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Major Histocompatibility Complex (MHC) class I molecules selectively bind peptides for presentation to cytotoxic T cells. The peptide-free state of these molecules is not well understood. Here, we characterize a disulfide-stabilized version of the human class I molecule HLA-A*02:01 that is stable in the absence of peptide and can readily exchange cognate peptides. We present X-ray crystal structures of the peptide-free state of HLA-A*02:01, together with structures that have dipeptides bound in the A and F pockets. These structural snapshots reveal that the amino acid side chains lining the binding pockets switch in a coordinated fashion between a peptide-free unlocked state and a peptide-bound locked state. Molecular dynamics simulations suggest that the opening and closing of the F pocket affects peptide ligand conformations in adjacent binding pockets. We propose that peptide binding is co-determined by synergy between the binding pockets of the MHC molecule.

The F pocket region of MHC class I molecules is highly flexible in the absence of peptides, leading to instability and denaturation. We present here the stabilization of class I molecules by a disulfide bond between residules 84 and 139. Disulfide-stabilized class I molecules can be folded in vitro with the help of small molecules with very low affinity such as dipeptides. Once the small molecules are removed, folded disulfide-stabilized class I molecules bind exogenous peptide rapidly and stoichiometrically. This allows for the production of peptide-free MHC multimers that may be converted into the desired peptide complex within minutes. Crystal structures, binding affinity measurements, and staining data show equivalence of TCR binding to wild type class I. The stabilization is principally accessible for all class I allotypes. [ABSTRACT FROM AUTHOR]