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.

Tyrosine sulfation in the second variable loop (V2) of HIV-1 gp120 stabilizes V2–V3 interaction and modulates neutralization sensitivity

“…Despite their definition as variable loops, the V2 and V3 regions of gp120 contain highly conserved domains (33). Because several lines of evidence suggest that V3 establishes direct interaction with V2 (12)(13)(14)(15)(16)(17)(18)(19)(20)(21), and the conserved base of V3 is the binding site for the N-terminal region of the CCR5 coreceptor (34), we looked for potential structural homology between V2 and CCR5. We recognized that the conserved central region of V2 contains two tyrosine residues (Tyr173 and Tyr177) with spacing identical to that of two tyrosines (Tyr10 and Tyr14) present in the N-terminal region of CCR5 (Fig.…”

“…Functional and antigenic interactions between V2 and V3 have long been recognized (21)(22)(23)(24)(25)(26)(27), and spatial proximity between these two loops has been documented by both cryo-EM studies (12)(13)(14)(15)(16)(17) and the recently solved crystal structure of a stabilized soluble SOSIP trimer at 4.7-Å resolution (18). Despite the lack of sulfated tyrosines (presumably because the soluble trimer was produced in HEK293 cells), the latter structure is consistent with our model because it shows the 173-177 segment of V2 directly juxtaposed to the CCR5-binding region at the base of V3, with the most conserved of the two sulfotyrosines, Tyr177, establishing van der Waals contacts with Ile420, Leu154, and Leu175, as well as an H-bond with Asn302, all residues that are proximal to the predicted binding pocket for the homologous sulfotyrosine (Tys14) of CCR5 (34).…”

“…However, despite these advances, many critical aspects related to the structural mechanisms of HIV-1 immune vulnerability and evasion remain unresolved. In particular, the fine molecular details of the interaction between the second and third variable loops (V2 and V3, respectively) of gp120, which are believed to play a critical role in stabilizing the prefusion envelope structure (19)(20)(21), are elucidated only partially. Functionally, V2 and V3 cooperate in the formation of quaternary epitopes targeted Significance Despite intensive efforts, the structure of the native HIV-1 envelope trimer-the sole relevant target for vaccine designhas remained elusive.…”

Conformation-Dependent Recognition of HIV gp120 by Designed Ankyrin Repeat Proteins Provides Access to Novel HIV Entry Inhibitors

“…1B, selection II). In a further strategy, we probed whether removal of the V1V2 domain, the largest and most flexible of the gp120 variable loop domains (53,54), improves the efficiency of gp120-specific DARPin selection (Fig. 1B, selection III, gp120⌬V1V2).…”

Physicochemical bases for protein folding, dynamics, and protein-ligand binding

“…Moreover, the higher conformational flexibility of the unliganded state compared to the CD4-bound state [80,81], suggests that the tier-0 well of the unliganded gp120 may have a more rugged bottom than that of the CD4-bound gp120. In the context of the functional viral spike in vivo, however, the unliganded conformation of gp120 can be constrained by interactions with gp41 and other subunits, thus providing advantages for HIV to evade immune surveillance [78]. The conformational stability enhanced by the interactions of gp120 with other molecules indicates that the FEL of the protein-solvent system is not static, but rather, dynamic, with the width and depth of the free energy wells and the height of the barriers being variable under the influence of solvent conditions (Figure 2).…”