Cryo-EM structures of HIV-1 trimer bound to CD4-mimetics BNM-III-170 and M48U1 adopt a CD4-bound open conformation

ARTICLE https://doi.org/10.1038/s41467-021-21816-x OPEN Cryo-EM structures of HIV-1 trimer bound to CD4-mimetics BNM-III-170 and M48U1 adopt a CD4-bound open conformation 1234567890():,; Claudia A. Jette 1, Christopher O. Barnes1, Sharon M. Kirk2, Bruno Melillo Pamela J. Bjorkman 1 ✉ 2, Amos B. Smith III2 & Human immunodeficiency virus-1 (HIV-1), the causative agent of AIDS, impacts millions of people. Entry into target cells is mediated by the HIV-1 envelope (Env) glycoprotein interacting with host receptor CD4, which triggers conformational changes allowing binding to a coreceptor and subsequent membrane fusion. Small molecule or peptide CD4-mimetic drugs mimic CD4’s Phe43 interaction with Env by inserting into the conserved Phe43 pocket on Env subunit gp120. Here, we present single-particle cryo-EM structures of CD4-mimetics BNMIII-170 and M48U1 bound to a BG505 native-like Env trimer plus the CD4-induced antibody 17b at 3.7 Å and 3.9 Å resolution, respectively. CD4-mimetic-bound BG505 exhibits canonical CD4-induced conformational changes including trimer opening, formation of the 4stranded gp120 bridging sheet, displacement of the V1V2 loop, and formation of a compact and elongated gp41 HR1C helical bundle. We conclude that CD4-induced structural changes on both gp120 and gp41 Env subunits are induced by binding to the gp120 Phe43 pocket. 1 Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA. 2 Department of Chemistry, University of Pennsylvania, Philadelphia, PA, USA. ✉email: NATURE COMMUNICATIONS | (2021)12:1950 | https://doi.org/10.1038/s41467-021-21816-x | www.nature.com/naturecommunications 1 ARTICLE H NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21816-x uman Immunodeficiency Virus 1 (HIV-1) is the causative agent of Acquired Immunodeficiency Syndrome (AIDS) and currently infects over 37.5 million people1. The entry of HIV-1 into host target cells is initiated by binding of the host receptor CD4 to the only viral protein on the surface of HIV-1, the envelope (Env) glycoprotein, a trimer of gp120-gp41 heterodimers2. Env binding to CD4 induces a well-characterized set of conformational changes3–6 that expose an occluded binding site in the gp120 V3 region for a co-receptor, either CCR5 or CXCR47. Upon co-receptor binding, Env undergoes further conformational changes resulting in the insertion of the gp41 fusion peptide into the target cell membrane, allowing the fusion of the viral and host membranes and entry of the HIV-1 genetic material into the target cell2. X-ray and cryo-EM structures of native-like soluble HIV-1 Env trimers (SOSIPs8) have defined a closed, prefusion state in which the V1V2 loops at the trimer apex shield the co-receptor binding site on the V3 region9, and a CD4-bound open state in which the gp120 subunits rotate outwards from the trimer axis, the V1V2 loops are displaced to the sides of the trimer, and the V3 loops are exposed3–6. A key interaction for exposure of the co-receptor binding site upon CD4 binding is the insertion of CD4 residue Phe43CD4 into a conserved, 150Å2 hydrophobic cavity at the junction between the gp120 inner domain, outer domain, and bridging sheet10. This interaction was first observed in crystal structures of monomeric gp120 cores complexed with CD410, which adopt a hallmark feature of CD4-bound Env trimers in the presence or absence of CD4: a 4-stranded anti-parallel β-sheet comprising β-strands β20, β21, β2, and β311. By contrast, SOSIP Env trimers in the closed, prefusion state contain a mixed parallel/anti-parallel 3-stranded β-sheet comprising strands β20, β21, and β312. Upon CD4 binding to an Env trimer, the loop between strands β20 and β21 is displaced, triggering changes that are propagated through the inner domain of gp120 and resulting in trimer opening, V1V2 displacement, and 4-stranded bridging sheet formation3–6. Identification of the importance of the gp120 Phe43 cavity for CD4 binding led to the development of cavityinteracting small molecule and peptide compounds called CD4 mimetic (CD4m) inhibitors13–20. Small molecule HIV-1 entry inhibitors that prevent HIV-1 trimer opening include BMS-378806 and a related family of compounds including BMS-626529, which bind orthogonally to the Phe43 opening beneath the Env β20-21 loop and extend into the base of the Phe43 cavity. Upon inhibitor binding, the Env trimer is kept closed by allosterically preventing CD4 binding by separating the bridging sheet and the inner domain of gp12020,21. CD4m compounds that open trimers by binding directly into the Phe43 pocket were initially identified with the discovery of NBD-556 and NBD-557, two small molecules that inhibit HIV-1 entry into cells expressing CD4 and a co-receptor, but enhance entry into cells that express a co-receptor in the absence of CD413. Subsequent studies showed that premature allosteric activation of trimer opening by these small molecules could inhibit viral entry after an initial period of increased activation22, leading to the modification of these compounds and the development of CD4m small molecule inhibitors such as BNM-III-170 that bind to the Phe43 pocket but prevent infection of cells lacking CD413,14. Members of this class were also shown to induce an intermediate Env conformation that can be stabilized by gp120 inner-domain-targeting Abs, permitting AntibodyDependent Cellular Cytotoxicity23. Concurrent with the development of small molecule CD4m inhibitors, peptide CD4m inhibitors were developed using scorpion toxin scyllatoxin scaffolds in which the CDR2-like loop of CD4 containing Phe43 had been grafted24–26. Unlike smallmolecule CD4m compounds, which primarily insert directly into 2 the Phe43 cavity with few external interactions, CD4m peptides contain a more extensive gp120 binding interface involving not only a synthetic Phe43-equivalent residue but also an equivalent to Arg59CD4, which forms a salt bridge with the highly-conserved Asp368gp12010 and an exposed C-terminal β-strand that forms hydrogen bonds with the β15 strand of gp120 immediately adjacent to the Phe43 cavity opening15. These peptides directly compete with CD4 binding and inhibit HIV-1 infection of cells24,25. Recent reports of structures of CD4-bound partially-open5 and fully-open but asymmetric Env trimers6 demonstrated that there are different conformations of open HIV-1 Env trimers. In addition, the structure of an Env trimer bound to the CD4binding site antibody b12 exhibited yet another open Env conformation4. Here we investigated the open conformation(s) of HIV-1 Env induced by two CD4m compounds: BNM-III-170, a small molecule, and M48U1, a peptide, both of which have been structurally characterized when bound to gp120 monomeric cores14,15. We report single-particle cryo-EM structures of complexes of these trimer-opening CD4m compounds with the BG505 SOSIP.664 trimer8 (hereafter BG505), which provide information about potential V1V2 displac (...truncated)