The HIV Fusion Peptide Adopts Intermolecular Parallel β-Sheet Structure in Membranes when Stabilized by the Adjacent N-Terminal Heptad Repeat: A 13C FTIR Study

The HIV gp41 protein mediates fusion with target host cells. The region primarily involved in directing fusion, the fusion peptide (FP), is poorly understood at the level of structure and function due to its toxic effect in expression systems. To overcome this, we used a synthetic approach to generate the N70 construct, whereby the FP is stabilized in context of the adjacent auto oligomerization domain. The amide I profile of unlabeled N70 in membranes reveals prominent α-helical contribution, along with significant β-structure. By truncating the N terminus (FP region) of N70, β-structure is eliminated, suggesting that the FP adopts a β-structure in membranes. To assess this directly, 13C Fourier-transformed infra-red analysis was carried out to map secondary structure of the 16 N-terminal hydrophobic residues of the fusion peptide (FP16). The 13C isotope shifted absorbance of the FP was filtered from the global secondary structure of the 70 residue construct (N70). On the basis of the peak shift induced by the 13C-labeled residues of FP16, we directly assign β-sheet structure in ordered membranes. A differential labeling scheme in FP16 allows us to distinguish the type of β-sheet structure as parallel. Dilution of each FP16-labeled N70 peptide, by mixing with unlabeled N70, shows directly that the FP16 β-strand region self-assembles. We discuss our structural findings in the context of the prevailing gp41 fusion paradigm. Specifically, we address the role of the FP region in organizing supramolecular gp41 assembly, and we also discuss the mechanism by which exogenous, free FP constructs inhibit gp41-induced fusion.