Doc Toxin Is a Kinase That Inactivates Elongation Factor Tu

• Type of Material: Article
• Authors: Jonathan W. Cruz, Rutgers State University; Francesca P. Rothenbacher, Rutgers State University; Tatsuya Maehigashi, Emory University; William S. Lane, Harvard University; Christine Dunham, Emory University; Nancy A. Woychik, Rutgers State University
• Language: English
• Date: 2014-03-14
• Publisher: American Society for Biochemistry and Molecular Biology
• Publication Version: Final Published Version
• Copyright Statement: © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
• Final Published Version (URL): http://dx.doi.org/10.1074/jbc.M113.544429
• Title of Journal or Parent Work: Journal of Biological Chemistry
• Volume: 289
• Issue: 11
• Start Page: 7788
• End Page: 7798
• Grant/Funding Information: This work was supported, in whole or in part, by National Institutes of Health Grants R01GM093279 (to C. M. D.) and R01GM095693 (to N. A. W.) from the NIGMS, National Science Foundation CAREER Award MCB 0953714 (to C. M. D.), and National Institutes of Health Training Grant T32AI007403, Virus-Host Interactions in Eukaryotic Cells (to J. W. C. and F. P. R., awarded to G. Brewer), from the NIAID.
• Keywords: Translation; Translation Elongation Factors; Ternary complex; Mechanism; Antitoxin systems; Adenylylation; Phosphorylation; Crystal structure; Antitoxin; Proteins; Post-translational Modification; Life Sciences & Biomedicine; Factor ef-tu; Biochemistry & Molecular Biology; Science & Technology; Substrate; Fic; Protein Synthesis; Prophage p1; GTPase; PHD; Ampylation

Abstract

The Doc toxin from bacteriophage P1 (of the phd-doc toxin-antitoxin system) has served as a model for the family of Doc toxins, many of which are harbored in the genomes of pathogens. We have shown previously that the mode of action of this toxin is distinct from the majority derived from toxin-antitoxin systems: it does not cleave RNA; in fact P1 Doc expression leads to mRNA stabilization. However, the molecular triggers that lead to translation arrest are not understood. The presence of a Fic domain, albeit slightly altered in length and at the catalytic site, provided a clue to the mechanism of P1 Doc action, as most proteins with this conserved domain inactivate GTPases through addition of an adenylyl group (also referred to as AMPylation). We demonstrated that P1 Doc added a single phosphate group to the essential translation elongation factor and GTPase, elongation factor (EF)-Tu. The phosphorylation site was at a highly conserved threonine, Thr-382, which was blocked when EF-Tu was treated with the antibiotic kirromycin. Therefore, we have established that Fic domain proteins can function as kinases. This distinct enzymatic activity exhibited by P1 Doc also solves the mystery of the degenerate Fic motif unique to the Doc family of toxins. Moreover, we have established that all characterized Fic domain proteins, even those that phosphorylate, target pivotal GTPases for inactivation through a post-translational modification at a single functionally critical acceptor site.