Elucidating binding modes of BRCT-modules

阐明 BRCT 模块的结合模式

• 批准号: 8798620
• 负责人: Jamaine S Davis
• 金额: $ 14.4万
• 依托单位: MEHARRY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-15 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8798620
• 关键词: Binding; Biochemical; C-terminal; Cancer Etiology; Cell Survival; Complex; Computer software; Crystallization; DNA Damage; DNA Repair; Data; Defect; Fostering; Genome Stability; Goals; Health; In Vitro; Individual; Investigation; Knowledge; Length; Maintenance; Malignant Neoplasms; Mediating; Mission; Molecular; Nuclear Protein; Outcome; Pathway interactions; Peptides; Phosphopeptides; Phosphoserine; Play; Positioning Attribute; Predisposition; Protein Binding; Proteins; Public Health; Regulation; Repair Complex; Research; Resolution; Roentgen Rays; Role; Serine; Signal Transduction; Specificity; Structure; Substrate Specificity; Surface Plasmon Resonance; Testing; Threonine; Transactivation; Work; analytical ultracentrifugation; base; improved; in vivo; innovation; insight; irradiation; killings; malignant breast neoplasm; neoplastic cell; novel; p53-binding protein 1; preference; programs; protein protein interaction; research study; response; screening; tumor

DESCRIPTION (provided by applicant): There is a fundamental gap in our understanding of how mechanisms of phosphoserine binding modules by BRCT domains enable the assembly of multiprotein DNA repair complexes. Overlooking this gap is an important problem because, until it is solved, understanding how defects in the regulation of DNA repair can potentially cause cancer will remain obscure. The long-term goal is to understand how full-length PTIP regulates the DNA damage response pathway. The objective of this particular application is to provide a molecular description of how PTIP-(BRCT)4 interactions help to regulate DNA repair. The central hypothesis is that (BRCT)4, is able to recognize a broader spectrum of phosphopeptide sequence motifs, that were initially determined, and it can mediate interactions with other BRCT-domain containing proteins. This hypothesis has been formulated based on preliminary data obtained in the applicant's lab. The rationale for the proposed research is that, once it is known how PTIP interactions relay DNA damage signals to regulate DNA repair in tumor cells, we can identify agents that disrupt these interactions thereby making them more sensitive to chemo- and irradiation therapies. This hypothesis will be tested by investigation of three aims: 1) determination of the X-ray crystal structure of the tandem pairs of C-terminal BRCT domains; 2) determination of the structural basis of substrate specificity of the tandem pair of BRCT domains; 3) determination of the mechanism of substrate preference of the tandem pairs of BRCT domains. Within the first aim, native crystals diffract to 2.8 A and the heavy atom derivative diffracts to 3.2 A. Automated software programs will be used to solve the structure. Within the second aim, we have purified sufficient amounts of protein for biochemical and structural investigations and will begin crystallization screening and direct peptide binding experiments using surface Plasmon resonance (SPR). Lastly, within the third aim, peptide arrays will be conducted to identify novel sequences recognized by (BRCT)4, and confirmed with SPR. This approach is innovative, in my opinion, because it will be the first comprehensive study in the field focused on the importance of four consecutive BRCT domains. The proposed research is significant, because it will elucidate the differences in binding mechanisms among BRCT domains, and fundamentally expand our understanding of protein recognition with BRCT-domain containing proteins. Ultimately, this knowledge can identify agents that disrupt BRCT domain interactions and thereby make tumor cells more sensitive to killing by chemo- and irradiation therapies.

描述（由申请人提供）：我们对磷酸丝氨酸结合模块通过BRCT结构域的机制如何能够组装多蛋白DNA修复复合物的理解存在根本性差距。忽视这一差距是一个重要的问题，因为在解决这一问题之前，理解DNA修复调控中的缺陷如何可能导致癌症仍然是一个模糊的问题。长期目标是了解全长PTIP如何调节DNA损伤反应途径。该特定应用的目的是提供PTIP-（BRCT）4相互作用如何帮助调节DNA修复的分子描述。中心假设是（BRCT）4能够识别最初确定的更广谱的磷酸肽序列基序，并且它可以介导与其他含有BRCT结构域的蛋白质的相互作用。该假设是根据申请人实验室获得的初步数据制定的。这项研究的基本原理是，一旦知道PTIP相互作用如何传递DNA损伤信号来调节肿瘤细胞中的DNA修复，我们就可以确定破坏这些相互作用的药物，从而使它们对化疗和放疗更敏感。 该假设将通过三个目的的研究来检验：1）确定C-末端BRCT结构域的串联对的X射线晶体结构; 2）确定BRCT结构域的串联对的底物特异性的结构基础; 3）确定BRCT结构域的串联对的底物偏好性的机制。在第一个目标内，天然晶体衍射到2.8 A，重原子衍生物衍射到3.2 A。自动化软件程序将用于解决结构。在第二个目标中，我们已经纯化了足够量的蛋白质用于生化和结构研究，并将开始结晶筛选和使用表面等离子体共振（SPR）进行直接肽结合实验。最后，在第三个目标中，将进行肽阵列以鉴定由（BRCT）4识别的新序列，并用SPR确认。在我看来，这种方法是创新的，因为它将是该领域第一个集中在四个连续BRCT领域重要性的综合研究。这项研究具有重要意义，因为它将阐明BRCT结构域之间结合机制的差异，并从根本上扩展我们对含BRCT结构域蛋白质识别的理解。最终，这些知识可以识别破坏BRCT结构域相互作用的药物，从而使肿瘤细胞对化疗和放疗的杀伤更敏感。