Circadian rhythms play an essential role in many biological processes and surprisingly only three prokaryotic proteins are required to constitute a true post-translational circadian oscillator. The evolutionary history of the three Kai proteins indicates that KaiC is the oldest member and central component of the clock, with subsequent additions of KaiB and KaiA to regulate its phosphorylation state for time synchronization. The canonical KaiABC system in cyanobacteria is well understood, but little is known about more ancient systems that possess just KaiBC, except for reports that they might exhibit a basic, hourglass-like timekeeping mechanism. Here, we investigate the primordial circadian clock in Rhodobacter sphaeroides (RS) that contains only KaiBC to elucidate its inner workings despite the missing KaiA. Using a combination X-ray crystallography and cryo-EM we find a novel dodecameric fold for KaiCRS where two hexamers are held together by a coiled-coil bundle of 12 helices. This interaction is formed by the C-terminal extension of KaiCRS and serves as an ancient regulatory moiety later superseded by KaiA. A coiled-coil register shift between daytime- and nighttime-conformations is connected to the phosphorylation sites through a long-range allosteric network that spans over 160 Å. Our kinetic data identify the difference in ATP-to-ADP ratio between day and night as the environmental cue that drives the clock and further unravels mechanistic details that shed light on the evolution of self-sustained oscillators.

中文翻译：

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从原始时钟到昼夜节律振荡器

昼夜节律在许多生物过程中起着重要作用，令人惊讶的是，只需要三种原核蛋白质即可构成真正的翻译后昼夜节律振荡器。三种 Kai 蛋白的进化历史表明，KaiC 是时钟中最古老的成员和核心成分，随后添加了 KaiB 和 KaiA 来调节其磷酸化状态以实现时间同步。蓝藻中的规范 KaiABC 系统已广为人知，但对仅拥有 KaiBC 的更古老系统知之甚少，除了有报道称它们可能表现出一种基本的、类似沙漏的计时机制。在这里，我们调查了仅包含 KaiBC 的球形红细菌 (RS) 中的原始生物钟，以阐明其内部运作机制，尽管缺少 KaiA。结合使用 X 射线晶体学和低温 EM，我们发现了 KaiCRS 的新型十二聚体折叠，其中两个六聚体由 12 个螺旋的盘绕螺旋束连接在一起。这种相互作用由 KaiCRS 的 C 端延伸形成，并作为一种古老的调节部分，后来被 KaiA 取代。白天和夜间构象之间的盘绕线圈寄存器转换通过跨越 160 Å 的远程变构网络连接到磷酸化位点。我们的动力学数据将昼夜 ATP 与 ADP 比率的差异确定为驱动时钟的环境线索，并进一步揭示了揭示自持振荡器演变的机制细节。这种相互作用由 KaiCRS 的 C 端延伸形成，并作为一种古老的调节部分，后来被 KaiA 取代。白天和夜间构象之间的盘绕线圈寄存器转换通过跨越 160 Å 的远程变构网络连接到磷酸化位点。我们的动力学数据将昼夜 ATP 与 ADP 比率的差异确定为驱动时钟的环境线索，并进一步揭示了揭示自持振荡器演变的机制细节。这种相互作用由 KaiCRS 的 C 端延伸形成，并作为一种古老的调节部分，后来被 KaiA 取代。白天和夜间构象之间的盘绕线圈寄存器转换通过跨越 160 Å 的远程变构网络连接到磷酸化位点。我们的动力学数据将昼夜 ATP 与 ADP 比率的差异确定为驱动时钟的环境线索，并进一步揭示了揭示自持振荡器演变的机制细节。