Solid-state nuclear magnetic resonance evidence for an extended beta strand conformation of the membrane-bound HIV-1 fusion peptide.

Solid-state nuclear magnetic resonance (NMR) spectroscopy was applied to the membrane-bound form of a synthetic peptide representing the 23-residue N-terminal fusion peptide domain of the HIV-1 gp41 envelope glycoprotein. 1D solid-state NMR line width measurements of singly 13C carbonyl labeled peptides showed that a significant population of the membrane-bound peptide is well-structured in its N-terminal and central regions while the C-terminus has more disorder. There was some dependence of line width on lipid composition, with narrower line widths and hence greater structural order observed for a lipid composition comparable to that found in the virus and its target T cells. In the more ordered N-terminal and central regions of the peptide, the 13C carbonyl chemical shifts are consistent with a nonhelical membrane-bound conformation. Additional evidence for a beta strand membrane-bound conformation was provided by analysis of 2D rotor-synchronized magic angle spinning NMR spectra of doubly 13C carbonyl labeled peptides. Lipid mixing and aqueous contents leakage assays were applied to demonstrate the fusogenicity of the peptide under conditions comparable to those used for the solid-state NMR sample preparation.

Solid state NMR measurements of conformation and conformational distributions in the membrane-bound HIV-1 fusion peptide.

The solid state NMR lineshape of a protein backbone carbonyl nucleus is a general diagnostic of the local conformational distribution in the vicinity of that nucleus. In addition, measurements of carbonyl chemical shifts and 2D exchange spectra provide information about the most probable conformation in the distribution. These types of solid state NMR methodologies have been applied to structural studies of the membrane-bound HIV-1 fusion peptide. This peptide is derived from a domain of the HIV-1 gp41 envelope protein, which is critical for viral-host cell-membrane fusion. Even in the absence of the rest of the envelope protein, the fusion peptide will fuse liposomes or erythrocytes. The solid state NMR measurements demonstrate that the center of the membrane-bound HIV-1 fusion peptide is structured, while the C-terminus is highly disordered. The structural distribution at the peptide center is lipid-dependent, with the greatest degree of structural homogeneity in a lipid environment whose composition reflects that of the target T cells. When bound to the lipid mixture, the peptide center is predominately beta sheet. The beta-sheet structure may be diagnostic of peptide oligomerization, which is thought to be a requirement for membrane fusion activity. Although the peptide partially disrupts bilayer orientational ordering in stacked glass-plate samples, 2H NMR demonstrates that the bilayers remain intact in the presence of the fusion peptide and are not micellized. The retention of the bilayer phase may relate to the biological requirement that the virus should fuse with, but not destroy, the target host cell membrane.