title: The p10 FAST protein fusion peptide functions as a cystine noose to induce cholesterol-dependent liposome fusion without liposome tubulation.
abstract: The reovirus p10 fusion-associated small transmembrane (FAST) proteins are the smallest known membrane fusion proteins, and evolved specifically to mediate cell-cell, rather than virus-cell, membrane fusion. The 36-40-residue ectodomains of avian reovirus (ARV) and Nelson Bay reovirus (NBV) p10 contain an essential intramolecular disulfide bond required for both cell-cell fusion and lipid mixing between liposomes. To more clearly define the functional, biochemical and biophysical features of this novel fusion peptide, synthetic peptides representing the p10 ectodomains of ARV and NBV were analyzed by solution-state NMR spectroscopy, circular dichroism spectroscopy, fluorescence spectroscopy-based hydrophobicity analysis, and liposome binding and fusion assays. Results indicate that disulfide bond formation promotes exposure of hydrophobic residues, as indicated by bis-ANS binding and time-dependent peptide aggregation under aqueous conditions, implying the disulfide bond creates a small, geometrically constrained, cystine noose. Noose formation is required for peptide partitioning into liposome membranes and liposome lipid mixing, and electron microscopy revealed that liposome-liposome fusion occurs in the absence of liposome tubulation. In addition, p10 fusion peptide activity, but not membrane partitioning, is dependent on membrane cholesterol.
authors.affiliation: Department of Microbiology & Immunology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada.
authors.forename: Tim
authors.initials: T
authors.lastname: Key
authors.affiliation: Department of Microbiology & Immunology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada; Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada.
authors.forename: Muzaddid
authors.initials: M
authors.lastname: Sarker
authors.affiliation: Department of Microbiology & Immunology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada.
authors.forename: Roberto
authors.initials: R
authors.lastname: de Antueno
authors.affiliation: Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada; Department of Chemistry, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada. Electronic address: jan.rainey@dal.ca.
authors.forename: Jan K
authors.initials: JK
authors.lastname: Rainey
authors.affiliation: Department of Microbiology & Immunology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada; Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada; Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada. Electronic address: roy.duncan@dal.ca.
authors.forename: Roy
authors.initials: R
authors.lastname: Duncan
mesh_terms: D000595:Amino Acid Sequence; D002784:Cholesterol / Q000737:chemistry*; D003553:Cystine / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D008081:Liposomes / Q000737:chemistry*; D008561:Membrane Fusion; D008969:Molecular Sequence Data; D018165:Orthoreovirus / Q000737:chemistry; D030101:Orthoreovirus, Avian / Q000737:chemistry; D017433:Protein Structure, Secondary; D014760:Viral Fusion Proteins / Q000138:chemical synthesis / Q000737:chemistry*
chemical_list: D008081:Liposomes; D014760:Viral Fusion Proteins; D003553:Cystine; D002784:Cholesterol
keywords: Fusion peptide; Liposome fusion; Membrane fusion; Viral fusion peptides