Identification of the HLA-DM binding site on HLA-DR molecules

HLA-DM removes CLIP and other loosely bound peptides from MHC class II molecules. The crystal structures of class II molecules and of HLA-DM have not permitted identification of their interaction sites. Libraries of randomly mutagenized DR3 α and β chains were screened for their ability to cause cell surface accumulation of DR3:CLIP complexes in EBV-B cells. Here we describe several novel HLA-DR mutations that fall into two categories based on their location and on the mechanism of action by which they cause CLIP accumulation. One group maps to the peptide binding groove of the HLA-DR molecule and generates high stability DR3:CLIP complexes. The other group of mutants are resistant to DM-catalyzed CLIP release and show reduced binding to HLA-DM. These mutants localize to a single lateral face of HLA-DR, which we propose interacts with DM during peptide exchange. Site-directed mutagenesis of residues at this interface has allowed refinement of the DM-binding site. We also demonstrate that a naturally occurring polymorphism at position β96 on HLA-DR molecules affects DM catalysis rates.