Stabilization of soluble, low-affinity HLA-DM/HLA-DR1 complexes by leucine zippers

The ectodomains of interacting membrane-bound proteins, when expressed as recombinant soluble molecules, often have low affinities for each other, hampering studies of their interaction. We reasoned that stabilization of unstable protein-protein complexes should aid our understanding of the structural and functional consequences of complex fori-nation, Here, we have used fusion with leucine zipper (LZ) domains to stabilize a complex formed between the class 11 major histocompatibility complex (MHC-II) protein, HLA-DR` (which binds peptides for presentation to CD4(-) T cells) and HLA-DM (which catalyzes peptide exchange of MHC-II molecules). To this end, the DM beta chain ectodomains were fused to acidic LZ domains (AcidP1 or Fos); similarly, the DR1 beta chain ectodomains were fused to basic LZ domains (BaseP1 or Jun), We expressed LZ-modified soluble DM or DR1 alphabeta dimers, or both, in insect cells and purified the secreted sDM-AcidP1 and sDR1-BaseP1 molecules as well as the complex, LZ modification greatly enhanced DM-catalyzed peptide binding to DR1 compared to unmodified soluble DM and DR1. We readily detected U-modified DM/DR complexes on native PAGE gels and by coimmunoprecipitation. Thus, fusion with artificial LZ domains can stabilize unstable protein-protein complexes for biochemical and structural studies of interactions within the complex. (C) 2002 Elsevier Science B.V All rights reserved.