Abstract
HIV-1 entry into cells is mediated by the fusion protein gp41. Cholesterol plays an important role in this virus-cell fusion, but molecular structural information about cholesterol-gp41 interaction is so far absent. Here, we present experimental and computational data about cholesterol complexation with gp41 in lipid bilayers. We focus on the C-terminal region of the protein, which comprises a membrane-proximal external region (MPER) and the transmembrane domain (TMD). We measured peptide-cholesterol contacts in virus-mimetic lipid bilayers using solid-state NMR spectroscopy, and augmented these experimental data with all-atom molecular dynamics simulations. 2D (19)F NMR spectra show correlation peaks between MPER residues and the cholesterol isooctyl tail, indicating that cholesterol is in molecular contact with the MPER-TMD trimer. (19)F-(13)C distance measurements between the peptide and (13)C-labeled cholesterol show that C17 on the D ring and C9 at the intersection of B and C rings are ~7.0 Å from the F673 side-chain 4-(19)F. At high peptide concentrations in the membrane, the (19)F-(13)C distance data indicate three cholesterol molecules bound near F673 in each trimer. Mutation of a cholesterol recognition amino acid consensus motif did not change these distances, indicating that cholesterol binding does not require this sequence motif. Molecular dynamics simulations further identify two hotspots for cholesterol interactions. Taken together, these experimental data and simulations indicate that the helix-turn-helix conformation of the MPER-TMD is responsible for sequestering cholesterol. We propose that this gp41-cholesterol interaction mediates virus-cell fusion by recruiting gp41 to the boundary of the liquid-disordered and liquid-ordered phases to incur membrane curvature.