Abstract
The cyanobacterial circadian clock oscillator is composed of three clock proteins--KaiA, KaiB, and KaiC, and interactions among the three Kai proteins generate clock oscillation in vitro. However, the regulation of these interactions remains to be solved. Here, we demonstrated that ATP regulates formation of the KaiB-KaiC complex. In the absence of ATP, KaiC was monomeric (KaiC(1mer)) and formed a complex with KaiB. The addition of ATP plus Mg(2+) (Mg-ATP), but not that of ATP only, to the KaiB-KaiC(1mer) complex induced the hexamerization of KaiC and the concomitant release of KaiB from the KaiB-KaiC(1mer) complex, indicating that Mg-ATP and KaiB compete each other for KaiC. In the presence of ATP and Mg(2+) (Mg-ATP), KaiC became a homohexameric ATPase (KaiC(6mer)) with bound Mg-ATP and formed a complex with KaiB, but KaiC hexamerized by unhydrolyzable substrates such as ATP and Mg-ATP analogs, did not. A KaiC N-terminal domain protein, but not its C-terminal one, formed a complex with KaiB, indicating that KaiC associates with KaiB via its N-terminal domain. A mutant KaiC(6mer) lacking N-terminal ATPase activity did not form a complex with KaiB whereas a mutant lacking C-terminal ATPase activity did. Thus, the N-terminal domain of KaiC is responsible for formation of the KaiB-KaiC complex, and the hydrolysis of the ATP bound to N-terminal ATPase motifs on KaiC(6mer) is required for formation of the KaiB-KaiC(6mer) complex. KaiC(6mer) that had been hexamerized with ADP plus aluminum fluoride, which are considered to mimic ADP-Pi state, formed a complex with KaiB, suggesting that KaiB is able to associate with KaiC(6mer) with bound ADP-Pi.