Structural Biology of the S. elongatus Circadian Clock

S. elongatus 昼夜节律钟的结构生物学

• 批准号: 8249840
• 负责人: MARTIN EGLI
• 金额: $ 32.74万
• 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/8249840
• 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。这些生物钟系统在恒定条件（恒定光照和温度）下产生精确的~24小时周期，在不同温度下几乎不变（温度补偿）。昼夜节律钟也显示出对白天和黑夜的夹带，主要由每日光/暗周期介导，使得内源性生物钟适当地定相于环境周期。这些特性--尤其是周期的长时间常数和温度补偿--很难用生物化学方法来解释。要充分了解这些不寻常的振荡器，就需要了解其分子组成部分的结构、功能和相互作用。哺乳动物生物钟非常复杂，需要几个相互连接的转录、翻译和翻译后反馈环（TTFL）来实现具有昼夜节律周期性的基因表达。我们研究了原核蓝藻，细长聚球藻，程序的许多过程，以符合最佳的日常周期，包括光合作用，固氮和基因表达的生物钟的组成部分。蓝藻的内源性昼夜节律系统对包括全局基因表达在内的细胞过程施加普遍的控制。事实上，整个染色体每天都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ATP存在的情况下用三种蓝藻蛋白质KaiA，KaiB和KaiC在体外重建！这些蛋白质相互作用以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振荡器单向滴答的一种棘轮机制。这种翻译后振荡器可能与TTFL相互作用，以产生体内的紧急昼夜节律行为。结合严格的结构，生物物理和生物化学的方法，这个系统将揭示生物计时的分子机制。KaiC同源六聚体形成时钟的中心齿轮，并且是自激酶和磷酸酶以及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X射线和中子散射（SAXS和SANS，分别），一系列生物物理和生物化学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），磷酸化环和推定的磷酸酶活性位点突变KaiC蛋白的结构和功能;（2）使用wt和P-位点突变KaiC优化蛋白质-蛋白质相互作用的KaiAC，KaiBC和KaiABC复合物的结构测定;和（3）确定时钟温度补偿的分子起源。 公共卫生相关性：一个全面的基于结构的计划，解剖蓝藻KaiABC昼夜节律钟的机制，这是一个非常有吸引力的生物化学和生物物理研究的目标，因为它可以在ATP存在的情况下从三种蛋白质在体外完全重建;激酶，磷酸酶，ATP酶，并且可能的核心时钟蛋白KaiC的ATP合酶活性和蛋白质-蛋白质相互作用将使用组合的诱变剂，功能和混合结构（X射线，EM，SAXS，SANS）的方法和温度补偿的分子起源，所有生物钟的标志将被确定。