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 formation. Here, we have used fusion with leucine zipper (LZ) domains to stabilize a complex formed between the class II major histocompatibility complex (MHC-II) protein, HLA-DR1 (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 LZ-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.
Busch
Robert
R
Department of Pediatrics, Stanford University Medical Center, Room 2120 CCSR Bldg., 269 Campus Drive, CA 94305-5164, USA. rbusch@stanford.edu
Pashine
Achal
A
Garcia
K Christopher
KC
Mellins
Elizabeth D
ED
D000818:Animals; D002460:Cell Line; D019281:Dimerization; D004331:Drosophila melanogaster; D005821:Genetic Techniques; D006681:HLA-D Antigens / Q000235:genetics*; D015798:HLA-DR1 Antigen / Q000235:genetics*; D010455:Peptides / Q000276:immunology; D012995:Solubility; D016335:Zinc Fingers / Q000235:genetics*
D006681:HLA-D Antigens; C084245:HLA-DM antigens; D015798:HLA-DR1 Antigen; D010455:Peptides