Solution structure, conformational dynamics, and CD4-induced activation in full-length, glycosylated, monomeric HIV gp120

The gp120 subunit of the HIV Env glycoprotein is responsible for receptor interactions leading to viral entry and is a primary target for neutralizing antibodies. Most structural studies have focused on the heavily truncated, deglycosylated gp120 core, leaving fundamental aspects of the glycoprotein that are responsible for immune evasion and receptor-induced activation unresolved. Here we investigate full-length, glycosylated HIV gp120 in unliganded and CD4-bound forms by using small-angle X-ray scattering to visualize global structural reorganization and hydrogen/deuterium exchange to track changes in local conformational dynamics. The studies revealed unliganded full-length gp120 to be considerably more dynamic, particularly at the CD4 binding site, than suggested by previous studies of the subunit core alone. The large V1/V2 loops, previously unmapped, are positioned to mask the coreceptor binding site in an orientation that recapitulates that observed in the Env trimer. CD4 binding shifts V1/V2 to unmask the coreceptor binding site and triggers profound dynamic changes in gp120 spanning from the binding site to the gp41-interactive face of gp120. These findings provide further insights on the structural basis of Env antigenicity and immunogenicity and of allosteric effects upon receptor binding.     

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.     

Molecular motions of human HIV-1 gp120 envelope glycoproteins

The HIV-1 gp120 exterior envelope glycoprotein undergoes a series of conformational rearrangements while sequentially interacting with the receptor CD4 and the 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 CD4 and antigen 17b, and the SIV gp120 core pre-bound by CD4 are known. Despite the wealth of knowledge on these static snapshots of molecular conformations, the details of molecular motions crucial to intervention remain elusive. We presented a comprehensive comparative analysis of dynamic behavior of gp120 in its CD4-complexed, CD4-free and CD4-unliganded states based on the homology models with modeled V3 and V4 loops. CONCOORD computer simulation was utilized to generate ensembles of feasible protein structures, which were subsequently analyzed by essential dynamics technique to identify preferred concerted motions. The revealed collective fluctuations are dominated by complex motional modes such as rotation/twisting, flexing/closing, and shortness/elongation between or within the inner, outer, and bridging-sheet domains. An attempt has been made to relate these modes to receptor/coreceptor association and neutralization avoidance. Covariance web analysis revealed four subdomains that undergo concerted motion in gp120. The structural components in gp120 that move in concert with CD4 were also identified, which may be the suitable target for inhibitor design to interrupt CD4-gp120 interaction. The differences in B-factors between the three gp120 states revealed certain structural regions that could be related either to CD4 association or to subsequent dissociation of gp120 from gp41. These dynamics data provide new insights into the structure-function relationship of gp120 and may aid in structure-based anti-HIV vaccine design.     

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.     

Lineage-Specific Differences between Human and Simian Immunodeficiency Virus Regulation of gp120 Trimer Association and CD4 Binding

Metastable conformations of the gp120 and gp41 envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) must be maintained in the unliganded state of the envelope glycoprotein trimer. Binding of gp120 to the primary receptor, CD4, triggers the transition to an open conformation of the trimer, promoting interaction with the CCR5 chemokine receptor and ultimately leading to gp41-mediated virus-cell membrane fusion and entry. Topological layers in the gp120 inner domain contribute to gp120-trimer association in the unliganded state and to CD4 binding. Here we describe similarities and differences between HIV-1 and SIVmac gp120. In both viruses, the gp120 N/C termini and the inner domain β-sandwich and layer 2 support the noncovalent association of gp120 with the envelope glycoprotein trimer. Layer 1 of the SIVmac gp120 inner domain contributes more to trimer association than the corresponding region of HIV-1 gp120. On the other hand, layer 1 plays an important role in stabilizing the CD4-bound conformation of HIV-1 but not SIVmac gp120 and thus contributes to HIV-1 binding to CD4. In SIVmac, CD4 binding is instead enhanced by tryptophan 375, which fills the Phe 43 cavity of gp120. Activation of SIVmac by soluble CD4 is dependent on tryptophan 375 and on layer 1 residues that determine a tight association of gp120 with the trimer. Distinct biological requirements for CD4 usage have resulted in lineage-specific differences in the HIV-1 and SIV gp120 structures that modulate trimer association and CD4 binding.     

