The aim of this project has been to understand the molecular mechanisms that underlie the biophysical interactions of MHC class I molecules with antigenic and self peptides as well as with T cell receptors. Since MHC molecules provide the critical cell surface signal in the initiation of the T cell response, detailed understanding of the structure, peptide binding, and T cell receptor binding of MHC molecules provides a basis for refining immunization strategy, for understanding autoimmune disease, and for developing rational molecular intervention in such illnesses. Our goals in this project have been to develop and refine methods for high level production of MHC molecules and T cell receptors in tissue culture cells, in bacterial expression systems and in transgenic mouse models. These studies provide the material for understanding binding interactions in vitro as well as for understanding the consequences of these interactions in whole animal model systems. Following up on previous attempts to exploit the single chain strategy for the production of well-folded MHC class I molecules, Dr. Kannan Natarajan has worked out a particularly efficient protocol, based on previously published procedures, for the production of two chain MHC class I molecule H-2Dd complexed with the light chain beta2- microglobulin and with any of a choice of antigenic peptides. This procedure has allowed the production of large amounts of homogeneous H-2Dd/P18-I10 complexes that have been shown to crystallize (in collaboration with Dr. R. Mariuzza, CARB, University of Maryland). Several large crystals have been obtained, and preliminary attempts to see if these crystals diffract x-rays, and if so, to what resolution, will be made shortly. To complement our understanding of how T cell receptors bind MHC/peptide complexes, Dr. Polakova and Plaksin have successfully produced two monoclonal antibodies that bind the same H-2Dd/P18-I10 complex that is seen by one of the T cell receptors that we have been studying. These monoclonal antibodies bind the MHC peptide complex with great specificity for both the MHC and the peptide, and thus analysis of their structure is expected to provide insight into the differences and similarities of the mechanisms by which antibodies and T cell receptors bind protein molecules. The genes encoding both the heavy and light chains of the two antibodies, as well as the alpha and beta chains of the T cell receptor have been sequenced, and remarkably, there exists a region in the CDR2 of the VH of both the antibodies that is identical to the aligned region of the Vbeta chain of the T cell receptor. With such molecular reagents in hand (T cell receptors, MHC/peptide complexes, and TCR-like monoclonal antibodies), we should have a unique opportunity to further explore the details of the initial binding reactions of the immune response.
| Natarajan, K; Li, H; Mariuzza, R A et al. (1999) MHC class I molecules, structure and function. Rev Immunogenet 1:32-46 |
| Plaksin, D; Chacko, S; Navaza, J et al. (1999) The X-ray crystal structure of a Valpha2.6Jalpha38 mouse T cell receptor domain at 2.5 A resolution: alternate modes of dimerization and crystal packing. J Mol Biol 289:1153-61 |
