Kinetic and Structural Insights Into the Mechanism of AMPylation by VopS Fic Domain

J Biol Chem. 2010 Jun 25;285(26):20155-63. doi: 10.1074/jbc.M110.114884. Epub 2010 Apr 21.

Abstract

The bacterial pathogen Vibrio parahemeolyticus manipulates host signaling pathways during infections by injecting type III effectors into the cytoplasm of the target cell. One of these effectors, VopS, blocks actin assembly by AMPylation of a conserved threonine residue in the switch 1 region of Rho GTPases. The modified GTPases are no longer able to interact with downstream effectors due to steric hindrance by the covalently linked AMP moiety. Herein we analyze the structure of VopS and its evolutionarily conserved catalytic residues. Steady-state analysis of VopS mutants provides kinetic understanding on the functional role of each residue for AMPylation activity by the Fic domain. Further mechanistic analysis of VopS with its two substrates, ATP and Cdc42, demonstrates that VopS utilizes a sequential mechanism to AMPylate Rho GTPases. Discovery of a ternary reaction mechanism along with structural insight provides critical groundwork for future studies for the family of AMPylators that modify hydroxyl-containing residues with AMP.

Publication types

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

MeSH terms

  • Adenosine Monophosphate / metabolism*
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Binding Sites / genetics
  • Catalysis
  • Crystallography, X-Ray
  • Host-Pathogen Interactions
  • Humans
  • Kinetics
  • Models, Molecular
  • Mutation
  • Nucleotidyltransferases / chemistry
  • Nucleotidyltransferases / genetics
  • Nucleotidyltransferases / metabolism*
  • Protein Binding
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Signal Transduction
  • Threonine / genetics
  • Threonine / metabolism
  • Vibrio Infections / metabolism
  • Vibrio Infections / microbiology
  • Vibrio parahaemolyticus / metabolism*
  • Vibrio parahaemolyticus / physiology
  • cdc42 GTP-Binding Protein / metabolism
  • rho GTP-Binding Proteins / genetics
  • rho GTP-Binding Proteins / metabolism

Substances

  • Bacterial Proteins
  • Threonine
  • Adenosine Monophosphate
  • Nucleotidyltransferases
  • cdc42 GTP-Binding Protein
  • rho GTP-Binding Proteins