The objective of the proposed research is to determine the three-dimensional structure and binding properties of Mycoplasma arthritidis-derived mitogen (MAM), a bacterial immunoregulatory protein implicated in the development of human rheumatoid arthritis. MAM is a soluble 26 kDa protein that binds to major histocompatibility complex (MHC) class II molecules and activates large numbers of T cells bearing particular T cell receptor (TCR) V beta chains. In this respect, MAM functions like conventional superantigens that cause disease by stimulating a strong nonspecific immune response. However, MAM does not share significant sequence homology with other superantigens and, in contrast to other superantigens, binding of MAM to the TCR is influenced by the CDR3 region of the TCR. In order to better understand the biology of this novel superantigen, the following specific aims will be pursued: 1) Determine the crystal structure of recombinant MAM (rMAM); 2) Determine the affmity and kinetic parameters of rMAM binding to TCR beta chains and MHC class II molecules; 3) Determine the crystal structures of rMAM when complexed with TCR beta chains and with MHC class II molecules. Small crystals of rMAM and of rMAM complexed with a TCR beta chain have already been produced. This study will resolve the three-dimensional structure of MAM as well as define its interaction with TCRs and MHC class II molecules. In the long term, the knowledge acquired will lay the groundwork for understanding the molecular basis of T cell activation by this unique superantigen, for determining its role in triggering and exacerbating autoimmune arthritis, and for developing structure-based therapies to treat disease.
