No abstract

ATP-induced KaiC hexamerization is necessary for the clock function of KaiC.

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...

No abstract

KaiB is a component of the circadian clock molecular machinery in cyanobacteria, which are the simplest organisms that exhibit circadian rhythms. Here we report the x-ray crystal structure of KaiB from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. The KaiB crystal diffracts at a resolution of 2.6 Å and includes four subunits organized as a dimer of dimers, each composed of two non-equivalent subunits. The overall shape of the tetramer is an elongated hexagonal plate, with a single positively charged cleft flanked by two negatively charged ridges whose surfaces includes several terminal chains. Site-directed mutagenesis of Synechococcus KaiB confirmed that alanine substitution of residues Lys-11 or Lys-43 in the cleft, or deletion of C-terminal residues 95-108, which forms part of the ridges, strongly weakens in vivo circadian rhythms. Characteristics of KaiB deduced from the x-ray crystal structure were also confirmed by physicochemical measurements of KaiB in solution. These data suggest that the positively charged cleft and flanking negatively charged ridges in KaiB are essential for the biological function of KaiB in the circadian molecular machinery in cyanobacteria.