Structural Biology of the S. elongatus Circadian Clock

S. elongatus 昼夜节律钟的结构生物学

• 批准号: 8073572
• 负责人: MARTIN EGLI
• 金额: $ 32.69万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8073572
• 关键词: ATP phosphohydrolase; Active Sites; Address; Affect; Back; Behavior; Binding; Biochemical; Biochemical Reaction; Biological; Biological Assay; Biological Clocks; Biological Process; Cell physiology; Cells; Chromosomes; Circadian Rhythms; Classification; Clock protein; Complement; Complex; Coupled; Cryoelectron Microscopy; Cyanobacterium; Electron Microscopy; Event; Exhibits; Feedback; Financial compensation; Funding; Gene Expression; Gene Expression Regulation; Genetic; Genetic Screening; Homo; Hybrids; Imagery; In Vitro; Individual; Investigation; Knowledge; Laboratories; Length; Light; Maps; Mediating; Methods; Modeling; Molecular; Molecular Structure; Mutation; Neutrons; Nitrogen Fixation Genes; Organism; Periodicity; Phase; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Photosynthesis; Process; Production; Property; Protein Dephosphorylation; Proteins; Reporting; Research; Resolution; Roentgen Rays; Role; Site; Site-Directed Mutagenesis; Solutions; Structure; Synechococcus; System; Techniques; Temperature; Ticks; Time; X-Ray Crystallography; autophosphorylation-dependent multifunctional protein kinase; base; circadian pacemaker; dimer; exhaust; in vivo; mutant; programs; promoter; protein complex; protein function; protein protein interaction; protein structure; public health relevance; reconstitution; structural biology; three dimensional structure

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. PUBLIC HEALTH RELEVANCE: A comprehensive structure-based program to dissect the mechanism of the cyanobacterial KaiABC circadian clock, a highly attractive target for biochemical and biophysical studies due to the fact that it can be fully reconstituted in vitro from three proteins in the presence of ATP; the kinase, phosphatase, ATPase, and potentially ATP synthase activities and protein-protein interactions of the core clock protein KaiC will be probed using a combined mutagenic, functional and hybrid structural (X-ray, EM, SAXS, SANS) approach and the molecular origins of temperature compensation, a hallmark of all biological clocks will be determined.

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）节律。这些生物钟系统在恒定条件（恒定光照和温度）下生成精确的约 24 小时周期，该周期在不同温度下几乎不变（温度补偿）。昼夜节律时钟还显示白天和黑夜的夹带，主要由每日光/暗周期介导，因此内源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——很难用生化方法解释。充分了解这些不寻常的振荡器需要了解其分子成分的结构、功能和相互作用。哺乳动物生物钟极其复杂，需要多个相互连接的转录、翻译和翻译后反馈环路 (TTFL) 来实现具有昼夜节律周期性的基因表达。我们研究了原核蓝细菌（Synechococcus elongatus）的生物钟组成部分，它对许多过程进行编程以最佳地符合日常周期，包括光合作用、固氮和基因表达。蓝细菌的内源昼夜节律系统对包括全局基因表达在内的细胞过程进行普遍控制。事实上，整个染色体每天都会经历拓扑变化和压缩的循环。值得注意的是，在 ATP 存在的情况下，仅用三种蓝藻蛋白 KaiA、KaiB 和 KaiC 即可在体外重建昼夜节律振荡器的生化机制！这些蛋白质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 振荡器单向滴答的棘轮机制。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。该系统严格的结构、生物物理和生化方法的结合将揭示生物计时的分子机制。 KaiC 同六聚体形成时钟的中心齿轮，是一种自激酶和β磷酸酶以及一种 ATP 酶。 KaiA 二聚体增强 KaiC 磷酸化，KaiB 二聚体拮抗 KaiA 的作用。我们将使用混合结构技术剖析 KaiABC 时钟的机制，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测定。三个具体目标是（1）磷酸化位点（P位点）、磷酸化环和假定的磷酸酶活性位点突变体KaiC蛋白的结构和功能； (2) 使用wt和P位突变体KaiCs确定KaiAC、KaiBC和KaiABC复合物的结构，以优化蛋白质-蛋白质相互作用； (3)确定时钟温度补偿的分子起源。 公共健康相关性：一个基于结构的综合程序，用于剖析蓝藻 KaiABC 昼夜节律钟的机制，它是生物化学和生物物理研究中极具吸引力的目标，因为它可以在 ATP 存在的情况下在体外从三种蛋白质完全重建；将使用诱变、功能和混合结构（X 射线、EM、SAXS、SANS）相结合的方法来探测核心时钟蛋白 KaiC 的激酶、磷酸酶、ATP 酶和潜在的 ATP 合酶活性以及蛋白质-蛋白质相互作用，并将确定温度补偿的分子起源，这是所有生物钟的标志。