Structural model of the circadian clock KaiB–KaiC complex and mechanism for modulation of KaiC phosphorylation

Abstract

The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro by the KaiA, KaiB and KaiC proteins in the presence of ATP. The principal clock component, KaiC, undergoes regular cycles between hyper‐ and hypo‐phosphorylated states with a period of ca. 24 h that is temperature compensated. KaiA enhances KaiC phosphorylation and this enhancement is antagonized by KaiB. Throughout the cycle Kai proteins interact in a dynamic manner to form complexes of different composition. We present a three‐dimensional model of the S. elongatus KaiB–KaiC complex based on X‐ray crystallography, negative‐stain and cryo‐electron microscopy, native gel electrophoresis and modelling techniques. We provide experimental evidence that KaiB dimers interact with KaiC from the same side as KaiA and for a conformational rearrangement of the C‐terminal regions of KaiC subunits. The enlarged central channel and thus KaiC subunit separation in the C‐terminal ring of the hexamer is consistent with KaiC subunit exchange during the dephosphorylation phase. The proposed binding mode of KaiB explains the observation of simultaneous binding of KaiA and KaiB to KaiC, and provides insight into the mechanism of KaiB's antagonism of KaiA.

Flexibility of the C-terminal, or CII, ring of KaiC governs the rhythm of the circadian clock of cyanobacteria

“…3A, panels 1-4), as also observed for S. elongatus proteins (16). This selective binding event, localized to the CII ring of KaiC (27), critically separates the phases of phosphorylation and dephosphorylation. However, as the phosphoforms of KaiC that do and do not bind KaiB have the same quaternary structure (26) (SI Appendix, Fig.…”

“…The highly conserved homologs from the thermophile T. elongatus, on the other hand, were superior with respect to expression, stability, and solubility. The T. elongatus and S. elongatus Kai proteins are highly similar with respect to sequence, structure, and biochemistry (21,27,(36)(37)(38)(39). Thus, the results herein were obtained on T. elongatus proteins, which will henceforth be referred to as simply KaiA, KaiB, and KaiC.…”

“…Arrows indicate peaks for free KaiC-EE497 (red peak) and the complex formed by KaiA-130C, KaiB, and KaiC-EE497 (black peak). The apparent molecular size of the complex (approximately 440 kDa) was close to the size expected for a complex composed of a KaiC-EE497 hexamer, two KaiA-130C dimers, and two KaiB dimers (460 kDa) (27,55). (C) SDS-PAGE gel of the elution peak indicated by the black arrow in B.…”

“…KaiB then sequesters KaiA from the A loops in a KaiCB(A) complex (15,(23)(24)(25), inducing KaiC autodephosphorylation (pSpT → pST → ST). The A loops, KaiB-binding site, and sites of phosphorylation (residues S431 and T432) are located in the C-terminal, or CII, ring of KaiC (26,27). It was assumed that large phosphorylation-dependent structural changes in the CII ring underpin the timing mechanism of this circadian oscillator.…”

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

“…KaiC forms a hexamer with a double ring structure, the CI and CII domain each forming one ring, stacked together (4,25,26). Free KaiB has been crystallized as a tetramer in four studies and has once been described as a dimer (27)(28)(29)(30)(31)(32). It was proposed to bind to KaiC as a tetramer (33), dimer (27), or, more recently, as a monomer (32).…”