title: Exchange of ADP with ATP in the CII ATPase domain promotes autophosphorylation of cyanobacterial clock protein KaiC.
abstract: The cyanobacterial circadian oscillator can be reconstituted in vitro. In the presence of KaiA and KaiB, the phosphorylation state of KaiC oscillates with a periodicity of ∼24 h. KaiC is a hexameric P-loop ATPase with autophosphorylation and autodephosphorylation activities. Recently, we found that dephosphorylation of KaiC occurs via reversal of the phosphorylation reaction: a phosphate group attached to Ser431/Thr432 is transferred to KaiC-bound ADP to generate ATP, which is subsequently hydrolyzed. This unusual reaction mechanism suggests that the KaiC phosphorylation rhythm is sustained by periodic shifts in the equilibrium of the reversible autophosphorylation reaction, the molecular basis of which has never been elucidated. Because KaiC-bound ATP and ADP serve as substrates for the forward and reverse reactions, respectively, we investigated the regulation of the nucleotide-bound state of KaiC. In the absence of KaiA, the condition in which the reverse reaction proceeds, KaiC favored the ADP-bound state. KaiA increased the ratio of ATP to total KaiC-bound nucleotides by facilitating the release of bound ADP and the incorporation of exogenous ATP, allowing the forward reaction to proceed. When both KaiA and KaiB were present, the ratio of ATP to total bound nucleotides exhibited a circadian rhythm, whose phase was advanced by several hours relative to that of the phosphorylation rhythm. Based on these findings, we propose that the direction of the reversible autophosphorylation reaction is regulated by KaiA and KaiB at the level of substrate availability and that this regulation sustains the oscillation of the phosphorylation state of KaiC.
authors.affiliation: Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan.
authors.forename: Taeko
authors.initials: T
authors.lastname: Nishiwaki-Ohkawa
authors.forename: Yohko
authors.initials: Y
authors.lastname: Kitayama
authors.forename: Erika
authors.initials: E
authors.lastname: Ochiai
authors.forename: Takao
authors.initials: T
authors.lastname: Kondo
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D056925:Circadian Rhythm Signaling Peptides and Proteins / Q000378:metabolism*; D000458:Cyanobacteria / Q000201:enzymology / Q000378:metabolism*; D010766:Phosphorylation; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D056925:Circadian Rhythm Signaling Peptides and Proteins; C114271:KaiC protein, cyanobacteria; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases
keywords: in vitro reconstitution; nucleotide exchange; self-sustained oscillation