A cooperative switch within the KaiC hexamer revealed by cryo-EM

The circadian clock of cyanobacteria is based on an approximately 24h rhythm in the phosphorylation level of KaiC, a hexameric ATPase. This oscillation can be reconstituted in vitro by incubating three proteins together with ATP. Like all chemical oscillators, this system must include a nonlinear, or switch-like, feedback loop, whose nature has been unclear. Here, by using single particle cryo-EM at near-atomic resolution we identified two major conformational states of KaiC subunits, denoted as the exposed state and the buried state, which may provide a structural basis of how the KaiC hexamer changes its conformation during the (day-night) phosphorylation-dephosphorylation cycle. We classify the abundance and pattern of exposed and buried states within hexamers for more than 160,000 KaiC particles. The statistics of the spatial arrangement of the two states in hexamers can be quantitatively fit by a simple statistical physics model with an interaction energy between neighboring subunits and a local field that depends on phosphorylation state. Our study shows that phosphorylation shifts the probability of each conformation and reveals that there is substantial cooperativity between neighboring subunits, which can allow a KaiC hexamer to respond in an ultrasensitive, switch-like manner to changes in the phosphorylation level.