title: Effect of nonpolar substitutions of the conserved Phe11 in the fusion peptide of HIV-1 gp41 on its function, structure, and organization in membranes.
abstract: The fusion domain of the HIV-1 envelope glycoprotein (gp120-gp41) is a conserved hydrophobic region located at the N-terminus of the transmembrane subunit (gp41). A prominent feature of this domain is a conserved five-residue "FLGFL" sequence at positions 8-12. Mutation of the highly conserved Phe(11) to Val (F11V), presumed not to significantly affect the hydrophobicity and the structure of this region, has been shown to decrease the level of syncytium formation and virus infectivity. Here we show that the substitution of Gly for Phe(11) (F11G) reduces cell-cell fusion activity by 80-90%. To determine the effect of these mutations on the properties of the fusion peptide, a 33-residue peptide (WT) identical to the extended fusion domain and its F11V and F11G mutants were synthesized, fluorescently labeled, and studied with respect to their function, structure, and organization in phospholipid membranes. The WT peptide alone induced fusion of both zwitterionic (PC/Chol) and negatively charged (PS/PC/Chol and POPG) vesicles, in contrast to a 23-mer fusion peptide lacking the C-terminal domain which has been shown to be inactive with PC vesicles but able to induce fusion of POPG vesicles which had been preaggragated with Ca(2+) or Mg(2+). The F11V peptide preserved 50% activity, and the F11G peptide was virtually inactive. ATR-FTIR spectroscopy indicated similar secondary structure of the peptides in multibilayers that was independent of membrane composition. Furthermore, all the peptides increased the extent of lipid disorder to a similar extent, but the kinetics of amide II H to D exchange was in the following order: F11G > F11V > WT. Fluorescence studies in the presence of membranes, as well as SDS-PAGE, revealed that the WT and F11V peptides self-associate to similar levels while F11G exhibited a decreased level of self-association. The data suggest that the FLGFL motif contributes to the functional organization of the HIV-1 fusion peptide and that the C-terminal domain following the fusion peptide contributes to the membrane fusion process.
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: M
authors.initials: M
authors.lastname: Pritsker
authors.forename: J
authors.initials: J
authors.lastname: Rucker
authors.forename: T L
authors.initials: TL
authors.lastname: Hoffman
authors.forename: R W
authors.initials: RW
authors.lastname: Doms
authors.forename: Y
authors.initials: Y
authors.lastname: Shai
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D002459:Cell Fusion / Q000235:genetics; D002460:Cell Line; D017124:Conserved Sequence / Q000235:genetics; D019286:Endopeptidase K / Q000378:metabolism; D017930:Genes, Reporter; D005998:Glycine / Q000235:genetics; D015700:HIV Envelope Protein gp41 / Q000737:chemistry / Q000235:genetics* / Q000502:physiology / Q000648:ultrastructure; D015497:HIV-1 / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D006801:Humans; D006868:Hydrolysis; D008561:Membrane Fusion / Q000235:genetics*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics* / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012996:Solutions; D017550:Spectroscopy, Fourier Transform Infrared; D013329:Structure-Activity Relationship; D014633:Valine / Q000235:genetics; D014760:Viral Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology / Q000648:ultrastructure
chemical_list: D015700:HIV Envelope Protein gp41; D010455:Peptides; D010743:Phospholipids; D012996:Solutions; D014760:Viral Fusion Proteins; D010649:Phenylalanine; D019286:Endopeptidase K; D014633:Valine; D005998:Glycine