Division of Biological Science, Graduate School of Science and Institute for Advanced Studies, Nagoya University, 464-8602 Nagoya, Japan;
Research Center of Integrative Molecular Systems, Institute for Molecular Science, National Institute of Natural Sciences, 444-8585 Okazaki, Japan.
Proc Natl Acad Sci U S A. 2020 Aug 25;117(34):20926-20931. doi: 10.1073/pnas.2005496117. Epub 2020 Aug 3.
The circadian clock of cyanobacteria consists of only three clock proteins, KaiA, KaiB, and KaiC, which generate a circadian rhythm of KaiC phosphorylation in vitro. The adenosine triphosphatase (ATPase) activity of KaiC is the source of the 24-h period and temperature compensation. Although numerous circadian mutants of KaiC have been identified, the tuning mechanism of the 24-h period remains unclear. Here, we show that the circadian period of in vitro phosphorylation rhythm of mutants at position 402 of KaiC changed dramatically, from 15 h (0.6 d) to 158 h (6.6 d). The ATPase activities of mutants at position 402 of KaiC, without KaiA and KaiB, correlated with the frequencies (1/period), indicating that KaiC structure was the source of extra period change. Despite the wide-range tunability, temperature compensation of both the circadian period and the KaiC ATPase activity of mutants at position 402 of KaiC were nearly intact. We also found that in vivo and in vitro circadian periods and the KaiC ATPase activity of mutants at position 402 of KaiC showed a correlation with the side-chain volume of the amino acid at position 402 of KaiC. Our results indicate that residue 402 is a key position of determining the circadian period of cyanobacteria, and it is possible to dramatically alter the period of KaiC while maintaining temperature compensation.
蓝藻的生物钟仅由三种生物钟蛋白组成，分别是 KaiA、KaiB 和 KaiC，它们在体外产生 KaiC 磷酸化的昼夜节律。KaiC 的三磷酸腺苷酶（ATPase）活性是 24 小时周期和温度补偿的来源。尽管已经鉴定出许多 KaiC 的生物钟突变体，但 24 小时周期的调谐机制仍不清楚。在这里，我们表明 KaiC 位置 402 的突变体在体外磷酸化节律的生物钟周期发生了巨大变化，从 15 小时（0.6 天）变为 158 小时（6.6 天）。没有 KaiA 和 KaiB 的 KaiC 位置 402 的突变体的 ATPase 活性与频率（1/周期）相关，表明 KaiC 结构是产生额外周期变化的来源。尽管具有广泛的可调性，但 KaiC 位置 402 的突变体的生物钟周期和 KaiC ATPase 活性的温度补偿几乎保持完整。我们还发现，KaiC 位置 402 的突变体的体内和体外生物钟周期以及 KaiC ATPase 活性与 KaiC 位置 402 的氨基酸侧链体积相关。我们的结果表明，残基 402 是决定蓝藻生物钟周期的关键位置，在保持温度补偿的同时，有可能显著改变 KaiC 的周期。
