CD8+ T cells recognize and kill infected and cancerous cells. This recognition is mediated by the T cell receptor complex that is expressed at the T cell surface. This complex structure contains the clonotypic T cell receptor (TCR), the four chains of the CD3 complex d, e, g, z, and CD8 accessory molecules, which are recruited upon activation. The ligand for this complex is the peptide-MHC class I molecule complex (p-MHC) that antigen presenting cells express at their cell surface. The specificity of recognition depends on the binding of the clonotypic TCR to p-MHC. CD8 is also important in this step. This project focuses on the molecular mechanism underlying this multicomponent recognition system. The extracellular domains of each of the components are expressed in a Drosophila expression system, purified and characterized by biochemical means. This system will be used to study the details of CD8/MHC class I interactions and to provide direct evidence for the binding of TCR to CD8. Using a similar approach, we will examine the binding of CD3 chains to each other and to the TCR ab dimer in an attempt to reconstitute the entire extracellular domain of the TCR complex, in vitro. The functional consequences of these associations will be measured directly by surface plasmon resonance on a bilayer membranes. These experiments will use a chelating cage-lipid which allows the capture and orientation of histidine-tagged molecules into bilayers. For all pertinent interactions, crystallization of single components and complexes will be attempted in collaboration with Dr. Ian Wilson. This collaboration will also lead to the mutagenesis of the ab TCR, based on the high resolution structure of TCR/MHC complexes. This mutagenesis will try to identify the structural features required for MHC restriction. The initiation of the production and optimization of new TCR/MHC pairs CD3 and CD8 will also be carried out in the shared protein production facility (shared resource) to provide both laboratories with protein for their immunological and crystallographic studies. Each new TCR will be biologically characterized to limit the effort to the most relevant systems.
The specific aims for the RO1 component are to 1) analyze CD8-pMHC interactions by mutagenesis and SPR and co-crystallization; 2) use SPR to explore direct interactions between TCR and CD8; 3) reconstitute CD3 subunits with TCR in the binding studies; 4) express CD3 subunits for crystallography; 5) do mutagenesis to confirm the interactions between pMHC and TCR suggested by crystallography; and 6) express and characterize new TCR/pMHC pairs.
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