Identification and characterization of a subpocket on the N-trimer of HIV-1 Gp41: implication for viral entry and drug target.

Abstract

Crystallographic studies of HIV-1 gp41 demonstrate a stable six-helix bundle (6-HB) folded by trimeric N and C-terminal heptad repeats (NHR and CHR), and a deep hydrophobic pocket (pocket-1) on the NHR helices (N-trimer); however, previous crystal structures of 6-HB core were determined by peptide fragments missing the downstream sequence of pocket-1; thus, the structural features of this site could not be observed. We recently determined several 6-HB structures containing the pocket-1 and its downstream site. Here, we focused to investigate the structural features of N-trimer previously uncharacterized. Biophysical, biochemical and functional approaches were combined to characterize the downstream residues of pocket-1. A subpocket (designated pocket-2) was visualized on the C-terminal portion of N-trimer, which is formed by a cluster of seven residues, including Leu587, Lys588 and Glu584 on one NHR helix and Tyr586, Val583, Ala582 and Arg579 of another NHR helix. Mutagenesis studies demonstrated that the pocket-2 residues play essential roles for HIV-1 Env-mediated cell entry and critically determine the antiviral activity of NHR-derived peptide fusion inhibitor T21. Further, the pocket-2 mutations dramatically impaired the thermostability and conformation of 6-HB structure and reduced the binding affinity of CHR-derived inhibitor HP23 that specifically targets the deep pocket-1. These data have provided important information for the structure-function relationship of HIV-1 gp41 and for the development of antiviral entry inhibitors.