Conformational Changes in IpaD from Shigella flexneri upon Binding Bile Salts Provide Insight into the Second Step of Type III Secretion

Shigella flexneri uses its type III secretion apparatus (TTSA) to inject host-altering proteins into targeted eukaryotic cells. The TTSA is composed of a basal body and an exposed needle with invasion plasmid antigen D (IpaD) forming a tip complex that controls secretion. The bile salt deoxycholate (DOG) stimulates recruitment of the translocator protein IpaB into the maturing TTSA needle tip complex. This process appears to be triggered by a direct interaction between DOC and IpaD. Fluorescence spectroscopy and NMR spectroscopy are used here to confirm the DOC IpaD interaction and to reveal that IpaD conformational changes upon DOG binding trigger the appearance of IpaB at the needle tip. Forster resonance energy transfer between specific sites on IpaD was used here to identify changes in distances between IpaD domains as a result of DOG binding. To further explore the effects of DOG binding on IpaD structure, NMR chemical shift mapping was employed. The environments of residues within the proposed DOG binding site and additional residues within the "distal" globular domain were perturbed upon DOG binding, further indicating that conformational changes occur within IpaD upon DOG binding. These events are proposed to be responsible for the recruitment of IpaB at the TTSA needle tip. Mutation analyses combined with additional spectroscopic analyses confirm that conformational changes in IpaD induced by DOG binding contribute to the recruitment of IpaB to the S. flexneri TTSA needle tip. These findings lay the foundation for determining how environmental factors promote TTSA needle tip maturation prior to host cell contact.