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

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 in complex with CHIP indicates conservation of the canonical E2/E3 interaction. E2∼ubiquitin conjugate formation assays and a mutant, isopeptide-linked E2∼ubiquitin conjugate structure demonstrate compatibility of the mutants with the E1 enzyme. Assays of CHIP auto-ubiquitination and CHIP-mediated ubiquitination of Hsp70 demonstrate full compatibility of the mutants with all components of the ubiquitination cascade. Thus, our structure-guided UbcH5b mutants retain native activity profiles and structures while improving the affinity for CHIP, thereby enabling future structural studies.