Rhythmic ring–ring stacking drives the circadian oscillator clockwise

Abstract

The oscillator of the circadian clock of cyanobacteria is composed of three proteins, KaiA, KaiB, and KaiC, which together generate a selfsustained ∼24-h rhythm of phosphorylation of KaiC. The mechanism propelling this oscillator has remained elusive, however. We show that stacking interactions between the CI and CII rings of KaiC drive the transition from the phosphorylation-specific KaiC-KaiA interaction to the dephosphorylation-specific KaiC-KaiB interaction. We have identified the KaiB-binding site, which is on the CI domain. This site is hidden when CI domains are associated as a hexameric ring. However, stacking of the CI and CII rings exposes the KaiB-binding site. Because the clock output protein SasA also binds to CI and competes with KaiB for binding, ring stacking likely regulates clock output. We demonstrate that ADP can expose the KaiB-binding site in the absence of ring stacking, providing an explanation for how it can reset the clock.dynamic allostery | NMR | protein structure | kinase | kinetics

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

“…were assessed on the isolated CI and CII domains and by negative-stain EM analysis of nanogold-labeled KaiCB (27,32,34,35). However, it was recently reported that KaiB does not interact with the isolated CII domain of KaiC, but interacts with an engineered monomeric form of the CI domain instead (36). It was suggested in this study that CI-CII stacking interactions wedge the CI domains apart, exposing the putative KaiB binding site.…”

Exchange of ADP with ATP in the CII ATPase domain promotes autophosphorylation of cyanobacterial clock protein KaiC

“…1 B and C), suggesting that CII is necessary not only for retaining ADP on itself but also for regulating the nucleotide-bound state of CI. A recent study showed that the KaiB-KaiC interaction is mediated by the CI domain and that the binding of ADP to CI is necessary for KaiC's association with KaiB (22). Previously, we and another group reported that the phosphorylation of S431 promotes the KaiB-KaiC interaction (16,17).…”

“…(i) The autophosphorylation sites are located at subunit interfaces of CII, and each site faces the ATP molecule on the neighboring subunit (14). (ii) KaiA acts on the CII domain to promote autophosphorylation and ATP hydrolysis (20)(21)(22), whereas KaiA has little effect on ATPase activity in the CI domain (22). (iii) ATP accounted for more than 95% of nucleotides bound to KaiC-CI, regardless of the presence or absence of KaiA ( Fig.…”

“…Phosphorylation of S431, which allows KaiC to enter the dephosphorylation phase (16,17), rigidifies the CII ring of KaiC (21), and enhances the stacking between the CI and CII rings (22). However, it is not fully understood how these conformational changes enhance the reversal of autophosphorylation.…”

“…S4A). Rigidification of the CII ring and enhanced stacking between the CI and CII rings (21,22) may inhibit exchange of bound ADP to external ATP. On the other hand, ATP consistently accounted for ∼60% of KaiC-AA-bound nucleotide, regardless of the presence or absence of KaiA (Fig.…”