The priming phosphorylation of KaiC is activated by the release of its autokinase autoinhibition

KaiC, a cyanobacterial circadian clock protein with autokinase activity, catalyzes the dual phosphorylation of its own S431 and T432 residues in a circadian manner in the presence of KaiA and KaiB. Priming phosphorylation at T432 is a key step that promotes secondary phosphorylation at S431. Although KaiA binding is considered essential for KaiC phosphorylation, the mechanisms underlying the activation and inactivation of priming phosphorylation remain elusive. We found that the priming phosphorylation proceeds even in the absence of KaiA, but is autoinhibited within KaiC, which decreases the rate constant to 0.019 h-1. The autoinhibition of KaiC and the mechanism underlying the release from autoinhibition by KaiA were examined by KaiC structural analysis, and by classical molecular dynamics and quantum mechanics / molecular mechanics simulations. We found that the side chain of T432 adopts two rotamers in dephosphorylated KaiC, one of which places T432 in a position suitable for a nucleophilic attack on the terminal phosphate of adenosine triphosphate (ATP). However, the nucleophilicity of T432 was insufficient to overcome an energy barrier of approximately 22 kcal mol-1 because the catalytic function of a nearby base, E318, was self-suppressed by hydrogen bonding to positively charged R385. Biochemical assays of KaiC mutants showed that the autoinhibition of KaiC autokinase activity is attenuated by conferring T432 high nucleophilicity through the KaiA-assisted release of R385 from E318 to E352. During the circadian cycle, R385 switches interacting partners to inactivate/activate the autokinase function and to ensure the unidirectionality of the KaiC phosphorylation cycle. Significance StatementKaiC, a central player in the circadian clock system of cyanobacteria, undergoes an ordered phosphorylation cycle in the presence of KaiA and KaiB. To elucidate the mechanism underlying the rhythmic regulation of the KaiC autokinase, we performed structural analyses, computational simulations, and biochemical assays of KaiC and its mutants. The results indicate that KaiC is essentially an autoinhibited autokinase, and the autoinhibition of primary phosphorylation at its T432 residue is attenuated by conferring it high nucleophilicity against the terminal phosphate of adenosine triphosphate. KaiA contributes to releasing the autoinhibition of KaiC in a morning phase by switching the interacting partners of R385 from a catalytic glutamate E318 to E352, as well as ensuring unidirectionality of the KaiC phosphorylation cycle.