title: R468A mutation in perfringolysin O destabilizes toxin structure and induces membrane fusion.
abstract: Perfringolysin O (PFO) belongs to the family of cholesterol-dependent cytolysins. Upon binding to a cholesterol-containing membrane, PFO undergoes a series of structural changes that result in the formation of a β-barrel pore and cell lysis. Recognition and binding to cholesterol are mediated by the D4 domain, one of four domains of PFO. The D4 domain contains a conserved tryptophan-rich loop named undecapeptide (E458CTGLAWEWWR468) in which arginine 468 is essential for retaining allosteric coupling between D4 and other domains during interaction of PFO with the membrane. In this report we studied the impact of R468A mutation on the whole protein structure using hydrogen-deuterium exchange coupled with mass spectrometry. We found that in aqueous solution, compared to wild type (PFO), PFOR468Ashowed increased deuterium uptake due to exposure of internal toxin regions to the solvent. This change reflected an overall structural destabilization of PFOR468Ain solution. Conversely, upon binding to cholesterol-containing membranes, PFOR468Arevealed a profound decrease of hydrogen-deuterium exchange when compared to PFO. This block of deuterium uptake resulted from PFOR468A-induced aggregation and fusion of liposomes, as found by dynamic light scattering, microscopic observations and FRET measurements. In the result of liposome aggregation and fusion, the entire PFOR468Amolecule became shielded from aqueous solution and thereby was protected against proteolytic digestion and deuteration. We have established that structural changes induced by the R468A mutation lead to exposure of an additional cholesterol-independent liposome-binding site in PFO that confers its fusogenic property, altering the mode of the toxin action.
authors.affiliation: Department of Biophysics, Institute of Biochemistry and Biophysics of the Polish Academy of Sciences, 5A Pawinskiego St., 02-106 Warsaw, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Kulma
authors.affiliation: Department of Biophysics, Institute of Biochemistry and Biophysics of the Polish Academy of Sciences, 5A Pawinskiego St., 02-106 Warsaw, Poland.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Kacprzyk-Stokowiec
authors.affiliation: Laboratory of Molecular Membrane Biology, Department of Cell Biology, Nencki Institute of Experimental Biology of the Polish Academy of Sciences, 3 Pasteur St., 02-093 Warsaw, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Kwiatkowska
authors.affiliation: Laboratory of Molecular Membrane Biology, Department of Cell Biology, Nencki Institute of Experimental Biology of the Polish Academy of Sciences, 3 Pasteur St., 02-093 Warsaw, Poland.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Traczyk
authors.affiliation: Laboratory of Molecular Membrane Biology, Department of Cell Biology, Nencki Institute of Experimental Biology of the Polish Academy of Sciences, 3 Pasteur St., 02-093 Warsaw, Poland. Electronic address: asobota@nencki.gov.pl.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Sobota
authors.affiliation: Department of Biophysics, Institute of Biochemistry and Biophysics of the Polish Academy of Sciences, 5A Pawinskiego St., 02-106 Warsaw, Poland; Institute of Genetics and Biotechnology, Department of Biology, Warsaw University, 1 Miecznikowa St., 02-185 Warsaw, Poland. Electronic address: michald@ibb.waw.pl.
authors.forename: Michał
authors.initials: M
authors.lastname: Dadlez
mesh_terms: D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000737:chemistry* / Q000472:pathogenicity; D041961:Deuterium Exchange Measurement; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism; D008561:Membrane Fusion; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D053835:Unilamellar Liposomes / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D008081:Liposomes; D011993:Recombinant Fusion Proteins; D053835:Unilamellar Liposomes; C020452:Clostridium perfringens theta-toxin
keywords: Cholesterol-dependent cytolysin; Hydrogen-deuterium exchange; Lipid bilayer; Liposome aggregation; Membrane fusion; Toxin