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

• Published in: Molecular cell 2017-09, Vol.67 (5), p.783-798.e20
• Main Authors: Shinohara, Yuta, Koyama, Yohei M., Ukai-Tadenuma, Maki, Hirokawa, Takatsugu, Kikuchi, Masaki, Yamada, Rikuhiro G., Ukai, Hideki, Fujishima, Hiroshi, Umehara, Takashi, Tainaka, Kazuki, Ueda, Hiroki R.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Animals; Binding Sites; casein kinase 1; Casein Kinase Idelta - chemistry; Casein Kinase Idelta - genetics; Casein Kinase Idelta - metabolism; Catalytic Domain; circadian clock; Circadian Clocks; Circadian Rhythm; Crystallography, X-Ray; enzyme design; enzyme mechanisms; enzyme simulation; Genotype; HEK293 Cells; Humans; Hydrolysis; Kinetics; Locomotion; Mice, Transgenic; Models, Biological; Molecular Docking Simulation; Molecular Dynamics Simulation; Mutation; Phenotype; Phosphorylation; Protein Binding; Protein Domains; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Serine; structural biology; Structure-Activity Relationship; Substrate Specificity; Suprachiasmatic Nucleus - enzymology; synthetic biology; system biology; Temperature; temperature compensation; Tissue Culture Techniques; Transfection
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2017.08.009

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. [Display omitted] •Temperature-sensitive affinity can counteract the speedup of phosphorylation•CKIδ agonist ATA is identified by inhibitor screening of dephosphorylation activity•K224D mutation significantly changed the temperature dependency of circadian rhythm•Temperature compensation is conferred by inserting CKI-specific domain around K224 The circadian clock is able to compensate for fluctuations in temperature, and Shinohara et al. reveal two underlying mechanisms: “lower substrate affinity to CKIδ-ATP complex” and “higher product affinity to CKIδ-ADP complex.” They also identify a key CKI-specific domain that is necessary for temperature compensation.