In this research, we delved into the intricate interactions within the CHIP-mediated ubiquitination pathway, focusing on the interplay among E1-ubiquitin-activating enzyme, E2-ubiquitin-conjugating enzyme (UbcH5b), and E3-ubiquitin ligase (CHIP) with Ubiquitin. The absence of available structures for CHIP in complexes with an E2~ubiquitin conjugate spurred our investigation, aiming to unravel the mechanistic insights and structure of the CHIP-E2-Ub complex. To address this, we developed a strategic plan. Stabile E2 mutants (M1K, D12R, C85K, and combinations) were engineered to identify variants binding strongly with both CHIP and ubiquitin. CD data analysis confirmed that all mutants maintained structural integrity, paving the way for downstream applications. In activity assays with SDS-PAGE, we probed which E2 mutant exhibited robust binding with ubiquitin. Notably, WT, M1K, and D12R mutants facilitated ubiquitin transfer to CHIP, while all C85K mutants exhibited impaired ubiquitin transfer. This led to the identification of C85K mutants as promising candidates for the desired three-protein complex. Further validation through Bio-Layer Interferometry (BLI) illuminated the E2 mutants’ interactions with CHIP. D12R C85K, M1K C85K, and M1K D12R C85K mutants emerged as the most favorable candidates with lower Kd values, indicating strong interactions with CHIP. Our findings support the candidacy of D12R C85K and M1K D12R C85K mutants for future structural studies. These insights not only address the challenge of cocrystallization but also contribute to understanding CHIP-mediated ubiquitination, holding implications for drug design in diseases associated with protein-protein interactions, such as neurodegenerative diseases and certain cancer types. Future directions include exploring disease mutations and their impact on the interactions of these crucial proteins.
Author: Jack Griffin, Maleesha Manage, Richard C. Page
Advisors: Richard C. Page, Department of Chemistry and Biochemistry
Meleesha Manage, Department of Chemistry and Biochemistry
You must be logged in to post a comment.