Abstract
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.
Highlights
Endogenous, entrainable oscillation with periods of ~24 h, known as the circadian rhythm, is found in many organisms
To examine the possible dependence of KaiA–KaiC interaction on KaiCbound nucleotide states, ATP and its nonhydrolyzable analog, adenylyl imidodiphosphate (AMPPNP), were used for the formation of the mutated KaiC hexamers, which were subjected to native mass spectrometry (MS) analysis
Whereas the KaiCAA hexamer mediated by AMPPNP had a constant molecular mass of 353,857 ± 10 D holding 12 AMPPNP molecules (Fig 1A and Table 1), the ATP-mediated KaiCAA hexamer preincubated for 5 h in the presence of 1 mM ATP exhibited two series of ion peaks (Fig 1B)
Summary
Endogenous, entrainable oscillation with periods of ~24 h, known as the circadian rhythm, is found in many organisms. The KaiC hexamer experiences autophosphorylation and dephosphorylation cycles in a 24-h period through interactions with KaiA and KaiB in the presence of ATP
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