Structural studies of the mechanisms for antigenic peptide binding of MHC class I molecule

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1.Using an improved refolding method in the absence of an antigenic peptide, we have successfully prepared large quantities of empty MHC molecules.2. The ^1H^<-15>N HSQC spectrum of the empty MHC molecule whose heavy chain was labeled with ^<15>N exhibited peaks corresponding to the peptide-bound MHC molecule and those characteristic of random coiled states, suggesting coexistence of preserved and unfolded regions within the molecule. Using a selective labeling technique of distinct amino acid residues, it was revealed that α3 domain maintains a native tertiary structure whereas α 1 and α 2 domains, which form the antigen binding site, are partially unfolded in the absence of an antigenic peptide.3.We have assigned 87 % ^1H^<-15>N TROSY peaks originating from the backbone amide groups of β_2m by combined use of a deuterate labeling technique. Using the assigned NMR signals as spectroscopic probes, we evaluated the effects of dissociation of the antigenic peptide on the β_2m structure. Based on the NMR data, we clarified that only the hydrophobic cluster located just bellow the antigen binding pocket and its vicinity are perturbed upon dissociation of the antigenic peptide.4.We have identified the binding sites of LILRB1and LILRB2 on the β_2m subunit embedded in the MHC class I molecule using NMR chemical-shift-perturbation data.5.We analyzed the interaction between MHC class I molecules and molecular chaperones calnexin and calreticulin by means of surface plasmon resonance. It was revealed that calnexin and calreticulin bind to MHC molecules even without the modification with sugar chains and that the affinities of these chaperones for empty MHC molecules were slightly higher than those for peptide-bound MHC molecules, suggesting that they recognize denatured surface area of the empty MHC molecules.