Structure and Interactions of HIV-1 gp41 CHR-NHR Reverse Hairpin Constructs Reveal Molecular Determinants of Antiviral Activity

• Published in: Journal of molecular biology 2024-08, Vol.436 (16), p.168650, Article 168650
• Main Authors: He, Li, McAndrew, Ryan, Barbu, Razvan, Gifford, Grant, Halacoglu, Cari, Drouin-Allaire, Camille, Weber, Lindsey, Kristensen, Line G., Gupta, Sayan, Chen, Yan, Petzold, Christopher J., Allaire, Marc, Li, Kathy H., Ralston, Corie Y., Gochin, Miriam
• Format: Article
• Language: English
• Subjects: BASIC BIOLOGICAL SCIENCES; covalent ligand; crystal structure; Crystallography, X-Ray; Gp41 derived antiviral; HIV Envelope Protein gp41 - chemistry; HIV Envelope Protein gp41 - genetics; HIV Envelope Protein gp41 - metabolism; HIV-1 - drug effects; Humans; lipid altered structure; Models, Molecular; Protein Conformation; Protein Folding; X-ray footprinting
• ISSN: 0022-2836; 1089-8638; 1089-8638
• DOI: 10.1016/j.jmb.2024.168650

[Display omitted] •N- and C-terminal helices of HIV gp41 coalesce into hairpin trimers upon fusion.•Mini-proteins containing truncated C-helix followed by N-helix are potent fusion inhibitors.•The mini-proteins adopt a 6-helix bundle structure with C-helices folded along the grooves of the N-helical trimer.•Folded C-helix augments bundle stability and protects against aggregation.•Biochemical and biophysical techniques revealed C-helix opening to expose N-helix as the active component.•Pockets in both N- and C-terminal halves of N helix contributed to antiviral potency.•Lipid induced unfolding allows the inhibitor to reach its target at the membrane. Engineered reverse hairpin constructs containing a partial C-heptad repeat (CHR) sequence followed by a short loop and full-length N-heptad repeat (NHR) were previously shown to form trimers in solution and to be nanomolar inhibitors of HIV-1 Env mediated fusion. Their target is the in situ gp41 fusion intermediate, and they have similar potency to other previously reported NHR trimers. However, their design implies that the NHR is partially covered by CHR, which would be expected to limit potency. An exposed hydrophobic pocket in the folded structure may be sufficient to confer the observed potency, or they may exist in a partially unfolded state exposing full length NHR. Here we examined their structure by crystallography, CD and fluorescence, establishing that the proteins are folded hairpins both in crystal form and in solution. We examined unfolding in the milieu of the fusion reaction by conducting experiments in the presence of a membrane mimetic solvent and by engineering a disulfide bond into the structure to prevent partial unfolding. We further examined the role of the hydrophobic pocket, using a hairpin-small molecule adduct that occluded the pocket, as confirmed by X-ray footprinting. The results demonstrated that the NHR region nominally covered by CHR in the engineered constructs and the hydrophobic pocket region that is exposed by design were both essential for nanomolar potency and that interaction with membrane is likely to play a role in promoting the required inhibitor structure. The design concepts can be applied to other Class 1 viral fusion proteins.