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

Huihui Chong,Xue Yao,Zonglin Qiu,Bo Qin,Ruiyun Han,Sandro Waltersperger,Meitian Wang,Sheng Cui,Yuxian He

doi:10.1074/jbc.m112.354126

Abstract

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 (621)QIWNNMT(627) 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 (621)QIWNNMT(627) motif does not maintain the α-helical conformation. Instead, residues Met(626) and Thr(627) form a unique hook-like structure (denoted as M-T hook), in which Thr(627) redirects the peptide chain to position Met(626) above the left side of the hydrophobic pocket on the NHR trimer. The side chain of Met(626) 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.

Highlights

CP621– 652 is a human immunodeficiency virus type 1 virus (HIV-1) fusion inhibitor peptide containing the gp41 621QIWNNMT627 motif
We previously identified that the 621QIWNNMT627 motif located at the upstream region of gp41 C-terminal heptad repeat regions (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, the molecular determinants underlying the stability and anti-HIV activity remained elusive
The 6-HB formed by CP621– 652 and T21 was purified by size-exclusion chromatography

Summary

Background

CP621– 652 is a HIV-1 fusion inhibitor peptide containing the gp41 621QIWNNMT627 motif. We previously identified that the 621QIWNNMT627 motif located at the upstream region of gp 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, the molecular determinants underlying the stability and anti-HIV activity remained elusive. Because of drug-resistance problem, a series of novel peptide-based fusion inhibitors were developed with improved anti-HIV potency and pharmacokinetic profiles (14 –18) These peptides have been derived primarily from residues 628 to 673 of the gp CHR, based on the peptide C34 sequence as a template in particular [17, 19], but little attention was drawn to the upstream sequence of the CHR.

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION