Project Summary The ability to encode time at the molecular level is universally used to synchronize biological processes into ~24-hour rhythms that coincide with the solar day for health and homeostasis. How do we measure time at the molecular level and use it as the basis for biological regulation? The goal of the current project is to use the simplest model system for circadian cycling to explore the structural basis for generating a molecular clock that keeps 24-hour time. Cyanobacteria have a biochemically tractable clock composed of three Kai (cycle) proteins that keep ~24-hour time in vitro, requiring only the addition of ATP as an energy source. KaiC is the central pacemaker of this clock, with two tandem ATPase domains (C1 and C2) that each assemble into hexameric rings connected by a flexible linker. Information about the time of day is transmitted between the rings to influence interactions with KaiA and KaiB, as well as with proteins that transmit information about the environment to the clock and control biological timing in vivo. Despite its relatively simple composition, the lack of structures for intermediate states formed by Kai protein complexes has limited our understanding of this molecular clock. The proposed research will provide structural, biochemical, and in vivo functional data in support of a new model for Kai protein interactions, leading to a major shift in our understanding of the cyanobacterial circadian oscillator. We are pursuing a hypothesis that competition for the KaiC C1 domain, a central hub for clock protein interactions, is essential for circadian timekeeping. Our approach is built on the discovery that KaiB undergoes a metamorphic transition to a new protein fold that is needed to regulate assembly of clock protein complexes with KaiC. Using a version of KaiB locked into its rare, active conformation, we present three new structures of clock protein complexes that demonstrate the vastly underappreciated role that the KaiC C1 domain plays in generating the molecular clock. In Aim 1, we will determine how the hexameric KaiC ATPase controls assembly of Kai protein complexes. In Aim 2, we will identify the structural basis for communication between the two ATPase rings of KaiC. In Aim 3, we will determine how competitive interactions at the KaiC C1 domain control clock signaling throughout the day and night. The proposed studies will demonstrate the structural basis by which KaiC integrates interactions with clock proteins to keep 24-hour time and transform our understanding of the cyanobacterial circadian clock.