似文献   

12.
13.
Peptide T, from the human immunodeficiency virus (HIV), whose sequence is Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr, has been shown to inhibit attachment of this virus to T cells and neural cells bearing the CD4 receptor. This peptide shares extensive homology with the 19–26 segment of ribonuclease A (RNase A), whose sequence is Ala-Ala-Ser-Ser-Ser-Asn-Tyr-Cys. Based on comparison of the structures of peptides occurring in proteins of known structure that are homologous to peptide T,viz, RNase A and endothiapepsin and on conformational energy calculations, we predicted that peptide T adopts a structure much like that for residues 19–26 in RNase A. A critical feature is a bend involving residues Thr 4-Asn 7 in peptide T corresponding to Ser 22-Tyr 25 in the RNase A peptide. Our proposed structure for peptide T has recently been confirmed by Cotelleet al. (Biochem. Biophys. Res. Commun. 171, 596–602). We now show directly that the RNase A peptide, with Met replacing Cys 26 to prevent disulfide exchange reactions, strongly induces monocyte-chemotaxis that is blocked by anti-CD4 monoclonal antibody. Both peptide T and RNase A fail to induce chemotaxis, however, in neutrophils which do not express surface CD4 receptors. These results suggest that both peptides interact with the CD4 receptor in inducing monocyte chemotaxis. We have also prepared cyclo-RNase A peptide with Met 26. Using molecular dynamics and conformational energy calculations, we find that the cyclic peptide cannot form a bend structure involving Ser 22-Tyr 25 that is superimposable on the RNase A bend. Indeed, we find that this peptide is inactive in inducing monocyte chemotaxis despite the fact that its amino acid sequence is identical to that of the open chain form. This result suggests that a correlation between the -bend structure of the RNase A peptide and peptide T and their abilities to bind to the CD4 receptor.  相似文献   

14.
The structure of the free form HIV gp120, critical for therapeutic agent development, is unavailable due to its high flexibility. Previous thermodynamic data, structural analysis and simulation results have suggested a large conformational change in the core domain upon CD4 binding. The bridging sheet, which consists of four beta-strands with beta20/21 nestling against the inner/outer domains and beta2/3 facing outward, more exposed to the solvent, was proposed to be unfolded in the native state. In order to test this proposition and to characterize the native conformations, we performed potential mean force (PMF) molecular dynamics (MD) simulations on the CD4-bound crystal structure. We pushed the bridging sheet away from the inner and outer domain to explore the accessible conformational space for the bridging sheet. In addition, we performed conventional MD simulations on structures with the bridging sheet partially unfolded to investigate the stability of the association between the inner and outer domains. Based on the free energy profiles, we find that the whole bridging sheet is unlikely to unfold without other concurrent conformational changes. On the other hand, the partial bridging sheet, beta strands 2/3, can switch its conformation from the folded to the unfolded state. Furthermore, relaxation of conformation with partially unfolded bridging sheet through MD simulations leads to a conformation with beta strands 20/21 quickly re-anchoring against the inner and outer domains. Such a conformation, although lacking some of the hydrophobic interactions present in the CD4-bound structure, displayed high stability as further indicated by other restrained MD simulations. The relevance of this conformation to the free form structure and the pathway for conformational change from the free form to the CD4-bound structure is discussed in detail in light of the available unliganded SIV gp120 crystal structure.  相似文献   

15.
CXCR4 belongs to the family of G protein-coupled receptors and mediates the various developmental and regulatory effects of the chemokine SDF-1alpha. In addition, CXCR4 acts as a co-receptor along with CD4 for the HIV-1 viral glycoprotein gp120. Recently, there has also been a small molecule described that antagonizes both SDF-1 and gp120 binding to CXCR4. The structural and mechanistic basis for this dual recognition ability of CXCR4 is unknown largely due to the technical challenges of biochemically producing the components of the various complexes. We expressed the human CXCR4 receptor using a modified baculovirus expression vector that facilitates a single step antibody affinity purification of CXCR4 to >80% purity from Hi5 cells. The recombinant receptor undergoes N-linked glycosylation, tyrosine sulfation and is recognized by the 12G5 conformation specific antibody against human CXCR4. We are able to purify CXCR4 alone as well as complexed with its endogenous ligand SDF-1, its viral ligand gp120, and a small molecule antagonist AMD3100 by ion-exchange chromatography. We anticipate that the expression and purification scheme described in this paper will facilitate structure-function studies aimed at elucidating the molecular basis for CXCR4 recognition of its endogenous chemokine and viral ligands.  相似文献   

