Pattanayek Rekha, Williams Dewight R, Pattanayek Sabuj, Xu Yao, Mori Tetsuya, Johnson Carl H, Stewart Phoebe L, Egli Martin
Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, TN 37232, USA.
EMBO J. 2006 May 3;25(9):2017-28. doi: 10.1038/sj.emboj.7601086. Epub 2006 Apr 20.
The cyanobacterial circadian clock can be reconstituted in vitro by mixing recombinant KaiA, KaiB and KaiC proteins with ATP, producing KaiC phosphorylation and dephosphorylation cycles that have a regular rhythm with a ca. 24-h period and are temperature-compensated. KaiA and KaiB are modulators of KaiC phosphorylation, whereby KaiB antagonizes KaiA's action. Here, we present a complete crystallographic model of the Synechococcus elongatus KaiC hexamer that includes previously unresolved portions of the C-terminal regions, and a negative-stain electron microscopy study of S. elongatus and Thermosynechococcus elongatus BP-1 KaiA-KaiC complexes. Site-directed mutagenesis in combination with EM reveals that KaiA binds exclusively to the CII half of the KaiC hexamer. The EM-based model of the KaiA-KaiC complex reveals protein-protein interactions at two sites: the known interaction of the flexible C-terminal KaiC peptide with KaiA, and a second postulated interaction between the apical region of KaiA and the ATP binding cleft on KaiC. This model brings KaiA mutation sites that alter clock period or abolish rhythmicity into contact with KaiC and suggests how KaiA might regulate KaiC phosphorylation.
通过将重组的KaiA、KaiB和KaiC蛋白与ATP混合，蓝藻生物钟可以在体外重建，产生具有约24小时周期的规则节律且温度补偿的KaiC磷酸化和去磷酸化循环。KaiA和KaiB是KaiC磷酸化的调节剂，其中KaiB拮抗KaiA的作用。在这里，我们展示了细长聚球藻KaiC六聚体的完整晶体学模型，该模型包括C端区域先前未解析的部分，以及对细长聚球藻和嗜热栖热菌BP-1 KaiA-KaiC复合物的负染色电子显微镜研究。定点诱变与电子显微镜相结合表明，KaiA仅与KaiC六聚体的CII半部分结合。基于电子显微镜的KaiA-KaiC复合物模型揭示了两个位点的蛋白质-蛋白质相互作用：柔性C端KaiC肽与KaiA的已知相互作用，以及KaiA顶端区域与KaiC上ATP结合裂隙之间的第二个假定相互作用。该模型使改变生物钟周期或消除节律性的KaiA突变位点与KaiC接触，并提出了KaiA可能调节KaiC磷酸化的方式。