The Highly Conserved Layer-3 Component of the HIV-1 gp120 Inner Domain Is Critical for CD4-Required Conformational Transitions

The trimeric envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) mediates virus entry into host cells. CD4 engagement with the gp120 exterior envelope glycoprotein subunit represents the first step during HIV-1 entry. CD4-induced conformational changes in the gp120 inner domain involve three potentially flexible topological layers (layers 1, 2, and 3). Structural rearrangements between layer 1 and layer 2 have been shown to facilitate the transition of the envelope glycoprotein trimer from the unliganded to the CD4-bound state and to stabilize gp120-CD4 interaction. However, our understanding of CD4-induced conformational changes in the gp120 inner domain remains incomplete. Here, we report that a highly conserved element of the gp120 inner domain, layer 3, plays a pivot-like role in these allosteric changes. In the unliganded state, layer 3 modulates the association of gp120 with the Env trimer, probably by influencing the relationship of the gp120 inner and outer domains. Importantly, layer 3 governs the efficiency of the initial gp120 interaction with CD4, a function that can also be fulfilled by filling the Phe43 cavity. This work defines the functional importance of layer 3 and completes a picture detailing the role of the gp120 inner domain in CD4-induced conformational transitions in the HIV-1 Env trimer.     

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.     

Homology models of the HIV‐1 attachment inhibitor BMS‐626529 bound to gp120 suggest a unique mechanism of action

HIV‐1 gp120 undergoes multiple conformational changes both before and after binding to the host CD4 receptor. BMS‐626529 is an attachment inhibitor (AI) in clinical development (administered as prodrug BMS‐663068) that binds to HIV‐1 gp120. To investigate the mechanism of action of this new class of antiretroviral compounds, we constructed homology models of unliganded HIV‐1 gp120 (UNLIG), a pre‐CD4 binding‐intermediate conformation (pCD4), a CD4 bound‐intermediate conformation (bCD4), and a CD4/co‐receptor‐bound gp120 (LIG) from a series of partial structures. We also describe a simple pathway illustrating the transition between these four states. Guided by the positions of BMS‐626529 resistance substitutions and structure–activity relationship data for the AI series, putative binding sites for BMS‐626529 were identified, supported by biochemical and biophysical data. BMS‐626529 was docked into the UNLIG model and molecular dynamics simulations were used to demonstrate the thermodynamic stability of the different gp120 UNLIG/BMS‐626529 models. We propose that BMS‐626529 binds to the UNLIG conformation of gp120 within the structurally conserved outer domain, under the antiparallel β20–β21 sheet, and adjacent to the CD4 binding loop. Through this binding mode, BMS‐626529 can inhibit both CD4‐induced and CD4‐independent formation of the “open state” four‐stranded gp120 bridging sheet, and the subsequent formation and exposure of the chemokine co‐receptor binding site. This unique mechanism of action prevents the initial interaction of HIV‐1 with the host CD4+ T cell, and subsequent HIV‐1 binding and entry. Our findings clarify the novel mechanism of BMS‐626529, supporting its ongoing clinical development. Proteins 2015; 83:331–350. © 2014 Wiley Periodicals, Inc.     

Importance of V3 Loop Flexibility and Net Charge in the Context of Co-Receptor Recognition. A Molecular Dynamics Study on HIV gp120

The entry of HIV-1 into a host cell requires the interaction of envelope glycoprotein gp120 with CD4 receptor as well as a co-receptor, which can be either CCR5 or CXCR4. The third variable loop (V3) of HIV-1 gp120 plays an important role in co-receptor selection (CCR5 or CXCR4) and also acts as an epitope for neutralizing antibodies against gp120. Here we have performed long time molecular dynamics simulations of two gp120 structures that are representatives of a R5 and X4 strains in the CD4-free and CD4-bound states. The results indicate some conserved features in both systems, such as the rigidity of the gp120 core, the conservation of the CD4 Phe43-gp120 binding cavity contacts, a high flexibility of the V3 loop particularly in the CD4 bound form. Analysis of the distribution of V3 loop's net charge shows it to be more positive for the gp120 sequences selecting CXCR4 co-receptor, letting us to propose that V3 loop net charge and flexibility are the two main elements in the co-receptor selection.     

