An evolutionary hotspot defines functional differences between CRYPTOCHROMES

Nat Commun. 2018 Mar 19;9(1):1138. doi: 10.1038/s41467-018-03503-6.


Mammalian circadian clocks are driven by a transcription/translation feedback loop composed of positive regulators (CLOCK/BMAL1) and repressors (CRYPTOCHROME 1/2 (CRY1/2) and PER1/2). To understand the structural principles of regulation, we used evolutionary sequence analysis to identify co-evolving residues within the CRY/PHL protein family. Here we report the identification of an ancestral secondary cofactor-binding pocket as an interface in repressive CRYs, mediating regulation through direct interaction with CLOCK and BMAL1. Mutations weakening binding between CLOCK/BMAL1 and CRY1 lead to acceleration of the clock, suggesting that subtle sequence divergences at this site can modulate clock function. Divergence between CRY1 and CRY2 at this site results in distinct periodic output. Weaker interactions between CRY2 and CLOCK/BMAL1 at this pocket are strengthened by co-expression of PER2, suggesting that PER expression limits the length of the repressive phase in CRY2-driven rhythms. Overall, this work provides a model for the mechanism and evolutionary variation of clock regulatory mechanisms.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • ARNTL Transcription Factors / chemistry
  • ARNTL Transcription Factors / genetics
  • ARNTL Transcription Factors / metabolism
  • Allosteric Site / genetics
  • Animals
  • CLOCK Proteins / chemistry
  • CLOCK Proteins / genetics
  • CLOCK Proteins / metabolism
  • Cell Line
  • Circadian Clocks / genetics
  • Cryptochromes / chemistry
  • Cryptochromes / genetics*
  • Cryptochromes / metabolism*
  • Evolution, Molecular*
  • HEK293 Cells
  • Humans
  • Insect Proteins / chemistry
  • Insect Proteins / genetics
  • Insect Proteins / metabolism
  • Mice
  • Mice, Knockout
  • Models, Molecular
  • Period Circadian Proteins / chemistry
  • Period Circadian Proteins / genetics
  • Period Circadian Proteins / metabolism
  • Protein Interaction Domains and Motifs / genetics
  • Structural Homology, Protein


  • ARNTL Transcription Factors
  • Cryptochromes
  • Insect Proteins
  • Period Circadian Proteins
  • CLOCK Proteins