作为唯一一个可以利用ATP作为能量源在体外利用其组成蛋白KaiA、KaiB和KaiC重组的昼夜节律振荡器,蓝藻昼夜节律振荡器可作为昼夜节律时钟转换详细机制研究的模型系统。当KaiB与KaiC形成夜间复合物以隔离KaiA时,发生昼夜转换,后者在白天与KaiC相互作用以促进KaiC自身磷酸化。然而,尽管KaiB与六聚体KaiC结合的结构可用,但对KaiB如何与KaiC形成复合物仍知之甚少。据推测,KaiB-KaiC结合受KaiB间的协同性调节。
该文中,研究人员利用自旋标记连续波电子顺磁共振波谱,鉴定并量化了KaiC结合KaiB的两个亚群,对应于化学计量比和亚化学计量比KaiBiC6复合物(i=1–5)中的“块状”和“边缘”KaiBC位点。研究人员提供了动力学证据,证明“边缘”KaiBC位点作为游离KaiB和“大块”KaiBC位点之间的桥梁和成核位点的中介作用。此外,研究人员还表明,“边缘”和“大块”位点的相对丰度取决于KaiC磷酸态和KaiA,同时证明了磷酸化状态控制的KaiB间协同性的概念。最后,研究人员证明了KaiC结合的KaiB的两个亚群之间的相互转换与KaiC磷酸化循环密切相关。以上发现丰富了人们对蓝藻生物钟机制的理解,并证明了EPR在阐明生物钟机制方面的作用。
附:英文原文
Title: A Night-Time Edge Site Intermediate in the Cyanobacterial Circadian Clock Identified by EPR Spectroscopy
Author: Gary K. Chow, Archana G. Chavan, Joel Heisler, Yong-Gang Chang, Ning Zhang, Andy LiWang, R. David Britt
Issue&Volume: January 3, 2022
Abstract: As the only circadian oscillator that can be reconstituted in vitro with its constituent proteins KaiA, KaiB, and KaiC using ATP as an energy source, the cyanobacterial circadian oscillator serves as a model system for detailed mechanistic studies of day–night transitions of circadian clocks in general. The day-to-night transition occurs when KaiB forms a night-time complex with KaiC to sequester KaiA, the latter of which interacts with KaiC during the day to promote KaiC autophosphorylation. However, how KaiB forms the complex with KaiC remains poorly understood, despite the available structures of KaiB bound to hexameric KaiC. It has been postulated that KaiB-KaiC binding is regulated by inter-KaiB cooperativity. Here, using spin labeling continuous-wave electron paramagnetic resonance spectroscopy, we identified and quantified two subpopulations of KaiC-bound KaiB, corresponding to the “bulk” and “edge” KaiBC sites in stoichiometric and substoichiometric KaiBiC6 complexes (i = 1–5). We provide kinetic evidence to support the intermediacy of the “edge” KaiBC sites as bridges and nucleation sites between free KaiB and the “bulk” KaiBC sites. Furthermore, we show that the relative abundance of “edge” and “bulk” sites is dependent on both KaiC phosphostate and KaiA, supporting the notion of phosphorylation-state controlled inter-KaiB cooperativity. Finally, we demonstrate that the interconversion between the two subpopulations of KaiC-bound KaiB is intimately linked to the KaiC phosphorylation cycle. These findings enrich our mechanistic understanding of the cyanobacterial clock and demonstrate the utility of EPR in elucidating circadian clock mechanisms.
DOI: 10.1021/jacs.1c08103
Source: https://pubs.acs.org/doi/10.1021/jacs.1c08103
JACS:《美国化学会志》,创刊于1879年。隶属于美国化学会,最新IF:14.612
官方网址:https://pubs.acs.org/journal/jacsat
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