Universal open MHC-I molecules for rapid peptide loading and enhanced complex stability across HLA allotypes

The polymorphic nature and intrinsic instability of class I major histocompatibility complex (MHC- I) and MHC- like molecules loaded with suboptimal peptides, metab-olites, 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 ((32 microglobulin, (32m) subunits for binding to the MHC- I heavy chain (HC) through an engineered disulfide bond bridging conserved epitopes across the HC/(32m 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 dynam-ics of the MHC- I structure, ranging from local changes in (32m -interacting sites of the peptide-binding groove to long -range effects on the & alpha;2-1 helix and & alpha;3 domain. The inter -chain 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 (3-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.

Automated summary

This study created stable MHC-I molecules capable of accepting low-to moderate-affinity peptides through an engineered disulfide bond bridging conserved epitopes. The effects of the disulfide bond on the conformation of the MHC-I structure were characterized using solution NMR. The structure-guided design provides a universal platform for screening antigenic epitope libraries and studying polyclonal TCR repertoires covering highly polymorphic HLA-I allotypes and oligomorphic nonclassical molecules.