elongatusKaiC. == Methodology and Principal Findings == The 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 -phosphate. 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 -phosphate. T432 is usually phosphorylated first because it lies consistently closer to P. 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 mutantsin vitroandin vivoimply a role in dephosphorylation. == Conclusions and Significance == We 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. == Introduction == In the cyanobacteriumSynechococcus elongatusthe KaiC, KaiA and KaiB proteins form a minimal circadian clockin vivothat is able to sustain a ca. 24-hour period in the absence of a transcription-translation oscillatory feedback loop[1]. Remarkably, the clock can be reconstitutedin vitrowith just the three Kai proteins and ATP[2]. Thein vitrotimer displays the hallmarks of all circadian oscillators, namely a period of approximately 24 hours, tuned to the daily light-dark cycle, and temperature compensation[3]. The discovery Meptyldinocap of thisin vitrooscillator paves the road to Meptyldinocap a rigorous biochemical, biophysical and structural characterization of a molecular clock[4]. KaiC comprises the core of the clock and acts as a kinase, phosphatase and ATPase[5][8]. KaiA enhances KaiC phosphorylation and in its absencein vitro, KaiC dephosphorylates over time, and KaiB antagonizes KaiA action[6],[7],[9][11]. Three-dimensional structures of the full-length cyanobacterial KaiA, KaiB and KaiC proteins have been reported during the past five years (reviewed in refs.[12]and[13]). KaiC is the result of a gene duplication[14]and forms a homo-hexamer of ca. 360 kDa molecular weight[15],[16]. ThekaiCgene displays similarities to therecAanddnaBfamilies[14], but a helicase activity for KaiC has not been established despite intense efforts[17]. The crystal structure of KaiC fromS. elongatusrevealed a hexamer in the shape of a double doughnut with approximate dimensions 100100 , whereby the N-terminal CI and C-terminal CII halves of subunits are joined by a 15-amino acid linker[18]. A total of twelve ATP molecules are bound between subunits in the upper and lower rings and C-terminal peptide tails that protrude from the dome-shaped surface of the CII hexamer give the KaiC double-doughnut an asymmetric appearance[19]. Both the KaiA and KaiB dimers contact only the KaiCII half and hybrid structural techniques have recently yielded 3-dimensional models of the KaiAC[19]and KaiBC complexes[20]and provided insights into the modes of action of the KaiA and KaiB proteins. Two phosphorylation sites (P-sites), T432 and S431 that are both located in the Meptyldinocap CII half were identified in KaiC[21],[22]. Over a 24-hour cycle phosphorylation proceeds in a rigid order TSpTSpTpSTpSTS[23],[24]. Rapid and repeated association of KaiA with KaiC results in the conversion from the hypo-phosphorylated (TS) to the hyper-phosphorylated (pTpS) form[25],[26]. KaiB binds preferably to the hyper-phosphorylated form and reverses Meptyldinocap KaiA’s action, whereby first T432 and then S431 are being dephosphorylated[23][26]. KaiB binding and dephosphorylation are accompanied by the exchange of KaiC subunits[25], a mechanism that is crucial to maintaining a stable oscillator[26],[27]. Rather than individual KaiC particles engaging in various protein-protein associations and moving essentially in lockstep from the hypo- to the hyper- and back to the hypo-phosphorylated form, the cyanobacterial minimal timer is usually characterized by a mixture of oscillating populations of free KaiC, KaiA and KaiB proteins and KaiAC and KaiBC as well as KaiABC complexes of different concentrations[25],[26]. Recent reports on KaiC and the mechanism of the KaiABC circadian clock take into account only two P-sites. However, we also found the T426A mutant ( = a426/S431/T432 = KaiaST= aST) to be arhythmic[21]. In the crystal structure S431 and T426 are very tightly spaced and the side chain of T426 engages in a H-bond conversation with S431 when the latter is usually phosphorylated[21](Fig. 1). We established that mutations of T426 alter the KaiC phosphorylation profilesin VCL vivoand that residue 426 needs to be phosphorylatable and not simply capable of forming a H-bond to pS431[28]. Moreover, like the KaiCaSTmutant, nST as well as eST abolish rhythmicity in strains expressing these mutants alone. Interestingly, when T426-mutant KaiCs are co-expressed with the wt enzyme, aST exhibits a dominant unfavorable effect,.