title: Nature of KaiB-KaiC binding in the cyanobacterial circadian oscillator.
abstract: In the cyanobacteria Synechococcus elongatus and Thermosynechococcus elongatus, the KaiA, KaiB and KaiC proteins in the presence of ATP generate a post-translational oscillator (PTO) that can be reconstituted in vitro. KaiC is the result of a gene duplication and resembles a double doughnut with N-terminal CI and C-terminal CII hexameric rings. Six ATPs are bound between subunits in both the CI and CII ring. CI harbors ATPase activity, and CII catalyzes phosphorylation and dephosphorylation at T432 and S431 with a ca. 24-h period. KaiA stimulates KaiC phosphorylation, and KaiB promotes KaiC subunit exchange and sequesters KaiA on the KaiB-KaiC interface in the final stage of the clock cycle. Studies of the PTO protein-protein interactions are convergent in terms of KaiA binding to CII but have led to two opposing models of the KaiB-KaiC interaction. Electron microscopy (EM) and small angle X-ray scattering (SAXS), together with native PAGE using full-length proteins and separate CI and CII rings, are consistent with binding of KaiB to CII. Conversely, NMR together with gel filtration chromatography and denatured PAGE using monomeric CI and CII domains support KaiB binding to CI. To resolve the existing controversy, we studied complexes between KaiB and gold-labeled, full-length KaiC with negative stain EM. The EM data clearly demonstrate that KaiB contacts the CII ring. Together with the outcomes of previous analyses, our work establishes that only CII participates in interactions with KaiA and KaiB as well as with the His kinase SasA involved in the clock output pathway.
authors: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA.
authors: Rekha
authors: R
authors: Pattanayek
authors: Kirthi Kiran
authors: KK
authors: Yadagiri
authors: Melanie D
authors: MD
authors: Ohi
authors: Martin
authors: M
authors: Egli
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D057906:Circadian Clocks; D056925:Circadian Rhythm Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D006639:Histidine / Q000378:metabolism; D008958:Models, Molecular; D049329:Nanostructures / Q000648:ultrastructure; D016624:Negative Staining; D009842:Oligopeptides / Q000378:metabolism; D011485:Protein Binding; D053838:Scattering, Small Angle; D055672:Static Electricity; D046940:Synechococcus / Q000378:metabolism*; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D056925:Circadian Rhythm Signaling Peptides and Proteins; C471213:His-His-His-His-His-His; C114270:KaiB protein, cyanobacteria; C114271:KaiC protein, cyanobacteria; D009842:Oligopeptides; D006639:Histidine
keywords: EM; Synechococcus elongatus; circadian clock; dephosphorylation; nanogold
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