PMID- 37310998 OWN - NLM STAT- MEDLINE DCOM- 20230615 LR - 20231106 IS - 1091-6490 (Electronic) IS - 0027-8424 (Print) IS - 0027-8424 (Linking) VI - 120 IP - 25 DP - 2023 Jun 20 TI - Universal open MHC-I molecules for rapid peptide loading and enhanced complex stability across HLA allotypes. PG - e2304055120 LID - 10.1073/pnas.2304055120 [doi] LID - e2304055120 AB - 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 (beta(2) microglobulin, beta(2)m) subunits for binding to the MHC-I heavy chain (HC) through an engineered disulfide bond bridging conserved epitopes across the HC/beta(2)m interface, to generate conformationally stable, peptide-receptive molecules named "open MHC-I." 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 moderate-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 beta(2)m-interacting sites of the peptide-binding groove to long-range effects on the alpha(2-1) helix and alpha(3) domain. The interchain disulfide bond stabilizes MHC-I molecules in an open conformation to promote peptide exchange across multiple human leukocyte antigen (HLA) allotypes, covering representatives from five HLA-A supertypes, six HLA-B supertypes, and oligomorphic HLA-Ib molecules. Our structure-guided design, combined with conditional beta-peptide ligands, provides a universal platform to generate ready-to-load MHC-I systems of enhanced stability, enabling a range of approaches to screen antigenic epitope libraries and probe polyclonal TCR repertoires covering highly polymorphic HLA-I allotypes, as well as oligomorphic nonclassical molecules. FAU - Sun, Yi AU - Sun Y AD - Center for Computational and Genomic Medicine, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA 19104. AD - Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104. FAU - Young, Michael C AU - Young MC AUID- ORCID: 0000-0002-8420-2149 AD - Center for Computational and Genomic Medicine, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA 19104. AD - Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104. FAU - Woodward, Claire H AU - Woodward CH AUID- ORCID: 0000-0002-0373-6318 AD - Center for Computational and Genomic Medicine, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA 19104. AD - Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104. FAU - Danon, Julia N AU - Danon JN AD - Center for Computational and Genomic Medicine, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA 19104. AD - Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104. FAU - Truong, Hau V AU - Truong HV AUID- ORCID: 0000-0002-0059-2793 AD - Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104. FAU - Gupta, Sagar AU - Gupta S AUID- ORCID: 0000-0002-2151-9018 AD - Center for Computational and Genomic Medicine, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA 19104. AD - Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104. FAU - Winters, Trenton J AU - Winters TJ AUID- ORCID: 0000-0001-8922-3013 AD - Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104. FAU - Font-Burgada, Joan AU - Font-Burgada J AUID- ORCID: 0000-0003-3372-418X AD - Cancer Signaling and Microenvironment Program, Fox Chase Cancer Center, Philadelphia, PA 19111. FAU - Burslem, George M AU - Burslem GM AUID- ORCID: 0000-0002-2812-983X AD - Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104. AD - Department of Cancer Biology and Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104. FAU - Sgourakis, Nikolaos G AU - Sgourakis NG AUID- ORCID: 0000-0003-3655-3902 AD - Center for Computational and Genomic Medicine, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA 19104. AD - Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104. LA - eng GR - U01 DK112217/DK/NIDDK NIH HHS/United States GR - R35 GM125034/GM/NIGMS NIH HHS/United States GR - R01 AI143997/AI/NIAID NIH HHS/United States GR - T32 GM132039/GM/NIGMS NIH HHS/United States GR - R35 GM142505/GM/NIGMS NIH HHS/United States GR - P30 CA006927/CA/NCI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20230613 PL - United States TA - Proc Natl Acad Sci U S A JT - Proceedings of the National Academy of Sciences of the United States of America JID - 7505876 RN - 0 (Histocompatibility Antigens) RN - 0 (Histocompatibility Antigens Class II) RN - 0 (Peptides) RN - 0 (Epitopes) RN - 0 (Disulfides) SB - IM UOF - bioRxiv. 2023 Mar 18;:. PMID: 36993702 MH - Humans MH - *Histocompatibility Antigens MH - *Histocompatibility Antigens Class II MH - Peptides/genetics MH - Major Histocompatibility Complex MH - Epitopes MH - Disulfides PMC - PMC10288639 OTO - NOTNLM OT - NMR OT - antigen processing and presentation OT - human leukocyte antigen OT - nonclassical MHC-I OT - peptide exchange COIS- Y.S., M.C.Y., and N.G.S. are listed as co-inventors in a provisional patent application related to this work. EDAT- 2023/06/13 19:15 MHDA- 2023/06/15 06:42 PMCR- 2023/06/13 CRDT- 2023/06/13 13:43 PHST- 2023/06/15 06:42 [medline] PHST- 2023/06/13 19:15 [pubmed] PHST- 2023/06/13 13:43 [entrez] PHST- 2023/06/13 00:00 [pmc-release] AID - 202304055 [pii] AID - 10.1073/pnas.2304055120 [doi] PST - ppublish SO - Proc Natl Acad Sci U S A. 2023 Jun 20;120(25):e2304055120. doi: 10.1073/pnas.2304055120. Epub 2023 Jun 13.