Structure and Activation Mechanism of Ubiquitin Conjugating Enzymes

描述（由申请人提供）：polyb链在底物蛋白上的组装需要泛素偶联酶E2和伙伴泛素蛋白连接酶E3的协同作用。已知RING-E3 gp78蛋白在er介导的降解中与Ubc7一起起作用。我们已经通过无锚定的k48特异性聚脲链合成表明，gp78的RING结构域是Ubc7中这种活性的有效激活剂。我们通过溶液核磁共振测量确定了这对相互作用对的RING:E2结合界面，并通过测定一个工程蛋白结构的2.2 a分辨率晶体结构，其中gp78环融合到Ubc7的n端。这些研究揭示了界面上相互作用的细节。我们手头有一组RING结构域，它们是Ubc7、E2-25K或两者的激活剂。除了激活功能外，RING结构域还可以分为四种主要的结构亚型。基于这些初步结果，我们提出了对RING介导的E2激活的详细了解，具体目的如下：1 .建立由RING结构域褶皱结构亚型形成的E2:RING结构域界面的性质。2。确定RING:E2界面中个体对相互作用如何促进选择性识别和E2激活。3。绘制蛋白质图谱：E2~Ub复合物中的蛋白质相互作用。公共卫生相关性：拟议的研究将导致关于泛素途径中蛋白质如何相互作用的新见解。这一途径已被成功利用于Velcade的开发中，Velcade是一种治疗骨髓瘤和淋巴瘤的抗癌药物。我们期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.