16.
Human immunodeficiency virus (HIV) entry into cells is initiated by the binding of its envelope glycoprotein (Env) gp120 to receptor CD4. Antibodies that bind to epitopes overlapping the CD4-binding site (CD4bs) on gp120 can prevent HIV entry by competing with cell-associated CD4; their ability to outcompete CD4 is a major determinant of their neutralizing potency and is proportional to their avidity. The breadth of neutralization and the likelihood of the emergence of antibody-resistant virus are critically dependent on the structure of their epitopes. Because CD4bs is highly conserved, it is reasonable to hypothesize that antibodies closely mimicking CD4 could exhibit relatively broad cross-reactivity and a high probability of preventing the emergence of resistant viruses. Previously, in a search for antibodies that mimic CD4 or the co-receptor, we identified and characterized a broadly cross-reactive HIV-neutralizing CD4bs human monoclonal antibody (hmAb), m18. Here, we describe the crystal structure of Fab m18 at 2.03 A resolution, which reveals unique conformations of heavy chain complementarity-determining regions (CDRs) 2 and 3 (H2 and H3). H2 is highly bulged and lacks cross-linking interstrand hydrogen bonds observed in all four canonical structures. H3 is 17.5 A long and rigid, forming an extended beta-sheet decorated with an alpha-turn motif bearing a phenylalanine-isoleucine fork at the apex. It shows striking similarity to the Ig CDR2-like C'C' region of the CD4 first domain D1 that dominates the binding of CD4 to gp120. Docking simulations suggest significant similarity between the m18 epitope and the CD4bs on gp120. Fab m18 does not enhance binding of CD4-induced (CD4i) antibodies, nor does it induce CD4-independent fusion mediated by the HIV Env. Thus, vaccine immunogens based on the m18 epitope structure are unlikely to elicit antibodies that could enhance infection. The structure can also serve as a basis for the design of novel, highly efficient inhibitors of HIV entry.  相似文献   

17.
The major causative agent for Acquired Immune Deficiency Syndrome (AIDS) is Human Immunodeficiency Virus-1 (HIV-1). HIV-1 is a predominant subtype of HIV which counts on human cellular mechanism virtually in every aspect of its life cycle. Binding of viral envelope glycoprotein-gp120 with human cell surface CD4 receptor triggers the early infection stage of HIV-1. This study focuses on the interaction interface between these two proteins that play a crucial role for viral infectivity. The CD4–gp120 interaction interface has been studied through a comprehensive protein–protein interaction network (PPIN) analysis and highlighted as a useful step towards identifying potential therapeutic drug targets against HIV-1 infection. We prioritized gp41, Nef and Tat proteins of HIV-1 as valuable drug targets at early stage of viral infection. Lack of crystal structure has made it difficult to understand the biological implication of these proteins during disease progression. Here, computational protein modeling techniques and molecular dynamics simulations were performed to generate three-dimensional models of these targets. Besides, molecular docking was initiated to determine the desirability of these target proteins for already available HIV-1 specific drugs which indicates the usefulness of these protein structures to identify an effective drug combination therapy against AIDS.  相似文献   

18.
Developing of multi-target HIV-1 entry inhibitors represents an important avenue of drug therapy. Two such inhibitors are hexa-arginine-neomycin-conjugate (NeoR6) and nona-d-arginine-neomycin-conjugate (Neo-r9). Our findings that NeoR6-resistant mutations appear in the gp120 constant regions; and NeoR6 is not CCR5 antagonist, but inhibits CXCR4 and CCR5 HIV-1 using isolates, led us to suggest that NeoR6 may inhibit HIV-1 entry by interfering with the CD4-gp120 binding. To support this notion, we constructed a homology model of unliganded HIV-1IIIB gp120 and docked NeoR6 and Neo-r9 to it, using a multistep docking procedure: geometric-electrostatic docking by MolFit; flexible ligand docking by Autodock3 and final refinement of the obtained complexes by Discover3. Binding free energies were calculated by MM-PBSA methodology. The model predicts competitive inhibition of CD4-gp120 binding by NeoR6 and Neo-r9. We determined plausible binding sites between constructed CD4-bound gp120 trimer and homology modeled membranal CXCR4, and tested NeoR6 and Neo-r9 interfering with this interaction. These models support our notion that another mechanism of anti-HIV-1 activity of NeoR6 is inhibition of gp120-CXCR4 binding. These structural models and interaction of NeoR6 and Neo-r9 with gp120 and CXCR4 provide a powerful approach for structural based drug design for selective targeting of HIV-1 entry and/or for inhibition of other retroviruses with similar mechanism of entry.  相似文献   

19.
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.  相似文献   

20.
The human monoclonal antibody 2G12 is a member of a small group of broadly neutralizing antibodies against human immunodeficiency virus type 1. 2G12 adopts a unique variable heavy domain-exchanged dimeric configuration that results in an extensive multivalent binding surface and the ability to bind with high affinity to densely clustered high mannose oligosaccharides on the “silent” face of the gp120 envelope glycoprotein. Here, we further define the amino acids responsible for this extraordinary domain-swapping event in 2G12.  相似文献   

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