Kinetic hierarchy of Kai protein complex formation governs the cyanobacterial circadian oscillator

Cyanobacterial circadian oscillations arise from a phosphorylation cycle of KaiC that is coupled to reversible complex formation with KaiA and KaiB. Although representative assemblies such as A2C6, B6C6, and A12B6C6 have been structurally characterized, quantitative understanding of their stoichiometry, affinity, and formation kinetics during the oscillation cycle remains limited. Here, we systematically quantified AC, BC and ABC complex formation using phosphorylation-mimetic KaiC and controlled protein mixing ratios by integrating analytical ultracentrifugation with small-angle X-ray/neutron scattering. A2C6 formation exhibits graded dependence on the phosphorylation state of KaiC and occurs rapidly in solution. In contrast, B6C6 formation behaves in a switch-like manner, showing strong selectivity for the hyperphosphorylation mimic and proceeding on a slow timescale ([~] 6 h). Upon B6C6 formation, KaiA rapidly associates with the complex to generate AnB6C6, with KaiA occupancy n determined by the mixing ratio through fast redistribution among coexisting complexes. This behavior enables dynamic allocation of KaiA among clock complexes. Together, these quantitative insights delineate a hierarchy of assembly dynamics--fast, graded AC exchange; slow, state-selective BC formation; and rapid KaiA redistribution--revealing a mechanistic basis for the dynamic regulation of the Kai oscillator.