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

Abstract

MHC class I “single-chain trimer” molecules, coupling MHC heavy chain, β2-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 predictions of peptide binding were often discordant with experiment 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.

The antibodies 3D12 and 4D12 recognise distinct epitopes and conformations of HLA-E

“…In this complex, the alpha 2 domain in the region of the 4D12 epitope shows significant distortion ( 46 ). Although the published structures of conventional MHC SCT show the expected conformation of the heavy chain (for example, see 47 ), there are no published crystal structures of HLA-E SCT. If distortion of the alpha 2 domain is common to all MHC-E complexes when the peptide sequence is extended (such as in SCT), 4D12 may stain cells expressing MHC-E SCT because this alpha 2 distortion holds the 4D12 epitope in a conformation that allows the antibody to bind.…”