Cooperative KaiA–KaiB–KaiC Interactions Affect KaiB/SasA Competition in the Circadian Clock of Cyanobacteria

Abstract

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.

Insight into cyanobacterial circadian timing from structural details of the KaiB–KaiC interaction

“…In addition, no direct evidence for an interaction between KaiB and KaiC-CI was demonstrated in the context of the full hexamer; only an interaction with an engineered monomeric variant of KaiC-CI was demonstrated. Our modeling data favor KaiB binding to the CII domain of KaiC, but we nevertheless still observe protection of CI in our HDX experiment as well, near a region (residues 116-123) that was recently suggested to be involved in KaiB binding (47).…”

Single mutations in sasA enable a simpler Δ cikA gene network architecture with equivalent circadian properties

“…Like CikA, KaiA has been shown to bind oxidized quinone, a major redox-responsive cofactor of the photosynthetic apparatus, which can directly reset the phase of the KaiABC oscillator in vitro (19). Because SasA and KaiB compete for the same binding site on KaiC (20), these mutations in SasA may affect KaiB-KaiC binding kinetics, and subsequently alter the temporal association of KaiA with the oscillator. An increased interaction between KaiA and KaiC could account for the hyperphosphorylation of KaiC in the cikA-null SasA mutant strains (Fig.…”

Intricate Protein-Protein Interactions in the Cyanobacterial Circadian Clock

“…A subsequent publication reported normal in vitro oscillations based on the KaiB 1-94 mutant, but attenuated amplitudes of oscillations and a lack of normal gene expression rhythms in cells expressing KaiB 1-94 (7). Both KaiB and KaiB* appear to favor the dimeric over the tetrameric state (7,42,60). As demonstrated by native MS, the quaternary structure adopted by KaiB, tetramer, dimer, or monomer, is influenced by both protein concentration and temperature, with lower concentrations and reduced temperatures favoring the monomer (44).…”

“…NMR studies of a KaiB mutant lacking the N-terminal tail (seven residues) in addition to the above C-terminal deletion and additional Y8A/Y94A mutations (KaiB*) mixed with a FLAG-tagged monomeric CI domain (CI*) revealed formation of a specific complex (Fig. 3D) (60). No complex is observed when CI* and KaiA lacking the N-terminal domain ( ⌬N KaiA) are mixed alone (Fig.…”

“…KaiA and KaiB do not form a complex according to native PAGE and fluorescence assays (29). However, KaiA ( ⌬N KaiA) and KaiB* do form a complex as demonstrated by NMR (60). Clearly, this observation argues against the KaiB C-terminal tail contacting KaiA.…”