Structural characterization of the circadian clock protein complex composed of KaiB and KaiC by inverse contrast-matching small-angle neutron scattering

Sci Rep. 2016 Oct 18;6:35567. doi: 10.1038/srep35567.

Abstract

The molecular machinery of the cyanobacterial circadian clock consists of three proteins: KaiA, KaiB, and KaiC. Through interactions among the three Kai proteins, the phosphorylation states of KaiC generate circadian oscillations in vitro in the presence of ATP. Here, we characterized the complex formation between KaiB and KaiC using a phospho-mimicking mutant of KaiC, which had an aspartate substitution at the Ser431 phosphorylation site and exhibited optimal binding to KaiB. Mass-spectrometric titration data showed that the proteins formed a complex exclusively in a 6:6 stoichiometry, indicating that KaiB bound to the KaiC hexamer with strong positive cooperativity. The inverse contrast-matching technique of small-angle neutron scattering enabled selective observation of KaiB in complex with the KaiC mutant with partial deuteration. It revealed a disk-shaped arrangement of the KaiB subunits on the outer surface of the KaiC C1 ring, which also serves as the interaction site for SasA, a histidine kinase that operates as a clock-output protein in the regulation of circadian transcription. These data suggest that cooperatively binding KaiB competes with SasA with respect to interaction with KaiC, thereby promoting the synergistic release of this clock-output protein from the circadian oscillator complex.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Circadian Clocks
  • Circadian Rhythm Signaling Peptides and Proteins / chemistry
  • Circadian Rhythm Signaling Peptides and Proteins / genetics
  • Circadian Rhythm Signaling Peptides and Proteins / metabolism*
  • Crystallography, X-Ray
  • Multiprotein Complexes / chemistry
  • Multiprotein Complexes / genetics
  • Multiprotein Complexes / metabolism*
  • Mutation / genetics
  • Phosphorylation / genetics
  • Phosphotransferases / metabolism*
  • Protein Binding
  • Protein Conformation
  • Protein Multimerization
  • Scattering, Small Angle
  • Synechococcus / physiology*

Substances

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