A broad spectrum anti-HIV inhibitor significantly disturbs V1/V2 domain rearrangements of HIV-1 gp120 and inhibits virus entry

Inhibition of human immunodeficiency virus (HIV) entry into target human cells is considered as a critical strategy for preventing HIV infection. Conformational shifts of the HIV-1 envelope glycoprotein (gp120) facilitates the attachment of the virus to target cells, therefore gp120 remains an attractive target for antiretroviral therapy development. Compound 18A has been recently identified as a broad-spectrum anti-HIV inhibitor. It was proposed that 18A disrupts rearrangements of V1/V2 region in gp120; however, the precise mechanism by which 18A interferes with the inherent motion of V1/V2 domain remains obscure. In this report, we elaborate on the binding mode of compound 18A to the closed conformation of a soluble cleaved gp120 and further examine the dynamic motion of V1/V2 region in both gp120 and the gp120–18A complex via all-atom molecular dynamics simulations. In this work, comparative molecular dynamic analyses revealed that 18A makes contact with Leu179, Ile194, Ile424, Met426 W427, E370 and Met475 in the main hydrophobic cavity of the unliganded gp120 and disrupts the restructuring of V1/V2 domain observed in apo gp120. The unwinding of α1 and slight inversion of β2 in gp120 leads to the shift of VI/V2 domain away from the V3 N-terminal regions and toward the outer domain. Stronger contacts between Trp425 and Trp112 rings may contribute to the reduced flexibility of α1 observed upon 18A binding thereby inhibiting the shifts of the V1/V2 region. Binding of 18A to gp120: (1) decreases the overall flexibility of the protein and (2) inhibits the formation a gp120 conformation that closely ressembles a CD4-bound-like conformation. Information gained from this report not only elaborates on important dynamic features of gp120, but will also assist with the future designs of potent gp120 inhibitors as anti-HIV.     

Involvement of the V1/V2 variable loop structure in the exposure of human immunodeficiency virus type 1 gp120 epitopes induced by receptor binding.

The binding of human immunodeficiency virus type 1 (HIV-1) to the cellular receptor CD4 has been suggested to induce conformational changes in the viral envelope glycoproteins that promote virus entry. Conserved, discontinuous epitopes on the HIV-1 gp120 glycoprotein recognized by the 17b, 48d, and A32 antibodies are preferentially exposed upon the binding of soluble CD4 (sCD4). The binding of the 17b and 48d antibodies to the gp120 glycoprotein can also be enhanced by the binding of the A32 antibody. Here we constructed HIV-1 gp120 mutants in which the variable segments of the V1/V2 and V3 structures were deleted, individually or in combination, while the 17b, 48d, and A32 epitopes were retained. The effects of the variable loop deletions on the function of the HIV-1 envelope glycoproteins and on the exposure of epitopes induced by sCD4 or A32 binding to the monomeric gp120 glycoprotein were examined. The variable-loop-deleted envelope glycoproteins were able to mediate virus entry, albeit at lower efficiencies than those of the wild-type glycoproteins. Thus, the V1/V2 and V3 variable sequences contribute to the efficiency of HIV-1 entry but are not absolutely required for the process. Neither the V1/V2 nor V3 loops were necessary for the increase in exposure of the 17b/48d epitopes induced by binding of the A32 monoclonal antibody. By contrast, induction of the 17b, 48d, and A32 epitopes by sCD4 binding apparently involves a movement of the V1/V2 loops, which in the absence of CD4 partially mask these epitopes on the native gp120 monomer. The results obtained with a mutant glycoprotein containing a deletion of the V1 loop alone indicated that the contribution of the V2 loop to these phenomena was more significant than that of the V1 sequences. These results suggest that the V1/V2 loops, which have been previously implicated in CD4-modulated, postattachment steps in HIV-1 entry, contribute to CD4-induced gp120 conformational changes detected by the 17b, 48d, and A32 antibodies.     

