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

Mol Cell. 2017 Sep 7;67(5):783-798.e20. 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.

Keywords: casein kinase 1; circadian clock; enzyme design; enzyme mechanisms; enzyme simulation; phosphorylation; structural biology; synthetic biology; system biology; temperature compensation.

Publication types

  • Video-Audio Media

MeSH terms

  • Adenosine Triphosphate / metabolism*
  • Animals
  • Binding Sites
  • Casein Kinase Idelta / chemistry
  • Casein Kinase Idelta / genetics
  • Casein Kinase Idelta / metabolism*
  • Catalytic Domain
  • Circadian Clocks*
  • Circadian Rhythm*
  • Crystallography, X-Ray
  • 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
  • Structure-Activity Relationship
  • Substrate Specificity
  • Suprachiasmatic Nucleus / enzymology*
  • Temperature*
  • Tissue Culture Techniques
  • Transfection


  • Serine
  • Adenosine Triphosphate
  • Casein Kinase Idelta