Faculty Collaboration Database

Identification of the major ubiquitin-binding domain of the Pseudomonas aeruginosa ExoU A2 phospholipase. J Biol Chem 2013 Sep 13;288(37):26741-52

Date

08/03/2013

Pubmed ID

23908356

Pubmed Central ID

PMC3772220

DOI

10.1074/jbc.M113.478529

Scopus ID

2-s2.0-84884182244   17 Citations

Abstract

Numerous Gram-negative bacterial pathogens use type III secretion systems to deliver effector molecules into the cytoplasm of a host cell. Many of these effectors have evolved to manipulate the host ubiquitin system to alter host cell physiology or the location, stability, or function of the effector itself. ExoU is a potent A2 phospholipase used by Pseudomonas aeruginosa to destroy membranes of infected cells. The enzyme is held in an inactive state inside of the bacterium due to the absence of a required eukaryotic activator, which was recently identified as ubiquitin. This study sought to identify the region of ExoU required to mediate this interaction and determine the properties of ubiquitin important for binding, ExoU activation, or both. Biochemical and biophysical approaches were used to map the ubiquitin-binding domain to a C-terminal four-helix bundle of ExoU. The hydrophobic patch of ubiquitin is required for full binding affinity and activation. Binding and activation were uncoupled by introducing an L8R substitution in ubiquitin. Purified L8R demonstrated a parental binding phenotype to ExoU but did not activate the phospholipase in vitro. Utilizing these new biochemical data and intermolecular distance measurements by double electron-electron resonance, we propose a model for an ExoU-monoubiquitin complex.

Author List

Anderson DM, Feix JB, Monroe AL, Peterson FC, Volkman BF, Haas AL, Frank DW

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
Francis C. Peterson PhD Professor in the Biochemistry department at Medical College of Wisconsin
Brian F. Volkman PhD Professor in the Biochemistry department at Medical College of Wisconsin

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

Bacterial Proteins
Circular Dichroism
Crystallography, X-Ray
Electrons
Glutathione Transferase
Magnetic Resonance Spectroscopy
Phospholipases A2
Point Mutation
Protein Binding
Protein Structure, Tertiary
Pseudomonas aeruginosa
Recombinant Proteins
Ubiquitin