Localized changes in the gp120 envelope glycoprotein confer resistance to human immunodeficiency virus entry inhibitors BMS-806 and #155

BMS-806 and the related compound, #155, are novel inhibitors of human immunodeficiency virus type 1 (HIV-1) entry that bind the gp120 exterior envelope glycoprotein. BMS-806 and #155 block conformational changes in the HIV-1 envelope glycoproteins that are induced by binding to the host cell receptor, CD4. We tested a panel of HIV-1 envelope glycoprotein mutants and identified several that were resistant to the antiviral effects of BMS-806 and #155. In the CD4-bound conformation of gp120, the amino acid residues implicated in BMS-806 and #155 resistance line the "phenylalanine 43 cavity" and a water-filled channel that extends from this cavity to the inner domain. Structural considerations suggest a model in which BMS-806 and #155 bind gp120 prior to receptor binding and, upon CD4 binding, are accommodated in the Phe-43 cavity and adjacent channel. The integrity of the nearby V1/V2 variable loops and N-linked carbohydrates on the V1/V2 stem indirectly influences sensitivity to the drugs. A putative binding site for BMS-806 and #155 between the gp120 receptor-binding regions and the inner domain, which is thought to interact with the gp41 transmembrane envelope glycoprotein, helps to explain the mode of action of these drugs.     

Conformational rearrangement within the soluble domains of the CD4 receptor is ligand-specific

Ligand binding induces shape changes within the four modular ectodomains (D1-D4) of the CD4 receptor, an important receptor in immune signaling. Small angle x-ray scattering (SAXS) on both a two-domain and a four-domain construct of the soluble CD4 (sCD4) is consistent with known crystal structures demonstrating a bilobal and a semi-extended tetralobal Z conformation in solution, respectively. Detection of conformational changes within sCD4 as a result of ligand binding was followed by SAXS on sCD4 bound to two different glycoprotein ligands: the tick saliva immunosuppressor Salp15 and the HIV-1 envelope protein gp120. Ab initio modeling of these data showed that both Salp15 and gp120 bind to the D1 domain of sCD4 and yet induce drastically different structural rearrangements. Upon binding, Salp15 primarily distorts the characteristic lobal architecture of the sCD4 without significantly altering the semi-extended shape of the sCD4 receptor. In sharp contrast, the interaction of gp120 with sCD4 induces a shape change within sCD4 that can be described as a Z-to-U bi-fold closure of the four domains across its flexible D2-D3 linker. Placement of known crystal structures within the boundaries of the SAXS-derived models suggests that the ligand-induced shape changes could be a result of conformational changes within this D2-D3 linker. Functionally, the observed shape changes in CD4 receptor causes dissociation of lymphocyte kinase from the cytoplasmic domain of Salp15-bound CD4 and facilitates an interaction between the exposed V3 loops of CD4-bound gp120 molecule to the extracellular loops of its co-receptor, a step essential for HIV-1 viral entry.     

Protein dynamics and motions in relation to their functions: several case studies and the underlying mechanisms

Proteins are dynamic entities in cellular solution with functions governed essentially by their dynamic personalities. We review several dynamics studies on serine protease proteinase K and HIV-1 gp120 envelope glycoprotein to demonstrate the importance of investigating the dynamic behaviors and molecular motions for a complete understanding of their structure–function relationships. Using computer simulations and essential dynamic (ED) analysis approaches, the dynamics data obtained revealed that: (i) proteinase K has highly flexible substrate-binding site, thus supporting the induced-fit or conformational selection mechanism of substrate binding; (ii) Ca²⁺ removal from proteinase K increases the global conformational flexibility, decreases the local flexibility of substrate-binding region, and does not influence the thermal motion of catalytic triad, thus explaining the experimentally determined decreased thermal stability, reduced substrate affinity, and almost unchanged catalytic activity upon Ca²⁺ removal; (iii) substrate binding affects the large concerted motions of proteinase K, and the resulting dynamic pocket can be connected to substrate binding, orientation, and product release; (iv) amino acid mutations 375 S/W and 423 I/P of HIV-1 gp120 have distinct effects on molecular motions of gp120, facilitating 375 S/W mutant to assume the CD4-bound conformation, while 423 I/P mutant to prefer for CD4-unliganded state. The mechanisms underlying protein dynamics and protein–ligand binding, including the concept of the free energy landscape (FEL) of the protein–solvent system, how the ruggedness and variability of FEL determine protein’s dynamics, and how the three ligand-binding models, the lock-and-key, induced-fit, and conformational selection are rationalized based on the FEL theory are discussed in depth.     

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.     

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.     

