The membrane topology of the fusion peptide region of influenza hemagglutinin determined by spin-labeling EPR.
Hemagglutinin (HA) is a homotrimeric surface glycoprotein of the influenza virus. In infection, it induces membrane fusion between viral and endosomal membranes at low pH. Each monomer consists of the receptor-binding HA1 domain and the membrane-interacting HA2 domain. It has been known that the NH2-terminal region of the HA2 domain, the so-called "fusion peptide", inserts into the target membrane and plays a crucial role in triggering fusion between the viral and endosomal membranes. A major portion of the HA2 domain (FHA2: aa 1 to 127) of influenza virus X-31, including the NH2-terminal fusion peptide region, was expressed in Escherichia coli. Through site-directed mutagenesis, eight cysteine (Cys) mutants in the fusion peptide region of HA2 (A5C, I6C, A7C, G8C, I10C, N12C, G13C, W14C) were generated and modified with a nitroxide spin label. Using spin-labeling electron paramagnetic resonance (EPR) techniques, we investigated the conformation, membrane topology and the local oligomeric state of the fusion peptide region in the membrane. EPR spectra showed that this region is likely to exist as a flexible monomer in the membrane at both neutral and fusogenic pH conditions. In addition, EPR power saturation methods allowed us to measure the depth in the membrane of the spin label at each mutation site. The resulting depth profile is consistent with an alpha-helix tilted approximately 25 degrees from the horizontal plane of the membrane with a maximum depth of 15 A from the phosphate group. The tilt and rotational orientation correlates well with a calculated amphiphilicity of this region.
Macosko
J C
JC
Department of Chemistry, University of California, Berkeley 94720, USA.
Kim
C H
CH
Shin
Y K
YK
D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry* / Q000235:genetics; D008561:Membrane Fusion; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D013113:Spin Labels; D014760:Viral Fusion Proteins / Q000737:chemistry* / Q000235:genetics
D003432:Cross-Linking Reagents; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013113:Spin Labels; D014760:Viral Fusion Proteins