Local and non-local impacts of intra-hexamer interactions on temperature compensation of KaiC

A hexameric clock protein KaiC exhibits a 24-hour phosphorylation cycle with a unique property termed temperature compensation. The period is kept constant over physiological temperatures through compensatory coordination of underlying elementary reactions. The temperature-compensated ATPase activity of KaiC is one such key reactions that potentially contribute to maintaining a constant circadian period. We identified four amino acid residues responsible for the temperature compensation in an N-terminal ATPase domain of KaiC. D82 and K172 were located in a primary site, and the ATPase activity of each alanine mutant showed a positive correlation with rising temperature. N62 and E69 constituted a secondary site, where each alanine replacement resulted in a negative correlation with the temperature. The primary site exerts a compensatory regulation over the ATPase cycle locally within the N-terminal domain. The secondary site prevents the ATPase activity from becoming over-compensated by suppressing another compensatory regulation mediated through a non-local interaction with a C-terminal domain of KaiC. Therefore, any imbalance between the local and non-local compensatory regulations in KaiC affects the temperature dependence of its phosphorylation rhythm.