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Background: Obliteration of insulin-producing beta cells is the major characteristic of type 1 diabetes (T1D), which is accompanied by hyperexpression of class I major histocompatibility complex (MHC) molecules by islet cells. Beta cell specific cytotoxic CD8+ T cells are important effectors in the elimination of beta cells in T1D. These T cells use their T cell receptor to interact with beta cell specific peptides presented by class I MHC molecules. These MHC molecules are thus prominent determinants of beta cell destruction in T1D. The MHC class I molecule HLA-B*39:06 is the most predisposing allele for T1D. To reveal the mechanism of how HLA-B*39:06 is related to beta cell destruction, we need to define its interactions with the peptides it presents. Thus, our study focuses on the structural analysis of HLA-B alleles in T1D, where HLA-B*39:06 is in the spotlight. Methods: Biochemistry and structural biology were the approaches used for conducting this study. A single-chain construct of HLA-B*39:06 having a disulfide trap between a high-binding model peptide (NRVMLPKAA) and HLA-B*39:06 was transfected into HEK 293 cells using polyethyleneimine. The secreted HLA-B*39:06 was purified from the culture supernatant by Ni-NTA+ and size exclusion chromatography. The protein was deglycosylated by PNGase F treatment and subjected to crystallization. Crystals were diffracted and data was indexed, scaled, and refined using molecular replacement. COOT was used to visualize the structures. Results: We have determined the structure of the MHC class I molecule HLA-B*39:06 by X-ray crystallography to 1.7Å resolution. Structural analysis of HLA-B*39:06 reveals that arginine at peptide position 2 (P2) and alanine at P8 serve as anchor residues. Methionine at P4 and lysine at P7 are solvent-exposed residues available for T cell receptor contact. In HLA-B*39:06, tryptophan at position 95 limits the size of the C-terminal F pocket, explaining why HLA-B*39:06 prefers to bind peptides having small amino acid residues like alanine at their C-terminus. Conclusions: Besides revealing the previously unknown structure of HLA-B*39:06, our study is the first instance of the use of a secreted, PNGase F-treated single-chain class I MHC molecule to obtain a crystal structure, further adding novelty to our study. Elucidating the structural determinants of HLA-B alleles with their beta cell-specific peptides will give further insight into the recognition mechanism between them, potentially suggesting immunotherapy approaches for T1D. Funding: Grant NIH R01 AI123730. Ruby Sharma is funded by AHA Award.
Sharma et al. (Fri,) studied this question.