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

J Immunol Methods. 2002 May 1;263(1-2):111-21. doi: 10.1016/s0022-1759(02)00034-0.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cell Line
  • Dimerization
  • Drosophila melanogaster
  • Genetic Techniques
  • HLA-D Antigens / genetics*
  • HLA-DR1 Antigen / genetics*
  • Peptides / immunology
  • Solubility
  • Zinc Fingers / genetics*

Substances

  • HLA-D Antigens
  • HLA-DM antigens
  • HLA-DR1 Antigen
  • Peptides