Conformations and Dynamics of PED/PEA-15 in Protein-Protein Interactions

描述(由申请人提供)：PED/PEA-15(富含糖尿病的磷酸蛋白/富含星形胶质细胞的磷酸蛋白，15 kDa)是一种小的非催化蛋白，如何调控许多不同的基本细胞过程，包括细胞凋亡、细胞外调节激酶(ERK)途径和葡萄糖代谢，目前还存在一个根本的空白。这一差距阻碍了人们对PED/PEA-15在肿瘤细胞存活、增殖和迁移、化疗耐药以及糖尿病发展中的作用的理解。其长期目标是阐明PED/PEA-15调控远距离生物通路的机制。总体目标是确定费用的功能角色 三联体，D-RxDL，几乎所有死亡效应结构域(DED)上的一个特征，与ERK2结合。根据初步数据提出的假设是，PED/PEA-15上的电荷三联体基序保持了DED的基本灵活性，促进了与参与各种生物途径的蛋白质相互作用时所需的构象变化。这项研究的基本原理是，一旦PED/PEA-15和ERK之间的相互作用得到充分表征，就有可能开发新的方法来操纵这种相互作用，以抑制肿瘤细胞的增殖和侵袭，提高抗癌药物的疗效。该项目的两个具体目标是：1)表征PED/PEA-15与ERK2结合后的结构和构象；2)阐明PED/PEA-15 DED上的电荷三联体在识别ERK2中的作用。在第一个目标下，将使用结合实验化学位移和残基偶极耦合(RDC)数据的新计算方法来计算PED/PEA-15在ERK2络合物中的构象。PED/PEA-15和ERK2之间的结合界面也将使用新的核磁共振技术来表征，包括新开发的基于RDC的技术来监测微妙的构象变化。在第二个目标下，将对PED/PEA-15的ERK2结合缺陷D74A突变体和同源ERK2结合蛋白Vanishin/PEA-15b进行结构和动力学研究，以揭示电荷三联体对结合能力的作用。突变体和Vanishin/PEA-15b都破坏了电荷三联体，并在一定程度上丧失了蛋白质的柔韧性。具有R-CxDL三联体的Vanishin/PEA-15b结构预计更接近ERK2结合构象，而具有D-RxAL三联体的D74A突变体锁定在自由构象，无法结合ERK2。这项研究具有创新性，因为现在电荷三联体的结构特征与维护功能联系在一起 蛋白质的灵活性，而不是直接参与与其他蛋白质的结合界面。研究中还使用了几种新技术，包括申请人实验室正在开发的一种技术。这项研究具有重要意义，因为它有望纵向推进和扩大对ERK通路中蛋白质-蛋白质相互作用的理解，最终有可能开发出新的ERK活性干预策略，用于预防和治疗常见癌症。

DESCRIPTION (provided by applicant): There is a fundamental gap in understanding how PED/PEA-15 (phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocyte, 15 kDa), a small, non-catalytic protein, can regulate a number of distinct fundamental cellular processes, including apoptosis, extracellular regulated kinase (ERK) pathway, and glucose metabolism. This gap prevents the comprehension of the roles of PED/PEA-15 in tumor cell survival, proliferation and migration, resistant to chemotherapy, as well as the development of diabetes. The long-term goal is to elucidate the mechanisms of PED/PEA-15 in modulating remote biological pathways. The overall objective is to identify the functional roles of the charge triad, D-RxDL, a characteristic feature on almost all death effector domains (DEDs), of PED/PEA-15 in binding to ERK2. The hypothesis, formulated on the basis of preliminary data, is that the charge-triad motif on PED/PEA-15 maintains the essential flexibility of the DED, facilitating necessary conformational changes upon interacting with proteins involved in various biological pathways. The rationale for the proposed research is that, once the interaction between PED/PEA-15 and ERK is fully characterized, novel approaches to manipulate the interaction can likely be developed to curb tumor cell proliferation and invasion, enhancing the efficacy of anticancer drugs. The two specific aims of the project are: 1) Characterize the structure and conformation of PED/PEA-15 upon binding to ERK2; and 2) Illustrate roles of charge triad on PED/PEA-15 DED in recognition of ERK2. Under the first aim, the conformation of PED/PEA-15 in the complex with ERK2 will be solved using novel computational protocols incorporating experimental chemical shift and residue dipolar coupling (RDC) data. The binding interface between PED/PEA-15 and ERK2 will also be characterized using novel NMR techniques, including the newly developed RDC-based technique to monitor subtle conformational changes. Under the second aim, structural and dynamic studies of the ERK2-binding deficient D74A mutant of PED/PEA-15 and the homologous ERK2-binding protein vanishin/PEA-15b will be performed to delineate the roles of the charge triad on the binding capability. Both the mutant and vanishin/PEA-15b have disrupted charge-triad, and putatively lose the protein flexibility to some extent. Vanishin/PEA-15b structure, with R-CxDL triad, is expected more close to the ERK2-bound conformation, while the D74A mutant, with D-RxAL triad, is locked at the free-form conformation, and unable to bind ERK2. This research is innovative, because structural feature of charge triad is now linked to the function of maintaining the flexibility of the protein, instead of directly involved in the binding interface with other proteins. Several novel techniques, including the one being developed in the applicant's lab, are also used in the research. The proposed research is significant, because it is expected to vertically advance and expand understanding of protein-protein interactions in ERK pathway, which ultimately has the potential to develop novel intervening strategies of the ERK activity in the prevention and treatment of common cancers.