Kinetic Characterization of Salmonella FliK-FlhB Interactions Demonstrates Complexity of the Type III Secretion Substrate-Specificity SwitchBiochemistry
AbstractThe bacterial flagellum is a complex macromolecular machine consisting of more than 20000 proteins, most of which must be exported from the cell via a dedicated Type III secretion apparatus. At a defined point in flagellar morphogenesis, hook completion is sensed and the apparatus switches substrate specificity type from rod and hook proteins to filament ones. How the switch works is a subject of intense interest. FIiK and F1hBs play central roles. In the present study, two optical biosensing methods were used to characterize FIiK-F1hB interactions using wild-type and two variant FlhBs from mutants with severe flagellar structural defects. Binding was found to be complex with fast and slow association and dissociation components. Surprisingly, wild-type and variant FlhBs had similar kinetic profiles and apparent affinities, which ranged between I and 10.5 μM, suggesting that the specificity switch is more complex than presently understood. Other binding experiments provided evidence for a conformational change after binding. Liquid chromatography-mass spectrometry (LC-MS) and NMR experiments were performed to identify a cyclic intermediate product whose existence supports the mechanism of autocatalytic cleavage at FlhB residue N269. The present results show that while autocatalytic cleavage is necessary for proper substrate specificity switching, it does not result in an altered interaction with FIiK. strongly suggesting the involvement of other proteins in the mechanism.
Digital Object Identifier (DOI)10.1021/bi100487p
Citation InformationMorris DP, Roush ED, Thompson JW, Moseley MA, Murphy JW, McMurry JL. 2010. Kinetic characterization of salmonella FliK-FlhB interactions demonstrates complexity of the type III secretion substrate-specificity switch. Biochemistry (N Y ) 49(30):6386-93.