ATP hydrolysis by KaiC promotes its KaiA binding in the cyanobacterial circadian clock system.

The cyanobacterial clock is controlled via the interplay among KaiA, KaiB, and KaiC, which generate a periodic oscillation of KaiC phosphorylation in the presence of ATP. KaiC forms a homohexamer harboring 12 ATP-binding sites and exerts ATPase activities associated with its autophosphorylation and dephosphorylation. The KaiC nucleotide state is a determining factor of the KaiB-KaiC interaction; however, its relationship with the KaiA-KaiC interaction has not yet been elucidated. With the attempt to address this, our native mass spectrometric analyses indicated that ATP hydrolysis in the KaiC hexamer promotes its interaction with KaiA. Furthermore, our nuclear magnetic resonance spectral data revealed that ATP hydrolysis is coupled with conformational changes in the flexible C-terminal segments of KaiC, which carry KaiA-binding sites. From these data, we conclude that ATP hydrolysis in KaiC is coupled with the exposure of its C-terminal KaiA-binding sites, resulting in its high affinity for KaiA. These findings provide mechanistic insights into the ATP-mediated circadian periodicity.