Understanding the binding mode and function of BMS-488043 against HIV-1 viral entry

A recently discovered small-molecule inhibitor, BMS-488043 (BMS-488), for the invasion of Human immunodeficiency virus Type 1 (HIV-1), shows a high activity against the entry of diversified HIV-1. Docking and molecular dynamic studies have been carried out to understand the binding mode of BMS-488 to gp120 as well as the effect of the small molecule on the conformational change of gp120 induced by CD4 binding. The results indicate that BMS-488 can accommodate in the CD4 binding pocket and interfere the CD4 binding in a noncompetitive mode. The piperazine group of BMS-488 prevents the bridging sheet formation of gp120 induced by the CD4 binding mainly through blocking the rotation of the Trp112 located on the α1 helix of gp120. The bridging sheet formation cannot be blocked for the W112A mutant of gp120 due to the reduced steric hindrance, in agreement with its significant resistance to the BMS inhibitor. The aza-indole ring is likely to interfere the exposure of gp41 by stacking within the β3-β5 and LB loops to disrupt the close packing of Pro212-His66-Phe210. The mode of action of BMS-488 also accommodates many mutagenesis results related to BMS-488 activity.     

Regions required for CD4 binding in the external glycoprotein gp120 of simian immunodeficiency virus.

The external domain of the envelope glycoprotein, gp120, of simian immunodeficiency virus (SIV) has been expressed as a mature secreted product using recombinant baculoviruses and the expressed protein, which has an observed molecular mass of 110 kDa, was purified by monoclonal antibody (MAb) affinity chromatography. N-terminal sequence analysis showed a signal sequence cleavage identity similar to that of the gp120s of both human immunodeficiency virus type 1 (HIV-1) and HIV type 2. The expressed molecule bound to soluble CD4 with an affinity that was approximately 10-fold lower than that of gp120 from HIV-1. A screening of the ability of SIV envelope MAbs to inhibit CD4 binding revealed two groups of inhibitory MAbs. One group is dependent on conformation, while the second group maps to a discrete epitope near the amino terminus. The particular role of the V3 loop region of the molecule in CD4 binding was investigated by the construction of an SIV-HIV hybrid in which the V3 loop of SIV was precisely replaced with the equivalent domain from HIV-1 MN. The hybrid glycoprotein bound HIV-1 V3 loop MAbs and not SIV V3 MAbs but continued to bind conformational SIV MAbs and soluble CD4 as well as the parent molecule.     

A novel human antibody against human immunodeficiency virus type 1 gp120 is V1, V2, and V3 loop dependent and helps delimit the epitope of the broadly neutralizing antibody immunoglobulin G1 b12

The V1/V2 and V3 loops are proximal to the CD4 binding site (CD4bs) of human immunodeficiency virus type 1 (HIV-1) gp120 and undergo conformational change upon CD4 receptor engagement by the HIV-1 envelope spike. Nearly all of the reported monoclonal antibodies (MAbs) against the CD4bs exhibit a very limited capacity to neutralize HIV-1. However, one such human MAb, immunoglobulin G1 (IgG1) b12, is uniquely able to neutralize primary isolates across subtypes with considerable potency. The molecular basis for the anti-HIV-1 activity of b12 is not fully understood but is relevant to vaccine design. Here we describe a novel human MAb, 4KG5, whose binding to monomeric gp120 is moderately enhanced by IgG1 b12. In sharp contrast, 4KG5 binding to gp120 is inhibited by soluble CD4 (sCD4) and by all other (n = 14) anti-CD4bs MAbs tested. 4KG5 is unable to recognize gp120 in which either V1, V2, or V3 has been deleted, and MAbs against the V2 or V3 loops inhibit the binding of 4KG5 to gp120. Moreover, 4KG5 is able to inhibit the binding of the CD4-induced MAbs 17b and X5 in the absence of sCD4, whereas 17b and X5 only weakly inhibit the binding of 4KG5 to gp120. Mutagenesis of gp120 provides further evidence of a discontinuous epitope of 4KG5 that is formed by the V1/V2 loop, the V3 loop, and a portion of the bridging sheet (C4). 4KG5 was isolated as a single-chain Fv from a phage display library constructed from the bone marrow of an HIV-1-seropositive subject (FDA2) whose serum neutralizes HIV-1 across subtypes. Despite its source, we observed no significant neutralization with 4KG5 against the autologous (R2) virus and several other strains of HIV-1. The results suggest a model in which antibody access to the CD4bs on the envelope spike of HIV-1 is restricted by the orientation and/or dynamics of the V1/V2 and V3 loops, and b12 avoids these restrictions.     

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.