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.
|Effective start/end date||8/1/08 → 7/31/13|
- National Institute of General Medical Sciences: $303,129.00
- National Institute of General Medical Sciences: $300,045.00
- National Institute of General Medical Sciences: $295,857.00
- National Institute of General Medical Sciences: $306,243.00
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