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

Yi Sun, Michael C. Young, Claire H. Woodward, Julia N. Danon, Hau Truong, Sagar Gupta, Trenton J. Winters, View ORCID ProfileGeorge Burslem, View ORCID ProfileNikolaos G. Sgourakis
doi: https://doi.org/10.1101/2023.03.18.533266
Yi Sun
1Center for Computational and Genomic Medicine, Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA, 19104, USA
2Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, 3401 Civic Center Blvd, Philadelphia, PA, 19104, USA
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Michael C. Young
1Center for Computational and Genomic Medicine, Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA, 19104, USA
2Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, 3401 Civic Center Blvd, Philadelphia, PA, 19104, USA
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Claire H. Woodward
1Center for Computational and Genomic Medicine, Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA, 19104, USA
2Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, 3401 Civic Center Blvd, Philadelphia, PA, 19104, USA
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Julia N. Danon
1Center for Computational and Genomic Medicine, Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA, 19104, USA
2Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, 3401 Civic Center Blvd, Philadelphia, PA, 19104, USA
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Hau Truong
3Department of Cancer Biology and Epigenetics Institute, Perelman School of Medicine
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Sagar Gupta
1Center for Computational and Genomic Medicine, Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA, 19104, USA
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Trenton J. Winters
2Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, 3401 Civic Center Blvd, Philadelphia, PA, 19104, USA
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George Burslem
2Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, 3401 Civic Center Blvd, Philadelphia, PA, 19104, USA
3Department of Cancer Biology and Epigenetics Institute, Perelman School of Medicine
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Nikolaos G. Sgourakis
1Center for Computational and Genomic Medicine, Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA, 19104, USA
2Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, 3401 Civic Center Blvd, Philadelphia, PA, 19104, USA
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  • ORCID record for Nikolaos G. Sgourakis
  • For correspondence: Nikolaos.Sgourakis@Pennmedicine.upenn.edu
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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 (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 Statement

Y.S., M.C.Y., and N.G.S. are listed as co-inventors in a provisional patent application related to this work.

Footnotes

  • Competing Interest Statement: Y.S., M.C.Y., and N.G.S. are listed as co-inventors in a provisional patent application related to this work.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted March 18, 2023.
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Universal open MHC-I molecules for rapid peptide loading and enhanced complex stability across HLA allotypes
Yi Sun, Michael C. Young, Claire H. Woodward, Julia N. Danon, Hau Truong, Sagar Gupta, Trenton J. Winters, George Burslem, Nikolaos G. Sgourakis
bioRxiv 2023.03.18.533266; doi: https://doi.org/10.1101/2023.03.18.533266
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Universal open MHC-I molecules for rapid peptide loading and enhanced complex stability across HLA allotypes
Yi Sun, Michael C. Young, Claire H. Woodward, Julia N. Danon, Hau Truong, Sagar Gupta, Trenton J. Winters, George Burslem, Nikolaos G. Sgourakis
bioRxiv 2023.03.18.533266; doi: https://doi.org/10.1101/2023.03.18.533266

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