{"doi":"10.1101/2025.05.01.651769","title":"Segmental Isotope Labelling of the Prion Protein: Identification of a Key Residue for Copper-Mediated Interdomain Structure","abstract":"Abstract The cellular prion protein is composed of two domains: a disordered N-terminal toxic effector domain and a three-helix C-terminal regulatory domain. Copper is thought to form a bridge between these two domains, inhibiting the protein’s inherent neurotoxicity. However, the molecular details of how copper interacts with the C-terminal regulatory surface are unclear. To assess the potential role of conserved C-terminal His residues in copper coordination, we applied sortase-mediated ligation to create an expressed, murine prion protein with segmental 15 N-labeling of the N-terminal domain. Pulsed EPR methods applied to a 1:1 protein:copper complex revealed both 14 N and 15 N couplings, consistent with simultaneous coordination of the two protein’s domains to the copper center. Mutagenesis studies localized C-terminal copper coordination to His176, present on the second α-helix. The cumulative EPR results reveal a copper coordination environment composed of three His residues from the protein’s N-terminal domain, along with His176. The feasibility of these findings was tested with AlphaFold 3 simulations. These results further refine the molecular details of the prion protein’s autoregulation, emphasizing the critical role of its copper cofactor. Moreover, this interdisciplinary work demonstrates how sortase-mediated ligation combined with pulsed EPR sensitive to distinct nuclear spin systems provides a new strategy for assessing metal ion binding to proteins.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":565199,"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":0.9524,"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":588685,"name":"Samuel Kaplan","orcid":"0000-0002-9903-6630","position":2,"is_corresponding":false},{"id":761164,"name":"Eefei Chen","orcid":"0000-0001-7411-7779","position":3,"is_corresponding":false},{"id":266178,"name":"Glenn L. Millhauser","orcid":"0000-0003-2442-7220","position":4,"is_corresponding":false},{"id":577099,"name":"Francesca A. Pavlovici","orcid":"0000-0002-1611-5035","position":0,"is_corresponding":true}],"reference_count":71,"raw_metadata":null,"created_at":"2026-07-19T02:56:28.709936Z","pmid":"40625640","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":[]}