Department of Biochemistry, Vanderbilt University, School of Medicine, Nashville, Tennessee, United States of America.
PLoS One. 2009 Nov 26;4(11):e7529. doi: 10.1371/journal.pone.0007529.
The 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.
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 gamma-phosphate. T432 is phosphorylated first because it lies consistently closer to Pgamma. 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.
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.
蓝藻聚球藻的生物钟可以通过三种蛋白质 KaiA、KaiB 和 KaiC 在体外重建。同型六聚体 KaiC 具有激酶、磷酸酶和 ATP 酶活性；KaiA 增强 KaiC 的磷酸化，而 KaiB 拮抗 KaiA。KaiC 亚基 C 端半胱氨酸残基 T432 和 S431 上的两个已知位点的磷酸化和去磷酸化遵循严格的顺序（TS-->pTS-->pTpS-->TpS-->TS），其起源尚不清楚。为了解决这一空白，并分析 KaiC 活性位点残基，特别是 T426 的作用，我们测定了聚球藻 KaiC 单突变和双 P 位突变体的晶体结构。
六个 KaiC 突变蛋白晶体结构中环区的构象存在细微差异，导致 Thr（或 Ala/Asn/Glu）和 Ser（或 Ala/Asp）残基与 ATP γ-磷酸之间的距离各不相同。T432 首先被磷酸化，因为它始终更靠近 Pγ。S431A 和 T432E/S431A 突变体的结构显示 T426 发生了磷酸化。该残基在结构中的环境以及 T426 体外和体内突变体的功能数据表明其在去磷酸化中的作用。
我们提供了 KaiC 中 T426 第三个磷酸化位点的证据。T426 和 S431 紧密相邻，一个 KaiC 亚基不能同时携带磷酸基团。与 wt 和其他突变体 KaiC 结构中磷酸化的 T432 和/或 S431 残基相比，两个 KaiC 突变体中 T426 磷酸化的亚基较少，这表明 T426 磷酸化可能不稳定。这些结构与大量 KaiC 突变蛋白的功能数据相结合，有助于解释为什么 S431 在失去其磷酸基团时滞后于 T432，并阐明 KaiC 激酶、ATP 酶和磷酸酶活性的机制。
