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

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Pages (from-to)20155-20163
Number of pages9
JournalJournal of Biological Chemistry
Volume285
Issue number26
DOIs
StatePublished - Jun 25 2010

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rho GTP-Binding Proteins
Adenosine Monophosphate
Kinetics
Vibrio
GTP Phosphohydrolases
Pathogens
Threonine
Hydroxyl Radical
Actins
Cytoplasm
Adenosine Triphosphate
Switches
Substrates
Infection

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Kinetic and structural insights into the mechanism of AMPylation by VopS Fic domain. / Luong, Phi; Kinch, Lisa N.; Brautigam, Chad A; Grishin, Nick V; Tomchick, Diana R; Orth, Kim A.

In: Journal of Biological Chemistry, Vol. 285, No. 26, 25.06.2010, p. 20155-20163.

Research output: Contribution to journalArticle

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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.",
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AU - Kinch, Lisa N.

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AU - Tomchick, Diana R

AU - Orth, Kim A

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