UC Merced

As an adaption to the solar day-night cycles, organisms have evolved endogenous circadian clocks in order to generate ~24-h rhythms in regulating their physiology and behavior. The goal of this study was to uncover mechanisms underlying the rhythm generation in the cyanobacterial clock, which, consisting of only three proteins, KaiA, KaiB, and KaiC, can be reconstituted in vitro. A wide array of biochemical and biophysical techniques including fluorescence spectroscopy, NMR, HDX-MS, and X-ray crystallography have been utilized for studying the transition of daytime (KaiA-KaiC) to nighttime (KaiA-KaiB-KaiC) complexes. In addition, genetic tools such as bioluminescence have been applied for functional validation in vivo. Specific findings to date include: (1) the day-to-night signal, KaiC phosphorylation at Ser431, is transmitted allosterically to a site over 60 Å away for initiating nighttime complex assembly; (2) KaiA cooperatively promotes the formation of the nighttime complex, in contrast to the view that KaiA is sequestered and inert at night; (3) KaiB is a metamorphic protein, forming a KaiA-KaiB-KaiC complex at night using its alternative thioredoxin-like fold; (4) KaiB inhibits KaiA through an unexpected mechanism in which KaiB disrupts KaiA-KaiC interactions by inducing a large conformational change on an α-helix of KaiA to block the binding site of KaiA for KaiC; (5) KaiB metamorphosis couples the circadian oscillator to clock output signaling by displacing the daytime output protein SasA, and engages the nighttime output protein CikA. In summary, this work reveals the role of allostery and large conformational changes in ~24-h rhythm generation in the cyanobacterial clock, creating a conceptual framework for understanding other clock systems.