A KaiA Conformational Switch Links Day-Night Transitions to Clock Robustness

Abstract Biological clocks require mechanisms that enable transition between temporal phases and buffer against environmental fluctuations. In the cyanobacterial KaiABC-based circadian oscillator, KaiA has been regarded as a daytime activator that is passively sequestered at night. Here we define KaiA as a conformationally regulated activator and an active catalyst of the night-to-dawn transition. We identify a single linker helix, α5, that acts as a structural switch, mediating a domain-driven “tug-of-war” that sets the open–closed equilibrium in KaiA. Structure-guided mutations that shift this balance in either direction abolish stable rhythmicity both in vitro and in cells. Also, we show that substoichiometric KaiA dismantles nighttime KaiB–KaiC complexes through interactions with the KaiC C-terminal tails and likely initiates an autocatalytic feedback loop that resets the phosphorylation cycle at dawn. Furthermore, we present two independent kinetic models that offer distinct, deterministic explanations for the clock’s low-temperature amplitude decline and failure at 20 °C.