Interaction of MxiG with the cytosolic complex of the type III secretion system controls Shigella virulence

Abstract

Gram-negative bacteria use the type 3 secretion system (T3SS) to colonize host cells. T3SSs are ring-shaped macromolecular complexes specific for the transport of effector molecules into host cells. It was recently suggested that a cytosolic ring-shaped protein complex delivers effector molecules to the T3SS. However, how transport of effector proteins is regulated is not known. Here, we report the high-resolution X-ray crystal structure of the whole cytosolic domain of MxiG (MxiG(1-126)), a major component of the inner T3SS rings in Shigella flexneri. MxiG(1-126) folds as an FHA domain, which specifically binds phosphorylated threonines. Indeed, MxiG(1-126) binds to Spa33, a cytoplasmic-ring component of Shigella, as revealed in pulldown studies. Surface plasmon resonance analysis showed specific interaction of MxiG with a Spa33 peptide only if phosphorylated. In total, 24 copies of the MxiG(1-126) crystal structure were fitted into the cryo-EM map of the Shigella T3SS. The phosphoprotein binding site of each MxiG molecule faces the channel of the T3SS, allowing interaction with cytosolic binding partners. Secretion assays and host cell invasion studies of complemented Shigella knockout cells indicated that the phosphoprotein binding of MxiG is essential for bacterial virulence. Our findings suggest that MxiG is involved in T3SS regulation.

Protein Export According to Schedule: Architecture, Assembly, and Regulation of Type III Secretion Systems from Plant- and Animal-Pathogenic Bacteria

“…Several YscJ family members also contain a predicted C-terminal transmembrane helix (10,492,504). YscD and homologs are lipoproteins with an N-terminal cytoplasmic and a C-terminal periplasmic domain and might form a multimeric ring structure next to the YscJ ring (25,142,293,492,516). Protein-protein interaction studies revealed that YscJ and YscD family members interact not only with each other but also with members of the YscC family of OM secretins (142,410,467,479) (Table 2).…”

Structure and Interactions of the Cytoplasmic Domain of the Yersinia Type III Secretion Protein YscD

“…MxiG lacks the conserved His but has a Ser (S61) and Arg (R39) (Fig. 2c, red arrowheads) that have been identified to be important for binding a threonine-phosphorylated peptide that corresponds to a sequence from the putative C-ring protein Spa33 (6,39). However, a different study using NMR titration reported no interaction between MxiGc and up to 50 mM phosphothreonine (37).…”

“…This includes Shigella flexneri MxiG, a member of the SPI-1 subfamily. The structure of the cytoplasmic domain of MxiG (called here MxiGc) was recently shown by NMR and X-ray crystallographic techniques to consist of an FHA domain (6,37). YscDc and MxiGc have 13% sequence identity and an RMSD of 3.08 Å between their two structures (86 C␣) (Fig.…”

“…YscD has distant but recognizable sequence identity to orthologs in the SPI-1 and SPI-2 subfamilies. This includes the SPI-1 ortholog MxiG, whose cytoplasmic domain was shown to have an FHA fold (6,37). L4 in MxiG lacks the conserved His but has two serines (S61 and S63), and L3 has an Arg (residue 39).…”

“…Comparison with other FHA domain proteins, including MxiG, a YscD ortholog from Shigella flexneri, whose cytoplasmic domain was recently shown to have an FHA fold by nuclear magnetic resonance (NMR) and X-ray crystallographic techniques (6,37), highlighted two loop regions as being potentially significant for function. Alanine substitutions at these two loops, L3 and L4, had no deleterious effects on protein structure or stability but abrogated T3S in a dominant negative manner.…”