Determination of the HLA-DM interaction site on HLA-DR molecules

HLA-DM removes CLIP and other loosely bound peptides from MHC class II mole cules. The crystal structures of class II molecules and of HLA-DM have not permitted identification of their interaction sites. Here, we describe muta tions in class II that impair interactions with DM. Libraries of randomly m utagenized DR3 alpha and beta chains were screened for their ability to cau se cell surface accumulation of CLIP/DR3 complexes in EBV-B cells. Seven mu tations were associated with impaired peptide loading in vivo, as detected by SDS stability assays. In vitro, these mutant DR3 molecules were resistan t to DM-catalyzed CLIP release and showed reduced binding to DM. All mutati ons localize to a single lateral face of HLA-DR, which we propose interacts with DM during peptide exchange.