AUTHOR=Finton Kathryn A. K. , Rupert Peter B. , Friend Della J. , Dinca Ana , Lovelace Erica S. , Buerger Matthew , Rusnac Domnita V. , Foote-McNabb Ulysses , Chour William , Heath James R. , Campbell Jean S. , Pierce Robert H. , Strong Roland K. 

TITLE=Effects of HLA single chain trimer design on peptide presentation and stability

JOURNAL=Frontiers in Immunology

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2023.1170462

DOI=10.3389/fimmu.2023.1170462

ISSN=1664-3224

ABSTRACT=<p>MHC class I “<italic>single-chain trimer</italic>” molecules, coupling MHC heavy chain, β<sub>2</sub>-microglobulin, and a specific peptide into a single polypeptide chain, are widely used in research. To more fully understand caveats associated with this design that may affect its use for basic and translational studies, we evaluated a set of engineered single-chain trimers with combinations of stabilizing mutations across eight different classical and non-classical human class I alleles with 44 different peptides, including a novel human/murine chimeric design. While, overall, single-chain trimers accurately recapitulate native molecules, care was needed in selecting designs for studying peptides longer or shorter than 9-mers, as single-chain trimer design could affect peptide conformation. In the process, we observed that <italic>predictions</italic> of peptide binding were often discordant with <italic>experiment</italic> and that yields and stabilities varied widely with construct design. We also developed novel reagents to improve the crystallizability of these proteins and confirmed novel modes of peptide presentation.</p>