Cooperative KaiA-KaiB-KaiC interactions affect KaiB/SasA competition in the circadian clock of cyanobacteria

J Mol Biol. 2014 Jan 23;426(2):389-402. doi: 10.1016/j.jmb.2013.09.040. Epub 2013 Oct 7.


The circadian oscillator of cyanobacteria is composed of only three proteins, KaiA, KaiB, and KaiC. Together, they generate an autonomous ~24-h biochemical rhythm of phosphorylation of KaiC. KaiA stimulates KaiC phosphorylation by binding to the so-called A-loops of KaiC, whereas KaiB sequesters KaiA in a KaiABC complex far away from the A-loops, thereby inducing KaiC dephosphorylation. The switch from KaiC phosphorylation to dephosphorylation is initiated by the formation of the KaiB-KaiC complex, which occurs upon phosphorylation of the S431 residues of KaiC. We show here that formation of the KaiB-KaiC complex is promoted by KaiA, suggesting cooperativity in the initiation of the dephosphorylation complex. In the KaiA-KaiB interaction, one monomeric subunit of KaiB likely binds to one face of a KaiA dimer, leaving the other face unoccupied. We also show that the A-loops of KaiC exist in a dynamic equilibrium between KaiA-accessible exposed and KaiA-inaccessible buried positions. Phosphorylation at the S431 residues of KaiC shift the A-loops toward the buried position, thereby weakening the KaiA-KaiC interaction, which is expected to be an additional mechanism promoting formation of the KaiABC complex. We also show that KaiB and the clock-output protein SasA compete for overlapping binding sites, which include the B-loops on the CI ring of KaiC. KaiA strongly shifts the competition in KaiB's favor. Thus, in addition to stimulating KaiC phosphorylation, it is likely that KaiA plays roles in switching KaiC from phosphorylation to dephosphorylation, as well as regulating clock output.

Keywords: 6-IAF; 6-iodoacetamidofluorescein; EM; NMR; cooperativity; electron microscopy; fluorescence; phosphorylation; protein.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / metabolism*
  • Circadian Clocks*
  • Circadian Rhythm Signaling Peptides and Proteins / metabolism*
  • Cyanobacteria / metabolism
  • Cyanobacteria / physiology*
  • Models, Biological
  • Molecular Sequence Data
  • Phosphorylation
  • Phosphotransferases / metabolism*
  • Protein Binding
  • Protein Multimerization*
  • Protein Processing, Post-Translational


  • Bacterial Proteins
  • Circadian Rhythm Signaling Peptides and Proteins
  • KaiA protein, cyanobacteria
  • KaiB protein, cyanobacteria
  • KaiC protein, cyanobacteria
  • Phosphotransferases
  • SasA protein, Synechococcus