PT  - JOURNAL ARTICLE
AU  - Yi Sun
AU  - Michael C. Young
AU  - Claire H. Woodward
AU  - Julia N. Danon
AU  - Hau Truong
AU  - Sagar Gupta
AU  - Trenton J. Winters
AU  - George Burslem
AU  - Nikolaos G. Sgourakis
TI  - Universal open MHC-I molecules for rapid peptide loading and enhanced complex stability across HLA allotypes
AID  - 10.1101/2023.03.18.533266
DP  - 2023 Jan 01
TA  - bioRxiv
PG  - 2023.03.18.533266
4099  - http://biorxiv.org/content/early/2023/03/18/2023.03.18.533266.short
4100  - http://biorxiv.org/content/early/2023/03/18/2023.03.18.533266.full
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 (β2 microglobulin, β2m) subunits for binding to the MHC-I heavy chain (HC) through an engineered disulfide bond bridging conserved epitopes across the HC/β2m 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 β2m interacting sites of the peptide binding groove to long-range effects on the α2-1 helix and α3 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 β2m association with the heavy chain by solution NMR and HDX-MS spectroscopy. We demonstrate that covalently linked β2m 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.Competing Interest StatementY.S., M.C.Y., and N.G.S. are listed as co-inventors in a provisional patent application related to this work.