Identification of a Third Period-tuning Site in Cyanobacterial Clock Protein KaiC

KaiC, a clock protein in cyanobacteria, cycles between dephosphorylated and phosphorylated states in a 24-hour period in the presence of KaiA and KaiB. We identified the 322nd residue of KaiC as a third example of period-tuning sites. 322nd-site-directed saturation mutagenesis resulted in a variety of KaiC mutants exhibiting either shortened or lengthened cycles. The tunable range of the periods was from approximately 11 to 78 h without significantly compromising temperature compensation. We conducted biochemical analyses of the 322nd variants and examined their predicted structural models. In contrast to another known period-tuning site, where the period decreases sharply as the side-chain volume increases due to mutations, the cycle lengths correlate only modestly with bulkiness at the 322nd residues. The 322nd residue is located in a C-terminal domain of KaiC and influences ATPase cycles in both the C-terminal domain and an N-terminal domain through its interaction with a flexible loop connecting the two domains. The structural models predict that placing less bulky but polar side chains, such as serine and threonine, at the 322nd position leads to the formation of a hydrogen-bonding network between that site and the loop. This reduces the mobility of the loop, resulting in the longer cycles due to decreases in the ATPase activity of the N-terminal domain. Conversely, placing bulky residues such as phenylalanine at the 322nd position appears to alter the loop structure, shortening the periods by enhancing the ATP activities of both the domains. The third period-tuning mechanism is distinct from other known mechanisms. Significance StatementA Kai-protein clock system serves as a model for studying how long circadian rhythms are achieved. We identified the 322nd residue of KaiC as a third example of period-tuning sites that allow tuning of the period in either long- and short-period directions. The third period-tuning mechanism differs from the two previously known types in several respects. Previous studies have suggested that the ATPase activity in an N-terminal domain of KaiC is the primary regulator of the period. On the other hand, the 322nd residues of KaiC can affect the period by activating the ATPase cycle in its C-terminal domain. Our findings will stimulate future studies on the period-tuning mechanism mediated by the ATPase activity in the C-terminal domain of KaiC.