{"doi":"10.1016/j.jbc.2025.108311","title":"Identifying the copper coordination environment between interacting neurodegenerative proteins: A new approach using pulsed EPR with 14N/15N isotopic labeling","abstract":"The trafficking and aggregation of neurodegenerative proteins often involve the interaction between intrinsically disordered domains, stabilized by the inclusion of physiological metal ions such as copper or zinc. Characterizing the metal ion coordination environment is critical for assessing the stability and organization of these relevant protein-protein interactions but is challenging given the lack of regular molecular order or global structure. The cellular prion protein (PrP C ) binds both monomers and aggregates of Alzheimer's amyloid-beta (Aβ), promoting A β internalization and aberrant signaling, respectively. Both proteins bind Cu 2+ with high affinity, opening the potential for copper to form an intermolecular bridge. We describe here a novel approach utilizing multiple EPR experiments to investigate the simultaneous Cu 2+ coordination of PrP C and A β in a 1:1:1 mixture. Uniformly 15 N-labeled PrP C is used in conjunction with natural abundance 14 N A β , the combination of which leads to distinct energy manifolds for paramagnetic Cu 2+ and is resolved by the pulsed EPR experiments ESEEM and HYSCORE. We develop acquisition parameters to simultaneously optimize 14 N (I = 1) and 15 N (I = ½) pulsed EPR signals and we also advance the theory of ESEEM and HYSCORE to quantitatively describe multiple 15 N imidazole coordination. This unique approach provides compelling evidence of a copper-stabilized ternary complex, with equatorial Cu 2+ coordination formed by one histidine imidazole from A β and three from PrP. Moreover, the methodologies developed here provide a framework for assessing the copper environment in other interacting neurodegenerative proteins.","journal":"Journal of Biological Chemistry","year":2025,"id":517891,"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":7,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9484,"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":868684,"name":"Kevin Singewald","orcid":"0000-0002-3041-5085","position":1,"is_corresponding":false},{"id":518877,"name":"Zikri Hasanbasri","orcid":"0000-0002-1824-9758","position":2,"is_corresponding":false},{"id":334945,"name":"R. David Britt","orcid":"0000-0003-0889-8436","position":3,"is_corresponding":false},{"id":266178,"name":"Glenn L. Millhauser","orcid":"0000-0003-2442-7220","position":4,"is_corresponding":false},{"id":470313,"name":"Amanda Smart","orcid":"0000-0001-7360-3353","position":0,"is_corresponding":true}],"reference_count":60,"raw_metadata":null,"created_at":"2026-07-19T02:49:04.756302Z","pmid":"39955064","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":[]}