Structure of DREAM a Calcium Sensor in Pain Control

描述(由申请人提供)：总体目标是阐明一种名为DREAM(下游调节元件拮抗剂调节剂)的钙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成员，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，仅在低钙水平下阻止下游靶标的转录。DREAM已被证明在基础钙水平抑制前强啡肽和c-fos基因的转录，但在细胞刺激或损伤引起的钙水平升高时不抑制转录。DREAM作为一种钙敏感转录抑制因子的重要性已在DREAM缺陷小鼠身上得到证实，这些小鼠由于前强啡肽表达上调而表现出非常显著的持续镇痛表型。重要的是，梦基因敲除小鼠没有表现出任何运动或行为异常。因此，DREAM通过控制前强啡肽的表达来调节疼痛的传递，并代表着一个有吸引力的治疗疼痛的机会。DREAM的原子级结构特征及其受钙离子调控的与靶DNA的相互作用对于开发通过特定靶向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DREAM在溶液中的C-末端钙结合结构域的结构。该结构对于了解钙诱导的EF-Hand基序内的构象变化如何控制DNA靶标的结合是重要的。第二个目标是测量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在针对梦想和止痛的药物的合理设计中增加结构数据。第三个目的是确定控制DNA靶标结合的DREAM的活性部位氨基酸残基。定点突变研究将确定重要的氨基酸残基和目标复合体的结构相互作用，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，然后将其用于通过X射线结晶学解决蛋白质-DNA复合体的原子分辨结构。

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.