A Highly Conserved gp120 Inner Domain Residue Modulates Env Conformation and Trimer Stability

Abstract

Previous studies have shown that highly conserved residues in the inner domain of gp120 are required for HIV-1 envelope glycoprotein ( We evaluated the contribution of the hydrophobicity of W69 to conformational changes of Env by replacing it with a series of residues with aliphatic or aromatic side chains of decreasing chain length. We have found that the hydrophobicity of residue 69 is important for Env processing, CD4 binding, and its transition to the CD4-bound conformation. The most deleterious effect was observed when W69 was replaced with alanine or glycine residues. However, the functions lost due to W69 mutations could be progressively restored with amino acids of increasing aliphatic chain length and fully recovered with residues bearing an aromatic ring. Interestingly, poor CD4 binding of W69A could be fully restored by introducing a compensatory mutation within layer 2 (S115W). Structural studies of HIV-1 gp120 core e W69A/S115W mutant bound to the CD4 peptide mimetic M48U1 and Fab of anti-cluster A antibody N60-i3 revealed no perturbations to the overall structure of the double mutant compared to the wild-type protein but identified higher mobility within the interface between layer 1 and layer 2, the bridging sheet region, and the CD4 binding site. HIV-1 envelope glycoproteins (Env) play a key role during the first step of viral infection. Mature HIV-1 Env trimers are the products of a proteolytic cleavage of gp160 precursor polypeptides into gp120 (SU) and gp41 (TM) subunits. Mature metastable Env is a consolidation of three gp120 with three gp41 transmembrane subunits related in a labile noncovalent manner (1). Binding of gp120 exterior subunit to its cellular receptor, CD4, initiates viral entry (2, 3). CD4 binding allows gp120 conformational rearrangement, which is required for its attachment to CCR5 or CXCR4 chemokine coreceptors (4-11), upon which further conformational changes expose the gp41 helical heptad repeat segment (HR1), resulting in the formation of the prehairpin intermediates followed by a transition to a six-helix bundle composed of HR1 and HR2. These conformational changes lead to viral and cellular membrane fusion (12)(13)(14). CD4-induced changes allow the trimer to switch from a metastable high-energy unliganded form to a low-energy stable state.Recent studies have shown that highly conserved residues in the inner domain of gp120 are required for HIV-1 Env transitions to the CD4-bound conformation and efficient CD4 binding (15,16). Moreover, among these residues, W69 was recently shown to be important for efficient Env recognition by antibody-dependent cellular cytotoxicity (ADCC)-mediating anti-cluster A antibodies and also by sera from HIV-1-infected individuals (17-20). Since W69 is located at the interface between layer 1 and layer 2 of the

Quaternary contact in the initial interaction of CD4 with the HIV-1 envelope trimer

“…Mutation of CD4-BS2 residues resulted in decreased CCR5 and 48d binding in the absence of sCD4 (Supplementary Fig. 6b), confirming previous mutagenesis studies that attributed this effect to reduced monomer flexibility caused by altered inter-layer interactions within the inner domain 7,31 . In contrast, in the presence of sCD4, binding to 48d was unaffected by CD4-BS2 mutations, demonstrating that sCD4 was able to induce the full range of conformational changes in the context of the mutated monomer, while binding to CCR5 was only moderately reduced, with the exception of mutant K207E that, as expected, lost the ability to bind CCR5 since this residue is part of the coreceptor-contact surface 32 (Supplementary Fig.…”

“…7). Thus, as suggested 7,31 , the slight reduction of CD4 and CCR5 binding observed in the context of monomeric gp120, as well as the reduced binding of mAb 48d in the absence of sCD4, may be due to reduced molecular flexibility and distant allosteric effects secondary to altered inter-layer adherence within the flexible inner domain of the gp120 glycoprotein.…”

“…Since mutation of specific residues in the gp120 inner domain, including H66, was previously reported to reduce sCD4 and coreceptor binding to monomeric gp120 7,31 , we investigated the effects of CD4-BS2 mutations in the context of gp120 monomers. The correct folding of the mutated gp120 proteins was confirmed by their reactivity with a panel of anti-gp120 mAbs (Supplementary Fig.…”

Residues in the gp41 Ectodomain Regulate HIV-1 Envelope Glycoprotein Conformational Transitions Induced by gp120-Directed Inhibitors

“…Processing was performed and association indices were determined by precipitation of radiolabeled cell lysates and supernatants with mixtures of sera from HIV-1-infected individuals, as previously described (7,26,46,52,53,79). The association index is a measure of the ability of the gp120 molecule to remain associated with the mutant Env trimer complex on the expressing cell, relative to that of the wild-type Env.…”

Induction of a Tier-1-Like Phenotype in Diverse Tier-2 Isolates by Agents That Guide HIV-1 Env to Perturbation-Sensitive, Nonnative States

“…Based on their neutralization by sera from HIV-infected individuals, primary isolates are classified using a tiered system; tier-1 Envs are globally sensitive, whereas tier-2 or tier-3 Envs (more commonly encountered in patients) are more resistant (17). Single-site mutations in gp120 or gp41 can render resistant Envs globally sensitive to antibodies (18)(19)(20)(21)(22)(23)(24)(25).…”