The proposed research is an investigation into molecular aspects of antigen selection and antigen presentation by class II major histocompatibility (MHC) proteins. The human class II MHC proteins HLA- DR1, DR3, and DQ8 will be expressed as empty molecules in insect cells and loaded in vitro with antigenic peptides. Three-dimensional structures will be determined by X-ray crystallography for two or more peptide complexes of DR1 and for peptide complexes of DR3 and DQ8. Currently the three- dimensional structure of only one class II MHC-peptide complex is known. The proposed structural determinations for peptide complexes of DR1 and DR3 will indicate the generality of the peptide binding mechanism observed in the published DR1-HA peptide structure and the source of the observed differences in peptide binding preferences for DR1 and DR3. The proposed structural determinations for peptide complexes of DR3 and DQ8 will identify any unusual features of these proteins that may be responsible for their disease association. In addition, these structures will provide a framework for interpretation of the large amount of data concerning antigen selection and presentation by other class II MHC proteins and will improve our ability to predict the peptide binding preferences of MHC proteins and the potential antigenicity of peptide and protein therapeutics. Detailed information about the peptide binding site will aid efforts to design structure-based antagonists and agonists of the MHC- antigen-T-cell receptor interactions. Class II MHC proteins will be expressed in E. coli and refolded in vitro, as an alternative to the insect cell expression system. Development of the E. coli expression system will provide a rich source of class II MHC proteins and class II- peptide complexes for crystallographic and other biophysical investigations of the peptide binding mechanism. Preliminary expression studies have provided DR1, DR3, and DQ8 proteins. Preliminary crystallographic studies have provided crystals of HLA-DR1 in complex with an endogenous peptide derived from HLA-A2, and initial phase information for determination of the structure of the DR1-A2 peptide complex. These crystals grow in a new crystal form that diffracts to higher resolution than previously reported class II MHC crystals. Preliminary refolding studies have produced active DR1 in approximately 30% yield after expression in E. coli and refolding in vitro.
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