SummaryFlhB, an integral membrane protein, gates the type III flagellar export pathway of Salmonella . It permits export of rod/hook-type proteins before hook completion, whereupon it switches specificity to recognize filament-type proteins. The cytoplasmic C-terminal domain of FlhB (FlhB C ) is cleaved between Asn-269 and Pro-270, defining two subdomains: FlhB CN and FlhB CC . Here, we show that subdomain interactions and cleavage within FlhB are central to substratespecificity switching. We found that deletions between residues 216 and 240 of FlhB CN permitted FlhB cleavage but abolished function, whereas a deletion spanning Asn-269 and Pro-270 abolished both. The mutation N269A prevented cleavage at the Flh-B CN -FlhB CC boundary. Cells producing FlhB(N269A) exported the same amounts of hook-capping protein as cells producing wild-type FlhB. However, they exported no flagellin, even when the fliC gene was being expressed from a foreign promoter to circumvent regulation of expression by FlgM, which is itself a filament-type substrate. Electron microscopy revealed that these cells assembled polyhook structures lacking filaments. Thus, FlhB(N269A) is locked in a conformation specific for rod/hook-type substrates. With FlhB(P270A), cleavage was reduced but not abolished, and cells producing this protein were weakly motile, exported reduced amounts of flagellin and assembled polyhook filaments.

The bacterial flagellum is a predominantly cell-external supermacromolecular construction whose structural components are exported by a flagellum-specific export apparatus. One of the export apparatus proteins, FlhB, regulates the substrate specificity of the entire apparatus; i.e. it has a role in the ordered export of the two main groups of flagellar structural proteins such that the cell-proximal components (rod-/hook-type proteins) are exported before the cell-distal components (filament-type proteins). The controlled switch between these two export states is believed to be mediated by conformational changes in the structure of the C-terminal cytoplasmic domain of FlhB (FlhB C ), which is consistently and specifically cleaved into two subdomains (FlhB CN and FlhB CC ) that remain tightly associated with each other. The cleavage event has been shown to be physiologically significant for the switch. In this study, the mechanism of FlhB cleavage has been more directly analyzed. We demonstrate that cleavage occurs in a heterologous host, Saccharomyces cerevisiae, deficient in vacuolar proteinases A and B. In addition, we find that cleavage of a slow-cleaving variant, FlhB C (P270A), is stimulated in vitro at alkaline pH. We also show by analytical gel-filtration chromatography and analytical ultracentrifugation experiments that both FlhB C and FlhB C (P270A) are monomeric in solution, and therefore self-proteolysis is unlikely. Finally, we provide evidence via peptide analysis and FlhB cleavage variants that the tertiary structure of FlhB plays a significant role in cleavage. Based on these results, we propose that FlhB cleavage is an autocatalytic process.A large percentage of the bacterial flagellar structure lies outside of the cell envelope, thus requiring that the vast majority of the subunits that compose the flagellum be exported from the cytosol across both the inner and outer membranes. Salmonella employs a type III export pathway to accomplish this (1, 2). It is a Sec-independent pathway that utilizes a flagellum-specific export apparatus to transport flagellar components across the inner membrane. These exported proteins then travel the length of the developing flagellum within an interior channel prior to their incorporation at the structure's cell-distal end (3-6) (the developing structure therefore facilitates export across the outer membrane). At least nine flagellar proteins are involved in the flagellumspecific export apparatus (7). Six are integral membrane components (FlhA, FlhB, FliO, FliP, FliQ, and FliR) postulated to be located within the basal body MS ring (8, 9), and three are soluble components: an ATPase (FliI) that drives export (10), a regulator of the ATPase (FliH) (11-13), and a general chaperone (FliJ) (14, 15).One of the integral membrane proteins, FlhB, has been found to play a central role in export substrate-specificity switching; i.e. regulation of the order in which flagellar subunits are exported, such that proteins incorporated into the cell-proximal rod and hook structure...

HAMP domains mediate signal transduction in over 7500 enzyme-coupled receptors represented in all kingdoms of life. The HAMP domain of the putative archaeal receptor Af1503 has a parallel, dimeric, four-helical coiled coil structure, but with unusual core packing, related to canonical packing by concerted axial rotation of the helices. This has led to the gearbox model for signal transduction, whereby the alternate packing modes correspond to signaling states. Here we present structures of a series of Af1503 HAMP variants. We show that substitution of a conserved small side chain within the domain core (A291) for larger residues induces a gradual transition in packing mode, involving both changes in helix rotation and bundle shape, which are most prominent at the C-terminal, output end of the domain. These are correlated with activity and ligand response in vitro and in vivo by incorporating Af1503 HAMP into mycobacterial adenylyl cyclase assay systems.

Bacterial transmembrane receptors regulate an intracellular catalytic output in response to extracellular sensory input. To investigate the conformational changes that relay the regulatory signal, we have studied the HAMP domain, a ubiquitous intracellular module connecting input to output domains. HAMP forms a parallel, dimeric, four-helical coiled coil, and rational substitutions in our model domain (Af1503 HAMP) induce a transition in its interhelical packing, characterized by axial rotation of all four helices (the gearbox signaling model). We now illustrate how these conformational changes are propagated to a downstream domain by fusing Af1503 HAMP variants to the DHp domain of EnvZ, a bacterial histidine kinase. Structures of wild-type and mutant constructs are correlated with ligand response in vivo, clearly associating them with distinct signaling states. We propose that altered recognition of the catalytic domain by DHp, rather than a shift in position of the phospho-accepting histidine, forms the basis for regulation of kinase activity.

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