Structure and Activation Mechanism of Ubiquitin Conjugating Enzymes

描述（由申请人提供）：polyUb链在底物蛋白上的组装需要泛素缀合酶E2和伴侣泛素蛋白连接酶E3的协同作用。已知RING-E3 gp78蛋白在ER介导的降解中与Ubc7起作用。我们已经用未锚定的K48特异性polyUb链合成表明，来自gp78的RING结构域是Ubc7中这种活性的有效激活剂。我们已经定义了环：E2结合界面的相互作用对溶液NMR测量和通过确定的2.2 A分辨率的晶体结构的工程蛋白质构建体，其中的gp78环融合的N-末端的Ubc7。这些研究揭示了界面相互作用的细节。我们手头上有一组RING结构域，它们是Ubc7，E2 - 25K或两者的激活剂。除了激活功能外，这些RING结构域可以分为四个主要的结构亚型。有了这些初步的结果，我们提出了实现一个详细的了解环介导的E2激活与以下具体目标：I。确定E2：RING结构域与RING结构域折叠的结构亚型形成的界面的性质。二.确定RING：E2界面中的个体成对相互作用如何有助于选择性识别和E2激活。三.绘制E2~Ub复合物中蛋白质间相互作用的图谱。公共卫生相关性：拟议的研究将导致对泛素途径中的蛋白质如何相互作用的新见解。这种途径已成功地用于开发Velcade，这是一种治疗骨髓瘤和淋巴瘤的抗癌药物。我们希望获得的知识将进一步提高这一药物开发途径的可及性。

DESCRIPTION (provided by applicant): The assembly of polyUb chains on substrate proteins requires the concerted action of a ubiquitin-conjugating enzyme E2 and a partner ubiquitin-protein ligase E3. The RING-E3 gp78 protein is known to function with Ubc7 in ER-mediated degradation. We have shown with unanchored K48-specific polyUb chain synthesis that the RING domain from gp78 is a potent activator of this activity in Ubc7. We have defined the RING:E2 binding interface of this interacting pair by solution NMR measurements and by determination of a 2.2 A resolution crystal structure of an engineered protein construct in which the gp78 RING is fused to the N-terminus of Ubc7. These studies reveal details of interaction at the interface. We have on hand a panel of RING domains that are activators of either Ubc7, E2-25K, or both. In addition to activation function, these RING domains can be segregated into four major structural subtypes. With these preliminary results, we proposed to achieve a detailed understanding of RING-mediated E2 activation with the following specific aims: I. To establish the nature of the E2:RING domain interface formed with structural subtypes of the RING domain fold. II. To determine how individual pair-wise interactions in a RING:E2 interface contribute to selective recognition and E2 activation. III. To map protein:protein interactions in E2~Ub complexes. Public Health Relevance: The proposed research will lead to novel insights on how proteins in the ubiquitin pathway interact with each other. This pathway has been successfully exploited in the development of Velcade, an anti-cancer drug to treat myeloma and lymphoma. We expect knowledge to be gained will further improve the accessibility of this pathway for drug development.