The Role of Hydrophobic Residues in the Kink Region of the Influenza Hemagglutinin Fusion Domain

The fusion peptide or fusion domain of influenza hemagglutinin is crucial for cell entry of this virus. Previous studies showed that this peptide adopts a boomerang-shaped structure in lipid model membranes at the pH of membrane fusion. To examine the role of the boomerang in fusion, I systematically changed residues in the kink region and examined their effects on structure and function. Among these, mutants W14A and F9A_I10A exhibited no fusion activities while F9A, I10A had no effect on fusion. Binding enthalpies of mutant peptides to model membranes and their ability to perturb lipid bilayer structures correlated well with the fusion activities of the corresponding full-length molecules. The structure of W14A determined by NMR and site-directed spin-labeling EPR features a flexible kink that points out of the membrane, in sharp contrast to the more ordered boomerang of the wild-type that points into the membrane. The structure of F9A and its docking in lipid bilayers are very similar to that of the wild-type. The more flexible kink region of the non-fusion mutants was further confirmed by double electron-electron resonance spectroscopy. We conclude that the hydrophobic residues Phe 9 and Ile 10 in the N-terminal arm and Trp 14 in the C-terminal arm of the kink region are critical to anchor the fusion domain in the membrane interface and to sustain a fixed-angle boomerang structure. This specific fixed-angle boomerang structure is required to support membrane fusion.

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