Functional and structural analyses of UbcH5 mutants with enhanced binding to the E3 ubiquitin ligase CHIP.

ABSTRACT: The E3 ubiquitin ligase CHIP ubiquitinates substrates in chaperone-dependent or -independent manners. Structural studies, particularly by cryo-electron microscopy, would aid in understanding the mechanisms governing CHIP-mediated ubiquitination. Key among necessary components is the E2 enzyme UbcH5b, which facilitates the transfer of ubiquitin from the E2∼ubiquitin conjugate to a target lysine residue. However, the affinity of CHIP for UbcH5b is approximately 4 μM, presenting a challenge for cryo-electron microscopy, which is typically conducted at concentrations below 5 μM. Herein, we report structure-guided UbcH5b mutants that substantially improve the affinity for CHIP. Bio-layer interferometry demonstrates a ten-fold improvement in affinity, while our crystal structure of mutant UbcH5b

SUBMITTER: Manage MM 

PROVIDER: S-EPMC12593235 | biostudies-literature | 2025 Nov

REPOSITORIES: biostudies-literature