Extended Data Fig. 7: The mechanism of SidJ-induced CaM-dependent self-AMPlyation and SdeA glutamylation.

a, SidJ induces self-AMPylation in a CaM-dependent manner. SidJ was incubated with ³²P-α-ATP and Mg²⁺, with or without CaM for 2 h at 37 °C. After separation by SDS–PAGE, the incorporation of ³²P-α-ATP was detected by autoradiography. b, SdeA glutamylation by SidJ interferes with SidJ self-AMPylation. SidJ was incubated with ³²P-α-ATP, Mg²⁺ and CaM for 2 h at 37 °C. l-glutamate, SdeA and SdeA(E860D) were supplemented as stated. After separation by SDS–PAGE, the incorporation of ³²P-α-ATP was detected by autoradiography. c, SdeA glutamylation by SidJ accelerates ATP hydrolysis and the release of AMP. SidJ was incubated with the indicated components for 2 h at 37 °C. Samples were analysed by HPLC. AMP and ATP were used as standards. In a–c, data shown are one representative from at least three independent experiments with similar results. d, Schematic model of SidJ-induced glutamylation and AMPylation. SidJ induces glutamylation on SdeA(E860D) when ATP and l-glutamate are supplemented into the reaction. In reactions in which l-glutamate or modifiable SdeA are not present, SidJ undergoes self-AMPylation.