Determinants of Lipid Mixing Membrane Fusion by HIV gp41

Abstract

A critical step in HIV infection involves membrane fusion between the enveloped virion and the target cell plasma membrane, catalyzed by the viral membrane protein gp41 at or near physiologic pH. Specific global gp41 conformations (i.e. six-helix-bundle and coiled-coil) and specific regions (i.e. fusion peptide) are implicated in steps of fusion catalysis. Our goal is to elucidate the molecular mechanism of gp41 catalyzed membrane fusion. One challenge to biophysical analysis of structure and function in hydrophobic gp41 is solubility. Low pH (∼3.0) is utilized to enhance protein solubility by significantly increasing overall positive charge. Interestingly, the cationic six-helix-bundle region enhances/inhibits lipid mixing of anionic vesicles at low/neutral pH, which correlates with close association (∼5A) between membrane headgroup phosphates and a large/small proportion or population of helical bundles based on solid-state NMR analysis. The apolar trimeric fusion-peptide region contributes to lipid mixing and is predominantly β-sheet (∼75%) with a large fraction of β-strands located close to (∼5-7A)/far from (>∼10A) membrane headgroup phosphates at residues Ala-1 and Ala-14/Leu-7 and Met-19 at low pH. Swapping to neutral pH does not affect fusion-peptide conformation, but does lower the fraction of β-strands close to the membrane surface. Partial shedding of fusion-peptides from membranes, poor protein solubility, and protein lipid mixing inactivity at neutral pH are reversed upon pH swap back to low, indicating irreversible aggregation is not occurring at neutral pH. Our solid-state NMR findings indicate that abrogation of lipid-mixing fusion function by gp41 in six-helix-bundle conformation at the physiologic pH of viral fusion is determined primarily by stearic and electrostatic barriers to close membrane apposition imposed by the six-helix-bundle region, and minimally by membrane location or conformation of the fusion-peptide region.