The circadian clock controls 24-h biological rhythms in our body, influencing many time-related activities such as sleep and wake. The simplest circadian clock is found in cyanobacteria, with the proteins KaiA, KaiB, and KaiC generating a self-sustained circadian oscillation of KaiC phosphorylation and dephosphorylation. KaiA activates KaiC phosphorylation by binding the A-loop of KaiC, while KaiB attenuates the phosphorylation by sequestering KaiA from the A-loop. Structural analysis revealed that magnesium regulates the phosphorylation and dephosphorylation of KaiC by dissociating from and associating with catalytic Glu residues that activate phosphorylation and dephosphorylation, respectively. High magnesium causes KaiC to dephosphorylate, whereas low magnesium causes KaiC to phosphorylate. KaiC alone behaves as an hourglass timekeeper when the magnesium concentration is alternated between low and high levels in vitro. We suggest that a magnesiumbased hourglass timekeeping system may have been used by ancient cyanobacteria before magnesium homeostasis was established. 

Metamorphic proteins, which can adopt multiple stable conformations, challenge the traditional understanding of protein structure and function. KaiB is a metamorphic protein that regulates the circadian clock, a central regulator governing gene expression in most light-perceiving organisms on Earth. An interesting aspect is that the circadian clock can be reconstituted in vitro by mixing Kai proteins (KaiA, KaiB, and KaiC) with ATP and Mg²⁺. The phosphorylation state of KaiC oscillates with a 24-hour period. The fold-switched form of KaiB binds to KaiC to activate the dephosphorylation of KaiC, while the other fold of KaiB dissociates from KaiC, allowing phosphorylation to be activated by the binding of KaiA to KaiC. To understand the metamorphic process of KaiB, we utilized AlphaFold2, a protein structure prediction program, and sequence alignments. We found that a proline residue determines the fold of KaiB. We also confirmed that mutating this proline to lysine changes KaiB to a fold-switched conformation. This validates that AlphaFold2 can be used for the study of metamorphic proteins.