Pattanayek Rekha, Williams Dewight R, Pattanayek Sabuj, Mori Tetsuya, Johnson Carl H, Stewart Phoebe L, Egli Martin
Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, TN 37232, USA.
EMBO J. 2008 Jun 18;27(12):1767-78. doi: 10.1038/emboj.2008.104. Epub 2008 May 22.
The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro by the KaiA, KaiB and KaiC proteins in the presence of ATP. The principal clock component, KaiC, undergoes regular cycles between hyper- and hypo-phosphorylated states with a period of ca. 24 h that is temperature compensated. KaiA enhances KaiC phosphorylation and this enhancement is antagonized by KaiB. Throughout the cycle Kai proteins interact in a dynamic manner to form complexes of different composition. We present a three-dimensional model of the S. elongatus KaiB-KaiC complex based on X-ray crystallography, negative-stain and cryo-electron microscopy, native gel electrophoresis and modelling techniques. We provide experimental evidence that KaiB dimers interact with KaiC from the same side as KaiA and for a conformational rearrangement of the C-terminal regions of KaiC subunits. The enlarged central channel and thus KaiC subunit separation in the C-terminal ring of the hexamer is consistent with KaiC subunit exchange during the dephosphorylation phase. The proposed binding mode of KaiB explains the observation of simultaneous binding of KaiA and KaiB to KaiC, and provides insight into the mechanism of KaiB's antagonism of KaiA.
在ATP存在的情况下，细长聚球藻的生物钟可通过KaiA、KaiB和KaiC蛋白在体外重建。生物钟的主要成分KaiC会在超磷酸化状态和低磷酸化状态之间进行有规律的循环，周期约为24小时，且具有温度补偿特性。KaiA会增强KaiC的磷酸化，而这种增强作用会被KaiB拮抗。在整个循环过程中，Kai蛋白以动态方式相互作用，形成不同组成的复合物。我们基于X射线晶体学、负染和冷冻电子显微镜、天然凝胶电泳及建模技术，提出了细长聚球藻KaiB-KaiC复合物的三维模型。我们提供了实验证据，证明KaiB二聚体与KaiC的相互作用面与KaiA相同，且会导致KaiC亚基C端区域的构象重排。六聚体C端环中扩大的中央通道以及由此导致的KaiC亚基分离与去磷酸化阶段的KaiC亚基交换一致。所提出的KaiB结合模式解释了KaiA和KaiB同时与KaiC结合的现象，并为KaiB对KaiA的拮抗机制提供了见解。
