The ATP-Mediated Regulation of KaiB-KaiC Interaction in the Cyanobacterial Circadian Clock

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 (KaiC1mer) and formed a complex with KaiB. The addition of ATP plus Mg2+ (Mg-ATP), but not that of ATP only, to the KaiB-KaiC1mer complex induced the hexamerization of KaiC and the concomitant release of KaiB from the KaiB-KaiC1mer complex, indicating that Mg-ATP and KaiB compete each other for KaiC. In the presence of ATP and Mg2+ (Mg-ATP), KaiC became a homohexameric ATPase (KaiC6mer) 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 KaiC6mer 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 KaiC6mer is required for formation of the KaiB-KaiC6mer complex. KaiC6mer 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 KaiC6mer with bound ADP-Pi.