Structural Biology of the S. elongatus Circadian Clock

描述（由申请人提供）：许多生物过程经历由自我维持的生化振荡器决定的每日（昼夜）节律。这些生物钟系统在恒定的条件下（恒定的光和温度）产生精确的~24小时周期，在不同的温度下几乎不变（温度补偿）。生物钟也显示出昼夜的影响，主要由每日的光/暗周期介导，因此内源性生物钟与环境周期相适应。这些特性——尤其是周期的长时间常数和温度补偿——很难用生物化学的方法来解释。充分了解这些不寻常的振荡器将需要了解其分子成分的结构，功能和相互作用。哺乳动物的生物钟极其复杂，需要多个相互连接的转录、翻译和翻译后反馈回路（TTFLs）来实现具有昼夜周期的基因表达。我们研究了原核蓝藻——长聚球菌（Synechococcus elongatus）生物钟的组成部分，它对包括光合作用、固氮和基因表达在内的许多过程进行了优化，以符合日常循环。蓝藻的内源性昼夜节律系统对包括全球基因表达在内的细胞过程具有普遍的控制。事实上，整个染色体每天都在经历拓扑变化和压缩的循环。值得注意的是，这种昼夜节律振荡器背后的生化机制可以在体外用三种蓝藻蛋白（KaiA， KaiB和KaiC）在ATP的存在下重建！这些蛋白相互作用促进构象变化和磷酸化事件，从而决定体外振荡的阶段。这些蛋白质的高分辨率结构表明，KaiABC振荡器存在一种单向摆动机制。这种翻译后振荡器可能与TTFL相互作用，在体内产生紧急的昼夜节律行为。结合严谨的结构、生物物理和生物化学方法来研究这个系统，将揭示生物计时的分子机制。KaiC同属六聚体构成生物钟的中心齿轮，是一种自激酶和-磷酸酶以及三磷酸腺苷酶。KaiA二聚体增强KaiA磷酸化，KaiB二聚体拮抗KaiA的作用。我们将使用混合结构技术，包括x射线晶体学，电子显微镜（EM），小角度x射线和中子散射（分别为SAXS和SANS），一系列生物物理和生化方法以及体内和体外功能分析来剖析KaiABC时钟的机制。三个具体目标是：(1)磷酸化位点（p位点）、磷酸化环和推定的磷酸酶活性位点突变KaiC蛋白的结构和功能；(2)利用wt和p位点突变型KaiCs对KaiAC、KaiBC和KaiABC复合物进行结构测定，优化蛋白-蛋白相互作用；(3)确定时钟温度补偿的分子起源。

DESCRIPTION (provided by applicant): Many biological processes undergo daily (circadian) rhythms that are dictated by self-sustained biochemical oscillators. These circadian clock systems generate a precise ~24 h period in constant conditions (constant light and temperature) that is nearly invariant at different temperatures (temperature compensation). Circadian clocks also show entrainment to day and night, predominantly mediated by the daily light/dark cycle, so that the endogenous biological clock is phased appropriately to the environmental cycle. These properties - especially the period's long time constant and temperature compensation - are difficult to explain biochemically. Full understanding of these unusual oscillators will require knowledge of the structures, functions, and interactions of their molecular components. Mammalian clocks are exceedingly complex and require several interconnecting transcriptional, translational and post-translational feedback loops (TTFLs) to achieve gene expression with circadian periodicity. We study the components of the biological clock in the prokaryotic cyanobacterium, Synechococcus elongatus, which programs many processes to conform optimally to the daily cycle, including photosynthesis, nitrogen fixation, and gene expression. The endogenous circadian system in cyanobacteria exerts pervasive control over cellular processes including global gene expression. Indeed, the entire chromosome undergoes daily cycles of topological changes and compaction. Remarkably, the biochemical machinery underlying this circadian oscillator can be reconstituted in vitro with just three cyanobacterial proteins, KaiA, KaiB, and KaiC in the presence of ATP! These proteins interact to promote conformational changes and phosphorylation events that determine the phase of the in vitro oscillation. The high-resolution structures of these proteins suggest a racheting mechanism by which the KaiABC oscillator ticks unidirectionally. This post-translational oscillator may interact with a TTFL to generate the emergent circadian behavior in vivo. The conjunction of rigorous structural, biophysical, and biochemical approaches to this system will reveal molecular mechanisms of biological timekeeping. The KaiC homo- hexamer forms the central cog of the clock and is an auto-kinase and -phosphatase and an ATPase. The KaiA dimer enhances KaiC phosphorylation and KaiB dimers antagonize KaiA's action. We will dissect the mechanism of the KaiABC clock using hybrid structural techniques, including X-ray crystallography, electron microscopy (EM), small angle X-ray and neutron scattering (SAXS and SANS, respectively), a range of biophysical and biochemical approaches as well as functional assays in vivo and in vitro. The three specific aims are (1) Structure and function of phosphorylation site (P-site), phosphorylation loop and putative phosphatase active-site mutant KaiC proteins; (2) Structure determinations of KaiAC, KaiBC and KaiABC complexes using both wt and P-site mutant KaiCs for optimization of protein-protein interactions; and (3) Determination of the molecular origins of the clock's temperature compensation.