Kinetic Characterization of Salmonella FliK−FlhB Interactions Demonstrates Complexity of the Type III Secretion Substrate-Specificity Switch

摘要

The bacterial flagellumis a complexmacromolecularmachine consisting ofmore than 20 000 proteins,nmost of which must be exported from the cell via a dedicated Type III secretion apparatus. At a defined point innflagellar morphogenesis, hook completion is sensed and the apparatus switches substrate specificity type from rodnand hook proteins to filament ones. How the switch works is a subject of intense interest. FliK and FlhB playncentral roles. In the present study, two optical biosensing methods were used to characterize FliK-FlhBninteractions using wild-type and two variant FlhBs from mutants with severe flagellar structural defects. Bindingnwas found to be complex with fast and slow association and dissociation components. Surprisingly, wild-type andnvariant FlhBs had similar kinetic profiles and apparent affinities, which ranged between 1 and 10.5 μM, suggestingnthat the specificity switch is more complex than presently understood. Other binding experiments providednevidence for a conformational change after binding. Liquid chromatography-mass spectrometry (LC-MS) andnNMR experiments were performed to identify a cyclic intermediate product whose existence supports thenmechanism of autocatalytic cleavage at FlhB residue N269. The present results show that while autocatalyticncleavage is necessary for proper substrate specificity switching, it does not result in an altered interactionwith FliK,nstrongly suggesting the involvement of other proteins in the mechanism.