Faculty Collaboration Database

Cooperative Substrate-Cofactor Interactions and Membrane Localization of the Bacterial Phospholipase A₂ (PLA₂) Enzyme, ExoU. J Biol Chem 2017 Feb 24;292(8):3411-3419

Date

01/11/2017

Pubmed ID

28069812

Pubmed Central ID

PMC5336173

DOI

10.1074/jbc.M116.760074

Scopus ID

2-s2.0-85013836015 (requires institutional sign-in at Scopus site)   25 Citations

Abstract

The ExoU type III secretion enzyme is a potent phospholipase A₂ secreted by the Gram-negative opportunistic pathogen, Pseudomonas aeruginosa Activation 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.

Author List

Tessmer MH, Anderson DM, Buchaklian A, Frank DW, Feix JB

Authors

Jimmy B. Feix PhD Professor in the Biophysics department at Medical College of Wisconsin
Dara W. Frank PhD Professor in the Microbiology and Immunology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Bacterial Proteins
Electron Spin Resonance Spectroscopy
Lipid Bilayers
Models, Molecular
Phospholipases A2
Protein Conformation
Pseudomonas aeruginosa
Ubiquitin