RT Journal Article SR Electronic T1 The S. Typhi effector StoD is an E3/E4 ubiquitin ligase which binds K48- and K63-linked diubiquitin JF Life Science Alliance JO Life Sci. Alliance FD Life Science Alliance LLC SP e201800272 DO 10.26508/lsa.201800272 VO 2 IS 3 A1 Melanie A McDowell A1 Alexander MP Byrne A1 Elli Mylona A1 Rebecca Johnson A1 Agnes Sagfors A1 Valerie F Crepin A1 Susan Lea A1 Gad Frankel YR 2019 UL https://www.life-science-alliance.org/content/2/3/e201800272.abstract AB Salmonella enterica (e.g., serovars Typhi and Typhimurium) relies on translocation of effectors via type III secretion systems (T3SS). Specialization of typhoidal serovars is thought to be mediated via pseudogenesis. Here, we show that the Salmonella Typhi STY1076/t1865 protein, named StoD, a homologue of the enteropathogenic Escherichia coli/enterohemorrhagic E. coli/Citrobacter rodentium NleG, is a T3SS effector. The StoD C terminus (StoD-C) is a U-box E3 ubiquitin ligase, capable of autoubiquitination in the presence of multiple E2s. The crystal structure of the StoD N terminus (StoD-N) at 2.5 Å resolution revealed a ubiquitin-like fold. In HeLa cells expressing StoD, ubiquitin is redistributed into puncta that colocalize with StoD. Binding assays showed that StoD-N and StoD-C bind the same exposed surface of the β-sheet of ubiquitin, suggesting that StoD could simultaneously interact with two ubiquitin molecules. Consistently, StoD interacted with both K63- (KD = 5.6 ± 1 μM) and K48-linked diubiquitin (KD = 15 ± 4 μM). Accordingly, we report the first S. Typhi–specific T3SS effector. We suggest that StoD recognizes and ubiquitinates pre-ubiquitinated targets, thus subverting intracellular signaling by functioning as an E4 enzyme.