Schematic of Shigella T3SS and location of MxiA_C mutations altering substrate selection.

(A) Shematic of Shigella T3SS. Needle components in blue-greens, transmembrane base dark purple, IMEA light purple, CEA components (C-ring and ATPase complex) in light blues. A curved arrow shows the proposed direction of proton flow during protein export, which may be coupled to ATP hydrolysis. Pseudomonas vT3SS and Salmonella fT3SS homologs mentioned in the text are shown in italics. (B) MxiA_C monomer (chain B [36]), putative oligomerisation domain (indicated by aas with charges shown) facing viewer. Grey, domain 4, facing cytoplasm; light blue, aa involved in chaperone binding within domain 2; mutations in both of these affect substrate selection in FlhA [44, 45]. Pink, location of mutations found in this work; top right N373, in an unstructured loop; bottom, I674. Orange, Q608 and green, location of two activating mutations identified by Rietsch [31] (homologous to Y587 and M667 in PcrD). Mutations in pink and orange aas are all surface located and affect interaction with MxiC. Right: cyan, chain A of oligomer. Assembly rotated left to show mutations do not cluster at oligomer interfaces (inside of nonamer ring facing viewer). (D) Schematic presenting the sequence homologies and functional correspondences between Shigella and Pseudomonas regulatory proteins mentioned in this work. Proteins are presented linearly, from N- to C-terminus; SS, secretion signal; CBD, chaperone binding domain; XB, X-bundle.