Structural and Kinetic Characterization of Protein Ampylation by VopS Fic Domain
Luong, Phi Hoang
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The bacterial pathogen Vibrio parahaemolyticus manipulates host signaling pathways by injecting type III effectors into the cytoplasm of the target cell. One of these effectors, VopS, blocks actin assembly by AMPylating 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. We describe features of the VopS crystal structure, including a hairpin element that is responsible for protein–protein interaction and residues involved in ATP binding. Steady-state analyses of VopS point mutants provide kinetic understanding on the functions of conserved residues for the AMPylation activity. Further mechanistic analysis of VopS with its two substrates, ATP and Cdc42, demonstrates that VopS utilizes a sequential mechanism to AMPylate Rho GTPases. The structure of VopS and its ternary reaction mechanism provide critical groundwork for future studies on AMPylators, a novel family of enzymes that modify hydroxyl-containing residues with AMP. We also developed molecular tools to facilitate the study of protein AMPylation in collaboration with Howard Hang at The Rockefeller University. An ATP analogue, N6pATP, was developed that utilizes click chemistry to allow for the detection of AMPylated proteins by fluorescent or biotin tags. N6pATP can be utilized in in vitro AMPylation reactions catalyzed by known AMPylators including Fic domain and adenylyltransferase domain proteins. Further, we showed that N6pATP can be used for the detection and purification of endogenous AMPylated proteins. Preliminary studies were performed on another effector protein of unknown function, VopQ from Vibrio parahaemolyticus. The protein sequence of VopQ does not resemble any known protein domains. Various constructs were made for VopQ, and here I describe the purification and crystallization of VopQ.