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

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, 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 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 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 β-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.

Conformational plasticity of RAS Q61 family of neoepitopes results in distinct features for targeted recognition

“…Differential scanning fluorimetry (DSF) revealed a moderately stable complex with melting temperature (T m ) of 53 °C (Supplementary Fig. 2c ), consistent with reports of pMHC-I complexes exhibiting T m ’s ranging from 41 to 67 °C 37 , 38 , 39 . We next carried out fluorescence anisotropy competition experiments to estimate the affinity of NRAS Q61K for the HLA-A*01:01/hβ 2 m complex 40 .…”

“…Thus, we sought to characterize how NRAS Q61K could affect the structure and stability of empty MHC-I complex. In vitro studies of peptide interactions with empty MHC-I complexes have been previously reported in the literature, and are often employed as a first approximation pMHC-I interactions in vivo 38 , 51 – 53 . To characterize how NRAS Q61K assembles with HLA-A*01:01, we first emptied HLA-A*01:01/hβ 2 m complexes of their in vitro refolded peptide by brief exposure to pH 12.5 followed by SEC purification (Fig.…”

Investigating the role of killer cell immunoglobulin‐like receptors and human leukocyte antigen genetic variants in hepatitis C virus infection

“…HLA‐A is widely expressed on the surface of eukaryotic cells, consisting of a heavy α chain and a β 2‐microglobulin non‐covalently bound together. Its function is to present endogenous epitopes derived from viral invasion or tumor cell aberrations to CD8+ T cells, triggering T cell‐mediated immune responses against damaged cells 18 . The investigative efforts led by Morgane et al demonstrates that the expression of KIR3DL2 is associated with acute‐type Adult T‐cell leukemia, and can be triggered by infection with human T‐cell leukemia virus type 1 19 .…”