title: Cooperative Substrate-Cofactor Interactions and Membrane Localization of the Bacterial Phospholipase A2(PLA2) Enzyme, ExoU.
abstract: The ExoU type III secretion enzyme is a potent phospholipase A2secreted by the Gram-negative opportunistic pathogen,Pseudomonas aeruginosaActivation of phospholipase activity is induced by protein-protein interactions with ubiquitin in the cytosol of a targeted eukaryotic cell, leading to destruction of host cell membranes. Previous work in our laboratory suggested that conformational changes within a C-terminal domain of the toxin might be involved in the activation mechanism. In this study, we use site-directed spin-labeling electron paramagnetic resonance spectroscopy to investigate conformational changes in a C-terminal four-helical bundle region of ExoU as it interacts with lipid substrates and ubiquitin, and to examine the localization of this domain with respect to the lipid bilayer. In the absence of ubiquitin or substrate liposomes, the overall structure of the C-terminal domain is in good agreement with crystallographic models derived from ExoU in complex with its chaperone, SpcU. Significant conformational changes are observed throughout the domain in the presence of ubiquitin and liposomes combined that are not observed with either liposomes or ubiquitin alone. In the presence of ubiquitin, two interhelical loops of the C-terminal four-helix bundle appear to penetrate the membrane bilayer, stabilizing ExoU-membrane association. Thus, ubiquitin and the substrate lipid bilayer act synergistically to induce a conformational rearrangement in the C-terminal domain of ExoU.
authors.affiliation: Department of Microbiology and Molecular Genetics; Center for Infectious Disease Research.
authors.forename: Maxx H
authors.initials: MH
authors.lastname: Tessmer
authors.affiliation: Department of Microbiology and Molecular Genetics; Center for Infectious Disease Research.
authors.forename: David M
authors.initials: DM
authors.lastname: Anderson
authors.affiliation: Department of Biophysics.
authors.forename: Adam
authors.initials: A
authors.lastname: Buchaklian
authors.affiliation: Department of Microbiology and Molecular Genetics; Center for Infectious Disease Research.
authors.forename: Dara W
authors.initials: DW
authors.lastname: Frank
authors.affiliation: Center for Infectious Disease Research; Department of Biophysics; National Biomedical EPR Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226. Electronic address: jfeix@mcw.edu.
authors.forename: Jimmy B
authors.initials: JB
authors.lastname: Feix
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D008051:Lipid Bilayers / Q000378:metabolism; D008958:Models, Molecular; D054467:Phospholipases A2 / Q000032:analysis / Q000378:metabolism*; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D008051:Lipid Bilayers; D025801:Ubiquitin; C111749:pseudomonas exoprotein A protein, Pseudomonas aeruginosa; D054467:Phospholipases A2
keywords: Pseudomonas aeruginosa (P. aeruginosa); electron paramagnetic resonance (EPR); phospholipase; protein structure; ubiquitin