{"doi":"10.1016/j.bbrc.2025.152873","title":"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 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.","journal":"Biochemical and Biophysical Research Communications","year":2025,"id":548021,"datarank":0.10397207708399181,"base_score":0.6931471805599453,"endowment":0.6931471805599453,"self_citation_contribution":0.10397207708399181,"citation_network_contribution":0.0,"self_endowment_contribution":0.10397207708399181,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":1,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.958,"is_data_producer":true,"deposit_databanks":{"PDB":["9YE9","9YEA"]},"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":218048,"name":"Jay C. Nix","orcid":"0000-0002-4041-4975","position":1,"is_corresponding":false},{"id":353430,"name":"Richard C. Page","orcid":"0000-0002-3006-3171","position":2,"is_corresponding":false},{"id":1440771,"name":"Maleesha Marakkala Manage","orcid":"0009-0006-4701-0793","position":0,"is_corresponding":true}],"reference_count":32,"raw_metadata":null,"created_at":"2026-07-19T02:53:53.962932Z","pmid":"41167006","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}