The identification and structure of the membrane-spanning domain of the Clostridium septicum alpha toxin.
Alpha toxin (AT) is a pore-forming toxin produced by Clostridium septicum that belongs to the unique aerolysin-like family of pore-forming toxins. The location and structure of the transmembrane domains of these toxins have remained elusive. Using deletion mutagenesis, cysteine-scanning mutagenesis and multiple spectrofluorimetric methods a membrane-spanning amphipathic beta-hairpin of AT has been identified. Spectrofluorimetric analysis of cysteine-substituted residues modified with an environmentally sensitive fluorescent probe via the cysteine sulfydryl showed that the side chains of residues 203-232 alternated between the aqueous milieu and the membrane core when the AT oligomer was inserted into membranes, consistent with the formation of an amphipathic transmembrane beta-hairpin. AT derivatives that contained deletions that removed up to 90% of the beta-hairpin did not form a pore but were similar to native toxin in all other aspects of the mechanism. Furthermore, a mutant of AT that contained an engineered disulfide, predicted to restrict the movement of the beta-hairpin, functioned similarly to native toxin except that it did not form a pore unless the disulfide bond was reduced. Together these studies revealed the location and structure of the membrane-spanning domain of AT.
Melton
Jody A
JA
Department of Microbiology and Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, USA.
Parker
Michael W
MW
Rossjohn
Jamie
J
Buckley
J Thomas
JT
Tweten
Rodney K
RK
D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics*; D003013:Clostridium / Q000737:chemistry* / Q000235:genetics*; D003461:Crystallography; D004220:Disulfides / Q000737:chemistry; D005973:Glutamine / Q000235:genetics; D008051:Lipid Bilayers; D008239:Lysine / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D001427:Bacterial Toxins; D004220:Disulfides; D008051:Lipid Bilayers; D008565:Membrane Proteins; C064270:hemolytic toxin, Clostridium septicum; D005973:Glutamine; D008239:Lysine