The Structural Study of the Fusion Peptide Analogs of Influenza Virus Hemagglutinin Implicates an a-helix is not sufficient for fusion activity

Chun-Hua Hsu1,2,3, Shih-Hsiung Wu2,3, Chinpan Chen1.

1Institute of Biomedical Sciences, 2Institute of Biological Chemistry, Academia Sinica, and 3Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan, R.O.C.

Infection by enveloped virus initially involves membrane fusion between viral and host cell membranes in which the fusion peptide plays a crucial role in triggering the reaction. To clarify how the fusion peptide exerts its specific functions, we have carried out structural studies of several fusion-peptide analogs of influenza virus hemagglutinin HA2: namely E5, G13L, L17A and so on. Fluorescence experiments in solution containing SDS exhibit a blue-shift for E5, revealing a strong hydrophobic interaction between analog and micelles. Circular dichroism (CD) data show that E5 displays a-helical structures either in 50% TFE or in SDS micelles, and possesses higher a-helical contents at its fusogenic pH. These biophysical methods reveal the fusion-peptide of HA and its analogs consist of almostly a-helical secondary structure as previouly described by several research groups. Our three-dimensional structure of the E5 peptide in SDS solution at pH 4.0, based on 372 restraints and generated using X-PLOR, demonstrates that Leu2-Glu11 and Trp14-Ile18 form an amphipathic a-helical conformation with Gly12-Gly13 forming a flexible hinge. At higher pH, it is observed that the C-terminal a-helix reduces dramatically. Comparing the structural data of E5 with the fusion activities of several fusion-peptide mutants, we suggest that the C-terminal helices of fusion peptide could be induced at fusogenic pH and the fusion activity requires the presence of both N- and C-terminal a-helices. Furthermore, the flexible hinge, Gly12-Gly13, also plays an important role that allow the C-terminal a-helices to span the lipid bilayer. Summing up the structural information, a model illustrating the structural change of the fusion peptide analog and how the analog interacts with the lipid bilayer at different pHs is proposed. The NMR studies of G13L and L17A further verify the accuracy of the proposed mechanism. The model provide a novel molecular insight into the involement of this protein segment into early events of fusion and a new idea for peptide design of drug/ODN( Oligo Deoxynucleotide ) deliverer.

 

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