Structures of KaiC Circadian Clock Mutant Proteins: A New Phosphorylation Site at T426 and Mechanisms of Kinase, ATPase and Phosphatase

Abstract

BackgroundThe circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro by three proteins, KaiA, KaiB and KaiC. Homo-hexameric KaiC displays kinase, phosphatase and ATPase activities; KaiA enhances KaiC phosphorylation and KaiB antagonizes KaiA. Phosphorylation and dephosphorylation of the two known sites in the C-terminal half of KaiC subunits, T432 and S431, follow a strict order (TS→pTS→pTpS→TpS→TS) over the daily cycle, the origin of which is not understood. To address this void and to analyze the roles of KaiC active site residues, in particular T426, we determined structures of single and double P-site mutants of S. elongatus KaiC.Methodology and Principal FindingsThe conformations of the loop region harboring P-site residues T432 and S431 in the crystal structures of six KaiC mutant proteins exhibit subtle differences that result in various distances between Thr (or Ala/Asn/Glu) and Ser (or Ala/Asp) residues and the ATP γ-phosphate. T432 is phosphorylated first because it lies consistently closer to Pγ. The structures of the S431A and T432E/S431A mutants reveal phosphorylation at T426. The environments of the latter residue in the structures and functional data for T426 mutants in vitro and in vivo imply a role in dephosphorylation.Conclusions and SignificanceWe provide evidence for a third phosphorylation site in KaiC at T426. T426 and S431 are closely spaced and a KaiC subunit cannot carry phosphates at both sites simultaneously. Fewer subunits are phosphorylated at T426 in the two KaiC mutants compared to phosphorylated T432 and/or S431 residues in the structures of wt and other mutant KaiCs, suggesting that T426 phosphorylation may be labile. The structures combined with functional data for a host of KaiC mutant proteins help rationalize why S431 trails T432 in the loss of its phosphate and shed light on the mechanisms of the KaiC kinase, ATPase and phosphatase activities.

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

“…KaiC autophosphorylation is probably sequential rather than concerted, as several crystal structures of S. elongatus KaiC were only partially phosphorylated (26). Accordingly, the observation that the autophosphorylation profile of KaiC-SA reached an attenuated steady-state level of approximately 56% (Fig.…”

“…Comparing the steadystate phosphorylation levels of KaiC-AT (approximately 83%) and KaiC-ET (approximately 0%) shows that phospho-S431 also inhibits autophosphorylation at T432. The virtually identical crystal structures of KaiC (26) suggest that these different kinetics profiles were not controlled by phosphorylation-dependent structural differences.…”

“…It was assumed that large phosphorylation-dependent structural changes in the CII ring underpin the timing mechanism of this circadian oscillator. Surprisingly, recent X-ray crystal structures of several different phosphomimics of KaiC were virtually identical (26). Thus, the mechanism driving the rhythm of phosphorylation of KaiC is not structurally based.…”

“…Thus, phospho-S431 restricts microsecond-millisecond time scale motions of the CII ring. Crystal structures of S. elongatus KaiC show that the phosphate group of phospho-S431 hydrogen bonds to residue H429 of the same subunit, which in turn interacts with D417 from the adjacent subunit, thereby stabilizing the CII ring, presumably (26). Although the crystal structures are of the S. elongatus homolog, residues D417 and H429 are conserved.…”

“…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.…”