Structure and Activation Mechanism of Ubiquitin Conjugating Enzymes

描述(由申请人提供)：在底物蛋白上组装多聚Ub链需要泛素结合酶E2和伙伴泛素蛋白连接酶E3的协同作用。已知RING-E3gp78蛋白与Ubc7一起在内质网介导的降解中发挥作用。我们已经用未锚定的K48特异性PolyUb链合成表明，来自gp78的环域是Ubc7中这一活性的有效激活剂。我们通过溶液核磁共振测量和测定了gp78环与Ubc7的N-端融合的工程蛋白结构的2.2A分辨率晶体结构，定义了该相互作用对的环：E2结合界面。这些研究揭示了界面上相互作用的细节。我们手头有一组环状结构域，它们是Ubc7、E2-25K或两者的激活剂。除了激活功能外，这些环状结构域还可以分为四种主要的结构亚型。根据这些初步结果，我们建议实现对环介导的E2激活的详细了解，具体目标如下：1.建立E2的性质：由环结构域折叠的结构亚型形成的环结构域界面。II.确定环中单个成对相互作用：E2界面如何有助于选择性识别和E2激活。定位蛋白质：E_2~Ub复合体中的蛋白质相互作用。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。这种途径已经被成功地用于开发VELCADE，这是一种治疗骨髓瘤和淋巴瘤的抗癌药物。我们预计，所获得的知识将进一步改善药物开发这一途径的可及性。

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.