Structure of DREAM a Calcium Sensor in Pain Control

描述（由申请人提供）：总体目标是阐明名为DREAM（下游调节元件拮抗剂调节剂）的Ca 2+敏感性转录阻遏蛋白的结构和作用机制。DREAM是EF-手超家族的神经元钙传感器亚类的成员，在脑中表达，在脑中它结合启动子区下游的特异性DNA序列（DRE，下游调节元件），并仅在低Ca 2+水平下阻断下游靶点的转录。DREAM已被证明在基础Ca 2+水平下抑制强啡肽原和c-fos基因的转录，但在由细胞刺激或损伤引起的升高的Ca 2+水平下不抑制转录。DREAM作为钙敏感性转录抑制因子的重要性已在DREAM缺陷小鼠中得到证实，DREAM缺陷小鼠由于强啡肽原表达上调而表现出非常显著的持续镇痛表型。重要的是，DREAM基因敲除小鼠没有表现出任何运动或行为异常。因此，DREAM通过控制强啡肽原的表达来调节疼痛的传递，并代表了一个有吸引力的治疗疼痛的机会。DREAM的原子水平结构表征及其与靶DNA的Ca 2+调节的相互作用对于开发旨在通过特异性靶向DREAM来消除疼痛的疗法至关重要。具体目标有4个：第一个目标是通过核磁共振（NMR）确定溶液中DREAM的C-末端钙结合结构域的结构。该结构对于理解EF-手基序内钙诱导的构象变化如何控制DNA靶的结合是重要的。第二个目标是测量DREAM与钙和DNA靶点结合的能量学和动力学。结合的热力学和动力学将增加靶向DREAM和阻滞疼痛的药物的合理设计中的结构数据。第三个目标是确定DREAM的活性位点氨基酸残基，这些氨基酸残基控制DNA靶点的结合。定点诱变研究将确定重要的氨基酸残基和结构相互作用的目标复合物，并可能揭示结构决定因素的重要药物设计。第四个目标是获得高质量的DREAM蛋白质晶体，然后将其用于通过X射线晶体学解决蛋白质-DNA复合物的原子分辨率结构。

DESCRIPTION (provided by applicant): The overall objectives are to elucidate the structure and mechanism of action of a Ca2+-sensitive transcriptional repressor protein named, DREAM (Downstream Regulatory Element Antagonist Modulator). DREAM, a member of the neuronal calcium sensor subclass of the EF-hand super family, is expressed in the brain where it binds to specific DNA sequences downstream of the promoter region (DRE, Downstream Regulatory Element) and blocks transcription of downstream targets only at low Ca2+ levels. DREAM has been shown to suppress the transcription of prodynorphin and c-fos genes at basal Ca2+ levels, but does not suppress transcription at elevated Ca2+ levels caused by cell stimulation or injury. The importance of DREAM as a calcium-sensitive transcriptional repressor has been demonstrated in DREAM-deficient mice that exhibit the very striking phenotype of ongoing analgesia due to upregulated expression of prodynorphin. Importantly, the DREAM knockout mice do not exhibit any motor or behavioral abnormalities. Hence, DREAM regulates pain transmission by controlling prodynorphin expression and represents an attractive therapeutic opportunity for managing pain. The atomic-level structural characterization of DREAM and its Ca2+-regulated interaction with target DNA are crucial for the development of therapeutics designed to eliminate pain by specifically targeting DREAM. The specific aims are 4-fold: The first aim is to determine the structure of the C-terminal calcium-binding domain of DREAM in solution by nuclear magnetic resonance (NMR). The structure is important for understanding how calcium-induced conformational changes within the EF-hand motifs control the binding of DNA targets. The second aim is to measure the energetics and kinetics of DREAM binding to calcium and DNA targets. The thermodynamics and kinetics of binding will augment the structural data in the rational design of drugs that target DREAM and block pain. The third aim is to determine the active-site amino acid residues of DREAM that control binding of DNA targets. Site directed mutagenesis studies will identify important amino acid residues and structural interactions of the target complex and may reveal structural determinants important for drug design. The fourth aim is to obtain high quality protein crystals of DREAM that will then be used to solve the atomic resolution structure of the protein-DNA complex by x-ray crystallography.