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（下游调控元件拮抗剂调节剂）的Ca2+敏感转录抑制蛋白的结构和作用机制。DREAM是EF-hand超家族神经元钙传感器亚类的一员，在大脑中表达，它与启动子区域下游的特定DNA序列（DRE，下游调控元件）结合，仅在低Ca2+水平时阻断下游靶标的转录。DREAM已被证明在基础Ca2+水平下抑制prodynorphin和c-fos基因的转录，但在细胞刺激或损伤引起的Ca2+水平升高时不抑制转录。DREAM作为钙敏感的转录抑制因子的重要性已经在DREAM缺乏的小鼠中得到证实，这些小鼠由于前啡肽的表达上调而表现出非常惊人的持续镇痛表型。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通过控制促啡肽的表达来调节疼痛的传递，为控制疼痛提供了一个有吸引力的治疗机会。DREAM的原子水平结构表征及其Ca2+调控与靶DNA的相互作用对于通过特异性靶向DREAM来消除疼痛的治疗方法的发展至关重要。具体目的有四个方面：第一个目的是通过核磁共振（NMR）测定溶液中DREAM的c端钙结合结构域的结构。该结构对于理解钙诱导的EF-hand基序内的构象变化如何控制DNA靶标的结合非常重要。第二个目的是测量DREAM与钙和DNA靶标结合的能量学和动力学。结合的热力学和动力学将为合理设计靶向DREAM和阻断疼痛的药物增加结构数据。第三个目的是确定DREAM控制DNA靶标结合的活性位点氨基酸残基。位点定向诱变研究将确定重要的氨基酸残基和靶复合物的结构相互作用，并可能揭示对药物设计重要的结构决定因素。第四个目标是获得高质量的DREAM蛋白质晶体，然后通过x射线晶体学用于解决蛋白质- 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.