Doc Toxin Is a Kinase That Inactivates Elongation Factor Tu

• Published in: The Journal of biological chemistry Vol. 289; no. 11; pp. 7788 - 7798
• Main Authors: Cruz, Jonathan W., Rothenbacher, Francesca P., Maehigashi, Tatsuya, Lane, William S., Dunham, Christine M., Woychik, Nancy A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 14.03.2014; American Society for Biochemistry and Molecular Biology
• Subjects: Adenylylation; Amino Acid Motifs; Anti-Bacterial Agents - chemistry; Antitoxin; Bacteriophage P1 - metabolism; Binding Sites; Cell Proliferation; Escherichia coli - metabolism; Escherichia coli Proteins - metabolism; Fic; Gene Expression Regulation, Bacterial; GTPase; Mass Spectrometry; Molecular Docking Simulation; Peptide Chain Elongation, Translational; Peptide Elongation Factor Tu - metabolism; Phosphorylation; Post-translational Modification; Protein Binding; Protein Processing, Post-Translational; Protein Structure, Tertiary; Protein Synthesis; Protein Synthesis and Degradation; Pyridones - chemistry; Recombinant Proteins - chemistry; RNA, Messenger - metabolism; Threonine - chemistry; Translation; Translation Elongation Factors; Viral Proteins - metabolism; Fic; Phosphorylation; Translation; Protein Synthesis; Post-translational Modification; Translation Elongation Factors; Adenylylation; Antitoxin; GTPase
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.544429

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. Background: Doc toxin, of the phd-doc toxin-antitoxin system, belongs to the Fic protein family found in all domains of life. Results: Doc inactivates elongation factor Tu by phosphorylation of a single amino acid. Conclusion: This phosphorylation event inhibits protein synthesis and thereby arrests cell growth. Significance: The phosphorylation activity of Doc toxin represents a new catalytic activity for members of the Fic protein family.