Molecular Shape Evolution of Cyanobacterial Circadian Clock Protein KaiC

Abstract A primitive form of clock protein KaiC has diverged into autonomous or passive time-measuring system in prokaryotes under selective pressures of day–night environmental changes caused by the rotation of Earth. However, the timing of such functional diversification and its structural basis remain unknown. Here we traced molecular shape evolution of older KaiCs by using X-ray solution scattering and structure prediction techniques. The result shows that the oldest ancestral KaiC emerged approximately 3.1 billion years ago as a moderately expanded and asymmetric double-ring hexamer, and subsequently evolved over a period of approximately 1 billion years into a compact and symmetric hexamer that is essential for achieving the self-sustained rhythmicity in extant cyanobacteria. In parallel with this compactification, the oldest KaiC branched into an oligomer composed of two hexamers approximately 0.5 billion years after its emergence. This is the direct experimental result demonstrating the early appearance of the prototypical dodecamer predicted by Kern and colleagues. It appears that this prototypical dodecamer gained a higher enzymatic activity during the next 0.4 billion years or so, and was passed down to non-cyanobacterial lineages as the passive timer capable of responding rapidly to environmental cues. Consequently, geological fluctuations over approximately 1 billion years since the earliest KaiC appeared caused the molecular shape of ancient KaiCs to evolve dramatically along the two distinct pathways. Significance Statement Molecular shape analysis has shed light on the 3-billion-year evolutionary history of clock protein KaiCs. The earliest KaiC emerged as a less compact and asymmetric hexamer, and later diverged along two distinct pathways. One led to the formation of a compact and symmetric hexamer as the core component of the self-sustained and temperature-compensated circadian oscillator found in modern cyanobacteria. The other reached the oligomerization of two hexamers as a prototype of the environmentally responsive timer found in non-cyanobacterial species. In the billion years following the birth of the oldest KaiC, ancient KaiCs adapted environmental alterations by dramatically evolving their molecular shape along the two directions.