Identification of the Major Ubiquitin-Binding Domain of the Pseudomonas Aeruginosa ExoU A2 Phospholipase

J Biol Chem. 2013 Sep 13;288(37):26741-52. doi: 10.1074/jbc.M113.478529. Epub 2013 Aug 1.

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.

Keywords: Bacterial Toxins; Phospholipase; Protein-Protein Interactions; Pseudomonas aeruginosa; Ubiquitin.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

  • Bacterial Proteins
  • Recombinant Proteins
  • Ubiquitin
  • pseudomonas exoprotein A protein, Pseudomonas aeruginosa
  • Glutathione Transferase
  • Phospholipases A2