{"doi":"10.1016/j.jbc.2025.110910","title":"A modular Uba1-nanobody fusion enables selective ubiquitin transfer to tagged E2 enzymes","abstract":"The ubiquitin-activating enzyme Uba1 initiates the ubiquitination cascade by activating ubiquitin and subsequently transferring it to a broad range of E2 conjugating enzymes via its C-terminal ubiquitin-fold domain. Tools to selectively redirect this transfer to defined E2s, and thus govern control of E2 enzyme usage, are limited. By replacing the ubiquitin-fold domain of Uba1 with the high-affinity nanobody VHH05, we create an engineered E1 enzyme (Uba1-VHH05) that selectively engages E2s fused to the 6e-tag, which is the epitope recognized by VHH05. This plug-and-play interface preserves native Uba1 catalytic activity while allowing ubiquitin loading to be directed toward user-defined E2s without altering other components of the cascade. We show that Uba1-VHH05 supports transfer of both WT ubiquitin and the activity-based probe Ub-Dha to a range of tagged E2s. This recapitulates endogenous in vitro activities, including polyubiquitin chain formation. This strategy enables precise dissection of E2-specific functions and offers a new tool to generate orthogonal ubiquitin cascades in vitro and ultimately in cells.","journal":"Journal of Biological Chemistry","year":2025,"id":580170,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9513,"is_data_producer":false,"deposit_databanks":null,"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":971920,"name":"Francesca D’Amico","orcid":"0000-0002-7104-2158","position":1,"is_corresponding":false},{"id":1490992,"name":"David A. Pérez Berrocal","orcid":"0000-0003-0472-2601","position":2,"is_corresponding":false},{"id":347199,"name":"Monique P. C. Mulder","orcid":"0000-0001-5386-7132","position":3,"is_corresponding":false},{"id":245647,"name":"Hidde L. Ploegh","orcid":"0000-0002-1090-6071","position":4,"is_corresponding":false},{"id":1113315,"name":"Gan Jin","orcid":"0000-0002-8785-3035","position":5,"is_corresponding":false},{"id":283307,"name":"Charlotte Wijne","orcid":null,"position":0,"is_corresponding":true}],"reference_count":55,"raw_metadata":null,"created_at":"2026-07-19T02:58:38.868285Z","pmid":"41203132","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":[]}