Hexamerization by the N-terminal domain and intersubunit phosphorylation by the C-terminal domain of cyanobacterial circadian clock protein KaiC
Hexamerization by the N-terminal domain and intersubunit phosphorylation by the C-terminal domain of cyanobacterial circadian clock protein KaiC
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DOI：
10.1016/j.bbrc.2006.07.143
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发表时间：
2006-09-29
影响因子：
3.1
通讯作者：
Ishiura, Masahiro
中科院分区：
文献类型：
--
作者：
Hayashi, Fumio;Iwase, Ryo;Ishiura, Masahiro
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。哑铃形结构的相对球形区域对应于KaiC的N-末端和C-末端结构域。以前，我们假设KaiC的N-末端结构域负责KaiC的ATP诱导的六聚化，而C-末端结构域负责KaiC的磷酸化（Hayashi等，2004，J. Biol. Chem. 279，52331-52337）。在这里，我们使用每个结构域的纯化蛋白质来测试该假设。我们制备了N-末端和C-末端结构域蛋白（分别为KaiC（N）和KaiC（C）），通过分析它们的ATP或5 '-腺苷酰亚氨基二磷酸（AMPPNP;不可水解的ATP类似物）诱导的六聚化、与KaiA的相互作用和磷酸化来检查它们的功能，并且我们证明了以下内容：（1）KaiCN具有比KaiC（C）更高的ATP或AMPPNP诱导的寡聚化活性。(2)KaiCc具有与KaiC（WT）相同的磷酸化活性，而KaiCN没有活性。(3)KaiCc与KaiA互动，而KaiCN则没有。(4)KaiCc与KaiA的相互作用不需要KaiC具有六聚体结构。(5)KaiCc与KaiA的相互作用增强了KaiCc的磷酸化。此外，我们提出了KaiC亚基间磷酸化的证据。KaiC（CatE 2）-由于催化Glu残基的突变而缺乏KaiC磷酸化活性，当其与KaiC（C）共孵育时被磷酸化。我们提出KaiC六聚体由六个具有六聚化活性的N-末端结构域形成的刚性环结构和六个具有亚基间磷酸化活性的C-末端结构域形成的柔性结构组成。(c)2006爱思唯尔公司All rights reserved.
Cyanobacterial clock protein KaiC has a hexagonal, pot-shaped structure composed of six identical dumbbell-shaped subunits. The opposing spherical regions of the dumbbell-shaped structures correspond to the N-terminal and C-terminal domains of KaiC. Previously, we hypothesized that the N-terminal domain of KaiC is responsible for the ATP-induced hexamerization of KaiC while the C-terminal domain is responsible for the phosphorylation of KaiC (Hayashi et al. 2004, J. Biol. Chem. 279, 52331-52337). Here, we tested that hypothesis using the purified protein of each domain. We prepared N-terminal and C-terminal domain proteins (KaiC(N) and KaiC(C), respectively), examined their function by analyzing their ATP- or 5'-adenylylimidodiphosphate (AMPPNP; an unhydrolyzable ATP analog)-induced hexamerization, interactions with KaiA, and phosphorylation, and we demonstrated the following: (1) KaiCN had higher ATP- or AMPPNP-induced oligomerization activity than KaiC(C). (2) KaiCc had phosphorylation activity as KaiC(WT) whereas KaiCN had no activity. (3) KaiCc interacted with KaiA whereas KaiCN did not. (4) The interactions of KaiCc with KaiA did not require that KaiC has a hexamer structure. (5) The interactions of KaiCc with KaiA enhanced the phosphorylation of KaiCc. Furthermore, we presented evidence for the intersubunit phosphorylation of KaiC. KaiC(CatE2)-, which lacks KaiC phosphorylation activity due to mutations of the catalytic Glu residues, was phosphorylated when it was co-incubated with KaiC(C). We propose that the KaiC hexamer consists of a rigid ring structure formed by six N-terminal domains with hexamerization activity and a flexible structure formed by six C-terminal domains with intersubunit phosphorylation activity. (c) 2006 Elsevier Inc. All rights reserved.