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.
Highlights
Doc toxin, of the phd-doc toxin-antitoxin system, belongs to the Fic protein family found in all domains of life
The Doc toxin from bacteriophage P1 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; P1 Doc expression leads to mRNA stabilization
Summary
Of the phd-doc toxin-antitoxin system, belongs to the Fic protein family found in all domains of life. 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 ( referred to as AMPylation). We have established that Fic domain proteins can function as kinases This distinct enzymatic activity exhibited by P1 Doc solves the mystery of the degenerate Fic motif unique to the Doc family of toxins. 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. The consensus for the Fic motif in all Doc toxins is slightly different, HXFX(D/ N)(A/G)NKR Alterations to this motif can result in changes to the enzymatic activity of the protein. Our functional data are complemented with molecular models that inform the mechanism of kirromycin-mediated phosphorylation inhibition and provide alternate mechanisms for Doc toxin binding to EF-Tu
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