Unliganded HIV-1 gp120 core structures assume the CD4-bound conformation with regulation by quaternary interactions and variable loops

Abstract

The HIV-1 envelope (Env) spike (gp120(3)/gp41(3)) undergoes considerable structural rearrangements to mediate virus entry into cells and to evade the host immune response. Engagement of CD4, the primary human receptor, fixes a particular conformation and primes Env for entry. The CD4-bound state, however, is prone to spontaneous inactivation and susceptible to antibody neutralization. How does unliganded HIV-1 maintain CD4-binding capacity and regulate transitions to the CD4-bound state? To define this mechanistically, we determined crystal structures of unliganded core gp120 from HIV-1 clades B, C, and E. Notably, all of these unliganded HIV-1 structures resembled the CD4-bound state. Conformational fixation with ligand selection and thermodynamic analysis of full-length and core gp120 interactions revealed that the tendency of HIV-1 gp120 to adopt the CD4-bound conformation was restrained by the V1/V2- and V3-variable loops. In parallel, we determined the structure of core gp120 in complex with the small molecule, NBD-556, which specifically recognizes the CD4-bound conformation of gp120. Neutralization by NBD-556 indicated that Env spikes on primary isolates rarely assume the CD4-bound conformation spontaneously, although they could do so when quaternary restraints were loosened. Together, the results suggest that the CD4-bound conformation represents a "ground state" for the gp120 core, with variable loop and quaternary interactions restraining unliganded gp120 from "snapping" into this conformation. A mechanism of control involving deformations in unliganded structure from a functionally critical state (e.g., the CD4-bound state) provides advantages in terms of HIV-1 Env structural diversity and resistance to antibodies and inhibitors, while maintaining elements essential for entry.

Fig. 1.

Unliganded structures of HIV-1 gp120 core. Crystal structures are displayed as Cα-ribbon diagrams with outer domains in gray and inner domains in magenta, blue or light blue, cyan, and green for HIV-1 clades B, C, and E, and SIV, respectively, and the region that in the CD4-bound conformation makes up the bridging sheet in red. The evolutionary relationships of these Env glycoproteins are represented by a dendrogram where the length of connections is proportional to evolutionary distance. [The four HIV-1 structures were determined here; the SIV structure was determined previously by Chen et al. (17).]

Fig. 2.

Comparison of unliganded HIV-1 gp120 core to previously determined gp120 structures. Despite substantial conformational diversity of the gp120 envelope glycoprotein, unliganded HIV-1 gp120 snaps into a conformation that closely resembles the CD4-bound state. (A) Ribbon diagrams, displayed as in Fig. 1. (B) Molecular surface representation colored by structural deviations from the HIV-1 clade E unliganded gp120 structure. The color scale ranges from dark blue to red for rmsds of 0 to >10 Å, respectively. Notably, conformational changes >100 Å are observed in the bridging sheet region between the b13-bound and unliganded forms of HIV-1 gp120.

Fig. 3.

Conformational diversity of HIV-1 gp120 in solution. The conformational diversity of gp120 in solution is sensitive to the presence of the variable loops. (A) Entropy of ligand interactions with full-length and truncated gp120s (for measurements involving the HIV-1 clade B gp120, the YU2 strain was used; for measurements involving the HIV-1 clade C gp120, the C1086 strain was used). (B) Conformational fixation followed by ligand selection. The ratio of ligand binding to cross-linked vs. untreated gp120s is shown for full-length and truncated forms of gp120. ps, recognition by pooled sera from HIV-1-infected individuals, which was used for normalization.

Fig. 4.

Conformational diversity of HIV-1 gp120 as assessed by the small molecule NBD-556 and mechanism of control. The unliganded state of HIV-1 gp120 exists as an equilibrium of conformations, with the core_e portion of gp120 displaying a strong intrinsic propensity to snap into the CD4-bound conformation when not restrained by variable loops or by interactions with gp41. (A) Structure of small molecule NBD-556 in complex with HIV-1 gp120 provides atomic-level details of its binding site, and also provides an explanation for its preference for the CD4-bound conformation of gp120. The surface of core_e gp120 is colored blue for inner domain, gray for outer domain, and red for bridging sheet, with the small molecule NBD-556 binding at a highly conserved pocket at the nexus of inner domain, outer domain, and bridging sheet minidomain. The NBD-556 is shown in stick representation, colored yellow for carbon, red for oxygen, blue for nitrogen, and green for chlorine. (B) Close-up rotated 90° about a vertical axis from A. (C) Assessment of gp120 conformation in solution. SPR measurements of NBD-556 binding to gp120 in core_e (Left) or full-length (Right) gp120 contexts and by direct binding (Upper) or by competition (Lower) indicates that the unliganded core_e has a greater propensity to assume the CD4-bound conformation than full-length gp120. (D) Assessment of gp120 conformation in the functional viral spike. Neutralization by NBD-556 or NBD-557 is strongly enhanced by gp41 mutants (Upper) and to a lesser extent by changes in the V1/V2 region (Lower).

Fig. 5.

Mechanism of gp120 conformation control. In full-length WT gp120 (Upper), interactions with gp41 and between the variable loops V1/V2 and V3 shift the conformational equilibrium of the gp120 core away from the CD4-bound conformation (equilibrium is denoted by size of blue arrows). The intrinsic propensity of core_e gp120 (Lower) to assume the CD4-bound conformation is revealed when the gp41-interactive region and variable loops are removed.