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.

蓝细菌生物钟振荡器（cyanobacterial circadian clock oscillator）由三种时钟蛋白——KaiA、KaiB与KaiC组成，三种Kai蛋白间的相互作用可在体外产生时钟振荡现象。然而，此类相互作用的调控机制仍有待阐明。本研究证实，三磷酸腺苷（ATP）可调控KaiB-KaiC复合物的形成。在无ATP的条件下，KaiC以单体形式（KaiC1mer）存在，并与KaiB形成复合物。向KaiB-KaiC1mer复合物中添加ATP与Mg²⁺的混合体系（Mg-ATP），而非单纯ATP，可诱导KaiC发生六聚化，并伴随KaiB从KaiB-KaiC1mer复合物中解离，这表明Mg-ATP与KaiB竞争性结合KaiC。在ATP与Mg²⁺共存（即Mg-ATP）的条件下，KaiC会转变为结合Mg-ATP的同源六聚体ATP酶（KaiC6mer），并与KaiB形成复合物；但使用ATP及Mg-ATP类似物等不可水解的底物诱导形成的KaiC六聚体，则无法与KaiB结合。仅KaiC的N端结构域（而非其C端结构域）可与KaiB形成复合物，这表明KaiC通过其N端结构域与KaiB发生相互作用。缺失N端ATP酶活性的突变型KaiC6mer无法与KaiB形成复合物，而缺失C端ATP酶活性的突变型KaiC6mer则可正常结合KaiB。综上，KaiC的N端结构域负责介导KaiB-KaiC复合物的形成，而结合在KaiC6mer上N端ATP酶基序的ATP水解过程，是KaiB-KaiC6mer复合物形成的必要条件。经ADP与氟化铝共同诱导形成的六聚体KaiC6mer（该组合被认为可模拟ADP-Pi结合状态）可与KaiB结合，这提示KaiB能够与结合有ADP-Pi的KaiC6mer发生相互作用。