TY - JOUR T1 - From primordial clocks to circadian oscillators JF - bioRxiv DO - 10.1101/2022.11.28.518275 SP - 2022.11.28.518275 AU - Warintra Pitsawong AU - Ricardo A. P. Pádua AU - Timothy Grant AU - Marc Hoemberger AU - Renee Otten AU - Niels Bradshaw AU - Nikolaus Grigorieff AU - Dorothee Kern Y1 - 2022/01/01 UR - http://biorxiv.org/content/early/2022/11/29/2022.11.28.518275.1.abstract N2 - Circadian rhythms play an essential role in many biological processes and surprisingly only three prokaryotic proteins are required to constitute a true post-translational circadian oscillator. The evolutionary history of the three Kai proteins indicates that KaiC is the oldest member and central component of the clock, with subsequent additions of KaiB and KaiA to regulate its phosphorylation state for time synchronization. The canonical KaiABC system in cyanobacteria is well understood, but little is known about more ancient systems that possess just KaiBC, except for reports that they might exhibit a basic, hourglass-like timekeeping mechanism. Here, we investigate the primordial circadian clock in Rhodobacter sphaeroides (RS) that contains only KaiBC to elucidate its inner workings despite the missing KaiA. Using a combination X-ray crystallography and cryo-EM we find a novel dodecameric fold for KaiCRS where two hexamers are held together by a coiled-coil bundle of 12 helices. This interaction is formed by the C-terminal extension of KaiCRS and serves as an ancient regulatory moiety later superseded by KaiA. A coiled-coil register shift between daytime- and nighttime-conformations is connected to the phosphorylation sites through a long-range allosteric network that spans over 160 Å. Our kinetic data identify the difference in ATP-to-ADP ratio between day and night as the environmental cue that drives the clock and further unravels mechanistic details that shed light on the evolution of self-sustained oscillators.Competing Interest StatementD.K. is co-founder of Relay Therapeutics and MOMA Therapeutics. The remaining authors declare no competing interests. ER -