Roles of two ATPase-motif-containing domains in cyanobacterial circadian clock protein KaiC
Roles of two ATPase-motif-containing domains in cyanobacterial circadian clock protein KaiC
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DOI：
10.1074/jbc.m406604200
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发表时间：
2004-12-10
影响因子：
4.8
通讯作者：
Ishiura, M
中科院分区：
文献类型：
--
作者：
Hayashi, F;Itoh, N;Ishiura, M
蓝细菌时钟蛋白KaiC具有由六个相同的哑铃形亚基组成的六边形壶形结构。每个亚基具有重复的结构域，并且每个结构域具有一组ATP酶基序。哑铃形的两个球形区域可能对应于两个域。我们通过分析ATP γ S结合的体外活性、AMPPNP诱导的六聚化、热稳定性和KaiC的磷酸化以及通过在野生型KaiC（KaiC（WT））和携带步行者基序A或推导的催化Glu残基中的突变的KaiC中的体内节律测定来检查两组ATP酶基序的作用。结果表明：KaiC亚基具有两种ATP结合位点，一个位于N端的高亲和力位点和一个位于C端的低亲和力位点; KaiC亚基的N端与六聚体的形成有关; KaiC亚基的C端与六聚体的稳定化和磷酸化有关。我们提出了以下反应机理。ATP优先结合到N-末端高亲和力位点，诱导KaiC的六聚化。然后，额外的ATP结合到C-末端低亲和力位点，稳定和磷酸化六聚体。我们讨论了这些KaiC突变对蓝藻细胞生物发光昼夜节律的影响。
Cyanobacterial clock protein KaiC has a hexagonal, pot-shaped structure composed of six identical dumb-bell-shaped subunits. Each subunit has duplicated domains, and each domain has a set of ATPase motifs. The two spherical regions of the dumbbell are likely to correspond to two domains. We examined the role of the two sets of ATPase motifs by analyzing the in vitro activity of ATPgammaS binding, AMPPNP-induced hexamerization, thermostability, and phosphorylation of KaiC and by in vivo rhythm assays both in wild type KaiC (KaiC(WT)) and KaiCs carrying mutations in either Walker motif A or deduced catalytic Glu residues. We demonstrated that 1) the KaiC subunit had two types of ATP-binding sites, a high affinity site in N-terminal ATPase motifs and a low affinity site in C-terminal ATPase motifs, 2) the N-terminal motifs were responsible for hexamerization, and 3) the C-terminal motifs were responsible for both stabilization and phosphorylation of the KaiC hexamer. We proposed the following reaction mechanism. ATP preferentially binds to the N-terminal high affinity site, inducing the hexamerization of KaiC. Additional ATP then binds to the C-terminal low affinity site, stabilizing and phosphorylating the hexamer. We discussed the effect of these KaiC mutations on circadian bioluminescence rhythm in cells of cyanobacteria.