Loop-Loop Interactions Regulate KaiA-Stimulated KaiC Phosphorylation in the Cyanobacterial KaiABC Circadian Clock
Loop-Loop Interactions Regulate KaiA-Stimulated KaiC Phosphorylation in the Cyanobacterial KaiABC Circadian Clock
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DOI：
10.1021/bi301691a
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发表时间：
2013-02-19
期刊：
影响因子：
2.9
通讯作者：
Johnson, Carl H.
中科院分区：
文献类型：
--
作者：
Egli, Martin;Pattanayek, Rekha;Johnson, Carl H.
在ATP存在下，长聚球菌KaiA、KaiB和KaiC蛋白产生一个翻译后振荡器，以温度补偿的方式运行，周期为24小时。KaiA二聚体刺激T432和S431， KaiB二聚体在每个亚基的两个位点上拮抗KaiA作用和KaiC六聚体的磷酸化，诱导KaiC亚基交换。目前对kaia刺激KaiC磷酸化的机制和kaib介导的KaiC去磷酸化的机制都没有详细的了解。我们在这里证明，A422V KaiC突变体揭示了前者的机制。先前有报道称，A422V对暗脉冲诱导的相位重置不太敏感，并且在体内具有KaiC磷酸化节律的振幅降低。A422映射到一个环（422环），它继续向磷酸化位点移动。通过拉动KaiC （a环）的c端肽，KaiA消除了相邻422环的约束，后者的灵活性增加间接促进了激酶活性。我们在晶体结构中发现，A422V KaiC在S431位点缺乏磷酸化，并且相对于野生型KaiC表现出微妙的局部构象变化。分子动力学模拟表明，在缺乏a环的情况下，422环的迁移率更高，并且野生型和突变型KaiCs在与磷酸化活性相关的其他区域的迁移率存在差异。通知KaiA二聚体结合的KaiC磷酸化位点的a -loop-422环接力从邻近的KaiC亚基传播到环，从而为协调一致的磷酸化变构机制提供支持。
The Synechococcus elongatus KaiA, KaiB, and KaiC proteins in the presence of ATP generate a post-translational oscillator that runs in a temperature-compensated manner with a period of 24 h. KaiA dimer stimulates T432 and S431, and KaiB dimers antagonize KaiA action and phosphorylation of KaiC hexamer at two sites per subunit, induce KaiC subunit exchange. Neither the mechanism of KaiA-stimulated KaiC phosphorylation nor that of KaiB-mediated KaiC dephosphorylation is understood in detail at present. We demonstrate here that the A422V KaiC mutant sheds light on the former mechanism. It was previously reported that A422V is less sensitive to dark pulse-induced phase resetting and has a reduced amplitude of the KaiC phosphorylation rhythm in vivo. A422 maps to a loop (422-loop) that continues toward the phosphorylation sites. By pulling on the C-terminal peptide of KaiC (A-loop), KaiA removes restraints from the adjacent 422-loop whose increased flexibility indirectly promotes kinase activity. We found in the crystal structure that A422V KaiC lacks phosphorylation at S431 and exhibits a subtle, local conformational change relative to wild-type KaiC. Molecular dynamics simulations indicate higher mobility of the 422-loop in the absence of the A-loop and mobility differences in other areas associated with phosphorylation activity between wild-type and mutant KaiCs. The A-loop-422-loop relay that informs KaiC phosphorylation sites of KaiA dimer binding propagates to loops from neighboring KaiC subunits, thus providing support for a concerted allosteric mechanism of phosphorylation.