Understanding the Local Backbone Dynamics of the pMHC and Kinetics of the TCR/pMHC Binding Interaction

Cytotoxic T cells destroy virally infected cells and tumor cells and are also implicated in transplant rejection. These cells are also known as CD8+ T cells, since they express the CD8 glycoprotein at their surface. The T cell receptor is a molecule found on the surface of T cells that is responsible for recognizing antigens presented by major histocompatability complex (MHC) proteins. MHC proteins are expressed on the surface of cells and display fragments of molecules from invading microbes or dysfunctional cells to the TCR.

Here, through the utilization of fluorescence anisotropy, we investigate MHC flexibility when different peptides are bound. The peptides presented by class I MHC HLA-A2 utilized in this study are as follows: Tax 9 (LLFGYPVYV), Tel1p (MLWGYLQYV), ELA (ELAGIGILTV), gp100 T2M (IMDQVPFSV) and Flu M1 (GILGFVFTL). To assess the changes in flexibility, we engineered a series of cysteine mutants in the α1 and α2 helix of the MHC complex. The samples were refolded in the presence of peptide, purified and consequently labeled with Alexa-Fluor 488 (Alexa488) for fluorescence studies. Steady-state fluorescence anisotropy measurements were performed, and we were able to show that the overall flexibility on the α1 and α2 helix regions of the MHC varies dependent on the peptide presented on the MHC complex.

Measurements of TCR-pMHC binding equilibrium and kinetic parameters have been obtained through the utilization of surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Unlike pure solution methods, SPR does not allow for detailed resolution of the kinetic mechanism for the TCR-pMHC interaction, which can be obtained from stopped-flow fluorescence anisotropy. The need for such an assay is essential to understanding the recognition mechanism of the TCR as well as enabling further investigation of TCR cross reactivity and specificity.

Fluorescence studies of the TCR-pMHC interaction were completed using a fluorescein derivative to label an MHC-free cysteine via maleimide chemistry. Current data with the A6 T cell receptor and the Tax 9 peptide presented by the class I MHC HLA-A2 shows that as the concentration of A6 increases, the pMHC is bound resulting in an increased anisotropy. The same trend is seen in various A6 constructs (A6 wild-type and A6 c134 zippered constructs, Cole/Sewell A6 wild-type and A6 c134) that have been studied. We have determined that upon ligation we see a change in anisotropy is observed using our fluorescein derivitized protein and determined the unique kinetic parameters and recognition mechanism of the A6/HLA-A2 binding interaction.