A Night-Time Edge Site Intermediate in the Cyanobacterial Circadian Clock Identified by EPR Spectroscopy

As the only circadian oscillator that can be reconstituted in vitro with its constituent proteins KaiA, KaiB, and KaiC using ATP as an energy source, the cyanobacterial circadian oscillator serves as a model system for detailed mechanistic studies of day-night transitions of circadian clocks in general. The day-to-night transition occurs when KaiB forms a night-time complex with KaiC to sequester KaiA, the latter of which interacts with KaiC during the day to promote KaiC autophosphorylation. However, how KaiB forms the complex with KaiC remains poorly understood, despite the available structures of KaiB bound to hexameric KaiC. It has been postulated that KaiB-KaiC binding is regulated by inter-KaiB cooperativity. Here, using spin labeling continuous-wave electron paramagnetic resonance spectroscopy, we identified and quantified two subpopulations of KaiC-bound KaiB, corresponding to the bulk and edge KaiBC sites in stoichiometric and substoichiometric KaiB,C 6 complexes (i = 1-5). We provide kinetic evidence to support the intermediacy of the edge KaiBC sites as bridges and nucleation sites between free KaiB and the bulk KaiBC sites. Furthermore, we show that the relative abundance of edge and bulk sites is dependent on both KaiC phosphostate and KaiA, supporting the notion of phosphorylation-state controlled inter-KaiB cooperativity. Finally, we demonstrate that the interconversion between the two subpopulations of KaiC-bound KaiB is intimately linked to the KaiC phosphorylation cycle. These findings enrich our mechanistic understanding of the cyanobacterial clock and demonstrate the utility of EPR in elucidating circadian clock mechanisms.

Automated summary

In this study, the authors used EPR spectroscopy to identify and quantify two subpopulations of KaiC-bound KaiB, providing kinetic evidence for their intermediacy as bridges and nucleation sites. Furthermore, they showed that the relative abundance of these subpopulations is dependent on the phosphorylation state of KaiC and the presence of KaiA. Finally, the authors demonstrated that the interconversion between these subpopulations is linked to the phosphorylation cycle of KaiC.