{"doi":"10.1101/2023.03.18.533266","title":"Universal open MHC-I molecules for rapid peptide loading and enhanced complex stability across HLA allotypes","abstract":"Abstract The polymorphic nature and intrinsic instability of class I major histocompatibility complex (MHC-I) and MHC-like molecules loaded with suboptimal peptides, metabolites, or glycolipids presents a fundamental challenge for identifying disease-relevant antigens and antigen-specific T cell receptors (TCRs), hindering the development of autologous therapeutics. Here, we leverage the positive allosteric coupling between the peptide and light chain (β 2 microglobulin, β 2 m) subunits for binding to the MHC-I heavy chain (HC) through an engineered disulfide bond bridging conserved epitopes across the HC/β 2 m interface, to generate conformationally stable, open MHC-I molecules. Biophysical characterization shows that open MHC-I molecules are properly folded protein complexes of enhanced thermal stability compared to the wild type, when loaded with low- to intermediate-affinity peptides. Using solution NMR, we characterize the effects of the disulfide bond on the conformation and dynamics of the MHC-I structure, ranging from local changes in β 2 m interacting sites of the peptide binding groove to long-range effects on the α 2-1 helix and α 3 domain. The interchain disulfide bond stabilizes empty MHC-I molecules in a peptide-receptive, open conformation to promote peptide exchange across multiple human leucocyte antigen (HLA) allotypes, covering representatives from five HLA-A, six HLA-B supertypes, and oligomorphic HLA-Ib molecules. Our structural design, combined with conditional β-peptide ligands, provides a universal platform for generating ready-to-load MHC-I systems of enhanced stability, enabling a range of approaches to screen antigenic epitope libraries and probe polyclonal TCR repertoires in the context of highly polymorphic HLA-I allotypes, as well as oligomorphic nonclassical molecules. Significance Statement We outline a structure-guided approach for generating conformationally stable, open MHC-I molecules with enhanced ligand exchange kinetics spanning five HLA-A, all HLA-B supertypes, and oligomorphic HLA-Ib allotypes. We present direct evidence of positive allosteric cooperativity between peptide binding and β 2 m association with the heavy chain by solution NMR and HDX-MS spectroscopy. We demonstrate that covalently linked β 2 m serves as a conformational chaperone to stabilize empty MHC-I molecules in a peptide-receptive state, by inducing an open conformation and preventing intrinsically unstable heterodimers from irreversible aggregation. Our study provides structural and biophysical insights into the conformational properties of MHC-I ternary complexes, which can be further applied to improve the design of ultra-stable, universal ligand exchange systems in a pan-HLA allelic setting.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2023,"id":394324,"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":3,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9507,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1038173,"name":"Michael C. Young","orcid":"0000-0002-8420-2149","position":1,"is_corresponding":false},{"id":864828,"name":"Claire H. Woodward","orcid":"0000-0002-0373-6318","position":2,"is_corresponding":false},{"id":1038174,"name":"Julia N. Danon","orcid":"0000-0001-6971-6582","position":3,"is_corresponding":false},{"id":636907,"name":"Hau V. Truong","orcid":"0000-0002-0059-2793","position":4,"is_corresponding":false},{"id":1038175,"name":"S. K. Gupta","orcid":"0000-0002-2151-9018","position":5,"is_corresponding":false},{"id":1038176,"name":"Trenton J. Winters","orcid":"0000-0001-8922-3013","position":6,"is_corresponding":false},{"id":107239,"name":"George M. Burslem","orcid":"0000-0002-2812-983X","position":7,"is_corresponding":false},{"id":314926,"name":"Nikolaos G. Sgourakis","orcid":"0000-0003-3655-3902","position":8,"is_corresponding":false},{"id":1038172,"name":"Yi Sun","orcid":"0000-0002-7821-4346","position":0,"is_corresponding":true}],"reference_count":64,"raw_metadata":null,"created_at":"2026-07-19T01:19:10.334330Z","pmid":"36993702","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":[]}