Transmembrane domain-mediated colocalization of HLA-DM and HLA-DR is required for optimal HLA-DM catalytic activity

HLA-DM catalyzes peptide loading and exchange reactions by MHC class II mol ecules. Soluble recombinant DM, lacking transmembrane and cytoplasmic domai ns, was observed to have 200- to 400-fold less activity compared with the f ull-length protein in assays measuring DM-catalyzed peptide dissociation fr om purified HLA-DR1 in detergent solutions. Additional studies with truncat ed soluble DR1 demonstrated that transmembrane domains in DR1 molecules are also required for optimal activity. The potential requirement for specific interaction between the transmembrane domains of DM and DR was ruled out i n experiments with chimeric DR1 molecules containing transmembrane domains from either DM or the unrelated protein CD80. These results suggested that the major role of the transmembrane domains is to facilitate colocalization of DM and DR in detergent micelles. The latter conclusion was further supp orted by the observation that HLA-DM-catalyzed peptide binding to certain m urine class II proteins Is increased by reducing the volume of detergent mi celles. The importance of membrane colocalization was directly demonstrated in experiments in which DM and DR were reconstituted separately or togethe r into membrane bilayers in unilamellar liposomes. Our findings demonstrate the importance of membrane anchoring in DM activity and underscore the pot ential importance of membrane localization in regulating peptide exchange b y class II molecules.