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Kinetic and structural insights into the mechanism of AMPylation by VopS Fic domain

Phi Luong1, Lisa N Kinch, Chad A Brautigam

  • 1Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

The Journal of Biological Chemistry
|April 23, 2010
PubMed

Insights

Vibrio parahemeolyticus VopS effector protein blocks host actin assembly by AMPylating Rho GTPases. Structural and kinetic analysis reveals its Fic domain mechanism for modifying hydroxyl-containing residues.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Biochemistry

Background:

  • Bacterial pathogens like *Vibrio parahemeolyticus* use type III effectors to disrupt host cell functions.
  • The VopS effector targets Rho GTPases, inhibiting actin assembly through a post-translational modification called AMPylation.

Purpose of the Study:

  • To elucidate the structure and catalytic mechanism of the VopS effector protein.
  • To understand the functional roles of conserved residues within the VopS Fic domain.
  • To determine the reaction mechanism by which VopS AMPylates Rho GTPases.

Main Methods:

  • Structural analysis of VopS.
  • Site-directed mutagenesis and steady-state kinetic analysis of VopS mutants.
  • Mechanistic studies involving VopS, ATP, and the substrate Cdc42.

Main Results:

  • The structure of VopS and its evolutionarily conserved catalytic residues were determined.
  • Kinetic analysis identified key residues essential for AMPylation activity.
  • VopS was shown to employ a sequential mechanism for AMPylating Rho GTPases, involving a ternary complex.

Conclusions:

  • Structural and mechanistic insights into VopS provide a foundation for understanding Fic domain-mediated AMPylation.
  • This work clarifies how VopS disrupts host actin dynamics by modifying Rho GTPases.
  • The findings pave the way for future research on AMPylators targeting hydroxyl-containing residues.

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