The pod components of the Shigella T3SS sorting platform accommodate multiple copies of Spa33 (SctQ)

The bacterial type III secretion system (T3SS) uses a membrane-embedded injectisome assembly to export effector proteins into host cells. While atomic-level structural details have been revealed for much of the T3SS apparatus, the model of the cytoplasmic sorting platform remains largely low-resolution. A central structural element of the sorting platform is the so-called "pod" protein, SctQ, which anchors the sorting platform to the inner membrane via interaction with the adaptor protein SctK, and connects to the central ATPase via the spoke protein SctL. SctQ proteins also interact with alternatively translated homodimers of their C-terminal SPOA2 domains. Low resolution electron density maps have provided an outline of the sorting platform architecture, and fluorescence microscopy studies have suggested a 1:4:2 SctK:SctQ:SctL stoichiometry. While there are experimental and AlphaFold structures of the individual components or complexes of sorting platform pod, there is currently no model for the pod structure that adequately fits the electron density or accounts for the proposed stoichiometry. Here we use AlphaFold to generate a model of the Shigella pod complex in which two copies of the SctQ protein, Spa33, bind the adaptor protein MxiK, with each copy of Spa33 bound to an alternately translated SPOA2-SPOA2 domain. We show through mutation of energetically critical interface residues, predicted by computational mutant scanning, that both Spa33 binding sites on MxiK are required for T3SS activity in Shigella flexneri, as well as binding of Spa33 to the SPOA2-SPOA2 homodimer. We find that this model fits well to the upper two-thirds of the pod electron density, albeit in a manner that places the protein components slightly closer to the inner membrane than traditionally presented. Further, cryogenic electron tomography of mutant injectisomes reveals the lack of a complete sorting platform and may suggest an alternative model featuring four copies of Spa33 that still fits the upper pod electron density but better explains unfilled density in the lower third of the pod.