Intermediate conformations of CD4-bound HIV-1 Env heterotrimers

Article Intermediate conformations of CD4-bound HIV-1 Env heterotrimers https://doi.org/10.1038/s41586-023-06639-8 Kim-Marie A. Dam1,3, Chengcheng Fan1,3, Zhi Yang1,2 & Pamela J. Bjorkman1 ✉ Received: 11 April 2023 Accepted: 13 September 2023 Published online: 22 November 2023 Open access Check for updates HIV-1 envelope (Env) exhibits distinct conformational changes in response to host receptor (CD4) engagement. Env, a trimer of gp120 and gp41 heterodimers, has been structurally characterized in a closed, prefusion conformation with closely associated gp120s and coreceptor binding sites on gp120 V3 hidden by V1V2 loops1–4 and in fully saturated CD4-bound open Env conformations with changes including outwardly rotated gp120s and displaced V1V2 loops3–9. To investigate changes resulting from substoichiometric CD4 binding, we solved single-particle cryo-electron microscopy (cryo-EM) structures of soluble, native-like heterotrimeric Envs bound to one or two CD4 molecules. Most of the Env trimers bound to one CD4 adopted the closed, prefusion Env state, with a minority exhibiting a heterogeneous partially open Env conformation. When bound to two CD4s, the CD4-bound gp120s exhibited an open Env conformation including a four-stranded gp120 bridging sheet and displaced gp120 V1V2 loops that expose the coreceptor sites on V3. The third gp120 adopted an intermediate, occluded-open state10 that showed gp120 outward rotation but maintained the prefusion three-stranded gp120 bridging sheet with only partial V1V2 displacement and V3 exposure. We conclude that most of the engagements with one CD4 molecule were insufficient to stimulate CD4-induced conformational changes, whereas binding two CD4 molecules led to Env opening in CD4-bound protomers only. The substoichiometric CD4-bound soluble Env heterotrimer structures resembled counterparts derived from a cryo-electron tomography study of complexes between virion-bound Envs and membrane-anchored CD4 (ref. 11), validating their physiological relevance. Together, these results illuminate intermediate conformations of HIV-1 Env and illustrate its structural plasticity. The HIV-1 Env glycoprotein, a heavily glycosylated homotrimer containing gp120 and gp41 subunits, mediates entry into host cells to initiate infection12. On the surface of virions, Env adopts a closed, prefusion conformation similar to that observed in soluble native-like Env trimer ectodomains1–4,13. The viral entry process is initiated when gp120s bind to the host receptor, CD4, at the CD4-binding site (CD4bs) located distal to the Env apex on the sides of each of the three gp120s5–9. This triggers conformational changes in gp120 that expose the gp120 V3 coreceptor binding site, which is occluded in the prefusion conformation beneath gp120 V1V2 loops5–9. Coreceptor binding results in further conformational changes that lead to insertion of the gp41 fusion peptide into the host cell membrane and fusion of viral and host membranes1,10. X-ray crystallography and single-particle cryo-EM structures have enabled characterization of soluble versions of HIV-1 Envs14 in closed, prefusion1,2, CD4-bound open5–7, and intermediate partially open conformations5,9,10. Several studies have demonstrated that the native-like soluble Envs (SOSIPs)14 used for structural studies resemble virion-bound Envs, indicating that these conformations may be relevant to the viral Env entry process3,4,14–17. The closed, prefusion Env conformation is characterized by gp120 V1V2 loops interacting around the trimer apex, thereby shielding the coreceptor binding sites on the V3 loops1,2,18. CD4-bound open Env trimer structures revealed receptor-induced changes in which the gp120 subunits rotated outwards, the V1V2 loops were displaced from the apex by approximately 40 Å to the sides of Env, and the coreceptor binding site on each V3 was exposed and became mostly disordered5–9 (Supplementary Video 1). This process also converted the closed, prefusion conformation three-stranded gp120 bridging sheet composed of the β20, β21 and β3 β-strands1 to a four-stranded antiparallel β-sheet in which strand β2, whose residues are located in a proximal helix in the closed, prefusion formation, is intercalated between strands β21 and β3 (refs. 1,5,6,9). Intermediate Env conformations include occluded-open5,10 and partially open conformations9,19. In the occluded-open conformation observed in trimer complexes with the CD4bs antibody b12 (ref. 5) and similar antibodies raised in vaccinated non-human primates10, the gp120 subunits were outwardly rotated from the central trimer axis as in CD4-bound open conformations, but V1V2 displacement and V3 exposure did not occur, and the prefusion three-stranded gp120 β-sheet was maintained5,10. In partially open Env conformations, CD4 binding led to the characteristic CD4-induced structural changes in gp120, but subsequent binding of Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA. 2Present address: Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA. 3These authors contributed equally: Kim-Marie A. Dam, Chengcheng Fan. ✉e-mail: 1 Nature | Vol 623 | 30 November 2023 | 1017 Article the gp120–gp41 interface antibody 8ANC195 led to partial closure of the gp120s9. A prevailing enigma about Env conformational changes and the role of CD4 in initiating the fusion process concerns whether the gp120/ gp41 protomers that form the Env trimer behave cooperatively or independently during receptor-induced transformations. This information would reveal how many CD4 receptor and CCR5 coreceptor molecules are needed to engage each Env trimer to initiate fusion and further explain Env function as it relates to virus infectivity, thereby informing the design of entry inhibitors and mechanisms of antibody neutralization and fusion. To investigate the role of receptor stoichiometry in CD4-induced conformational changes in HIV-1 Env, we designed soluble Env heterotrimers that can bind only one or only two CD4 receptors for comparisons with Env homotrimers binding either zero CD4s (closed, prefusion trimers) or three CD4s (fully saturated CD4-bound open trimers). Using single-particle cryo-EM, we solved structures of one or two CD4s bound to the clade A BG505 trimer14 to 3.4 and 3.9 Å, respectively. We found that binding one CD4 primarily resulted in a closed, prefusion Env conformation that showed only subtle indications of CD4-induced changes. Binding two CD4 molecules induced an asymmetric, partially open Env conformation in which the gp120 subunits resembled open (for CD4-bound protomers) and occluded-open (for the unliganded protomer) conformations, whereas the three gp41 subunits were structurally different from each other. Together, these results illustrate intermediate Env conformations and inform our understanding of the events that lead to HIV-1 fusion. Heterotrimer Env construct design A solub (...truncated)