Discovery of Critical Residues for Viral Entry and Inhibition through Structural Insight of HIV-1 Fusion Inhibitor CP621–652*

The core structure of HIV-1 gp41 is a stable six-helix bundle (6-HB) folded by its trimeric N- and C-terminal heptad repeats (NHR and CHR). We previously identified that the ⁶²¹QIWNNMT⁶²⁷ motif located at the upstream region of gp41 CHR plays critical roles for the stabilization of the 6-HB core and peptide CP621–652 containing this motif is a potent HIV-1 fusion inhibitor, however, the molecular determinants underlying the stability and anti-HIV activity remained elusive. In this study, we determined the high-resolution crystal structure of CP621–652 complexed by T21. We find that the ⁶²¹QIWNNMT⁶²⁷ motif does not maintain the α-helical conformation. Instead, residues Met⁶²⁶ and Thr⁶²⁷ form a unique hook-like structure (denoted as M-T hook), in which Thr⁶²⁷ redirects the peptide chain to position Met⁶²⁶ above the left side of the hydrophobic pocket on the NHR trimer. The side chain of Met⁶²⁶ caps the hydrophobic pocket, stabilizing the interaction between the pocket and the pocket-binding domain. Our mutagenesis studies demonstrate that mutations of the M-T hook residues could completely abolish HIV-1 Env-mediated cell fusion and virus entry, and significantly destabilize the interaction of NHR and CHR peptides and reduce the anti-HIV activity of CP621–652. Our results identify an unusual structural feature that stabilizes the six-helix bundle, providing novel insights into the mechanisms of HIV-1 fusion and inhibition.