Temperature-Sensitive Substrate and Product Binding Underlie Temperature-Compensated Phosphorylation in the Clock

• DOI: 10.1016/j.molcel.2017.08.009
• Publication Date: 2017-09-19T20:02:29Z
• Authors: Yuta Shinohara; Yohei M. Koyama; Maki Ukai-Tadenuma; Takatsugu Hirokawa; Masaki Kikuchi; Rikuhiro G. Yamada; Hideki Ukai; Hiroshi Fujishima; Takashi Umehara; Kazuki Tainaka; Hiroki R. Ueda
• Keywords: 0301 basic medicine; Binding Sites; Genotype; Hydrolysis; Mice, Transgenic; Molecular Dynamics Simulation; Crystallography, X-Ray; Models, Biological; Circadian Rhythm; Molecular Docking Simulation; Kinetics; 03 medical and health sciences; Adenosine Triphosphate; HEK293 Cells; Phenotype; Casein Kinase Idelta; Catalytic Domain; Circadian Clocks; Mutation; Animals; Humans; Locomotion

Temperature compensation is a striking feature of the circadian clock. Here we investigate biochemical mechanisms underlying temperature-compensated, CKIδ-dependent multi-site phosphorylation in mammals. We identify two mechanisms for temperature-insensitive phosphorylation at higher temperature: lower substrate affinity to CKIδ-ATP complex and higher product affinity to CKIδ-ADP complex. Inhibitor screening of ADP-dependent phosphatase activity of CKIδ identified aurintricarboxylic acid (ATA) as a temperature-sensitive kinase activator. Docking simulation of ATA and mutagenesis experiment revealed K224D/K224E mutations in CKIδ that impaired product binding and temperature-compensated primed phosphorylation. Importantly, K224D mutation shortens behavioral circadian rhythms and changes the temperature dependency of SCN's circadian period. Interestingly, temperature-compensated phosphorylation was evolutionary conserved in yeast. Molecular dynamics simulation and X-ray crystallography demonstrate that an evolutionally conserved CKI-specific domain around K224 can provide a structural basis for temperature-sensitive substrate and product binding. Surprisingly, this domain can confer temperature compensation on a temperature-sensitive TTBK1. These findings suggest the temperature-sensitive substrate- and product-binding mechanisms underlie temperature compensation.