MHC-I molecules disulfide-linked to antigenic peptides offer a simple approach to stable MHC-I/peptide complexes

Abstract

Abstract A variety of engineering approaches have been used to develop MHC/beta2m/peptide complexes with improved stability for structural studies and binding to T cell receptors and other MHC-I ligands. To generate MHC-I molecules covalently linked to antigenic peptides we considered X-ray structures of the H2-Dd molecule bound to the HIV IIIB derived peptide, P18-I10, RGPGRAFVTI. Computer analysis suggested that mutation of an H2-Dd residue to cysteine in the peptide binding groove would provide an MHC heavy chain that could be refolded with P18-I10 derived peptides substituted with cysteine at peptide position P5. Using standard E. coli expression as inclusion bodies and in vitro refolding protocols, we show that a set of cysteine-substituted peptides derived from P18-I10 including the decamer P18-C5-I10 (RGPGCAFVTI) and octamer P18-C5-V8 (RGPGCAFV) could be efficiently refolded with cys-mutated H2-Dd heavy chain and beta2-microglobulin. These molecules were analyzed by antibody binding studies and by X-ray crystallography. The P18-C5-I10/cys-mut-H2-Dd complex has a structure almost identical to that of the parental P18-I10/H2-Dd complex, while the complex with the octamer P18-C5-V8 repositions the peptide such that the C-terminal valine residue occupies the F pocket of H2-Dd. Such a strategy for producing covalently coupled MHC/peptide complexes offers a new approach to generate stable complexes for structural and imaging applications that should be widely applicable.