title: Induced conformational changes in the activation of the Pseudomonas aeruginosa type III toxin, ExoU.
abstract: ExoU is a 74-kDa, water-soluble toxin injected directly into mammalian cells through the type III secretion system of the opportunistic pathogen, Pseudomonas aeruginosa. Previous studies have shown that ExoU is a Ca(2+)-independent phospholipase that requires a eukaryotic protein cofactor. One protein capable of activating ExoU and serving as a required cofactor was identified by biochemical and proteomic methods as superoxide dismutase (SOD1). In these studies, we carried out site-directed spin-labeling electron paramagnetic resonance spectroscopy to examine the effects of SOD1 and substrate liposomes on the structure and dynamics of ExoU. Local conformational changes within the catalytic site were observed in the presence of substrate liposomes, and were enhanced by the addition of SOD1 in a concentration-dependent manner. Conformational changes in the C-terminal domain of ExoU were observed upon addition of cofactor, even in the absence of liposomes. Double electron-electron resonance experiments indicated that ExoU samples multiple conformations in the resting state. In contrast, addition of SOD1 induced ExoU to adopt a single, well-defined conformation. These studies provide, to our knowledge, the first direct evidence for cofactor- and membrane-induced conformational changes in the mechanism of activation of ExoU.
authors.affiliation: Center for Infectious Disease Research, Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
authors.forename: Marc A
authors.initials: MA
authors.lastname: Benson
authors.forename: Steven M
authors.initials: SM
authors.lastname: Komas
authors.forename: Katherine M
authors.initials: KM
authors.lastname: Schmalzer
authors.forename: Monika S
authors.initials: MS
authors.lastname: Casey
authors.forename: Dara W
authors.initials: DW
authors.lastname: Frank
authors.forename: Jimmy B
authors.initials: JB
authors.lastname: Feix
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain / Q000187:drug effects*; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D011550:Pseudomonas aeruginosa; D013113:Spin Labels; D013482:Superoxide Dismutase / Q000494:pharmacology; D000072105:Superoxide Dismutase-1
chemical_list: D001426:Bacterial Proteins; D013113:Spin Labels; C111749:pseudomonas exoprotein A protein, Pseudomonas aeruginosa; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1