Physical basis for T cell receptor binding and activity

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DESCRIPTION (provided by applicant): Visualization of TCR-peptide/MHC complexes through crystallographic methods has provided enormous insight into the nature of TCR-peptide/MHC interactions. However, structures are static and do not reveal the molecular mechanisms of how the T cell receptor binds its ligand. Structures do not indicate the rates at which TCRs and peptide/MHC molecules associate and dissociate or the extent to which details within the interface contribute to the stability of the complex. Structures provide little information about the dynamic motions the proteins undergo upon forming the complex. Binding kinetics and thermodynamics have been correlated with the type and potency of T cell response, and loop mobility has been implicated in cross-reactivity and ligand discrimination. Here, a detailed investigation of TCR-peptide/MHC interactions addressing questions left unanswered by crystallography will be performed. Specifically, the interactions between multiple T cell receptors that all recognize the Tax peptide presented by HLA-A2 with will be studied. Two of these receptors, A6 and B7, are known to form almost entirely different contacts with ligand, yet the overall structures and functional responses of the two receptor complexes are extremely similar. Preliminary sequence data for other Tax/HLA-A2 specific receptors suggests that they likewise bind with different amino acids. This indicates that there are multiple ways in which a given receptor can recognize a ligand. Examination of multiple receptors specific for the same ligand should provide significant insight into how TCRs bind peptide/MHC, as detailed comparisons, correlations, and ultimately generalizations can be made. Experiments to be performed include determination of binding thermodynamics, kinetics, and activation energies for at least seven TCR-Tax/HLA-A2 interactions using titration calorimetry, surface plasmon resonance, and analytical ultracentrifugation. Mutational analyses will be performed, allowing determination of "hot spots" or "focal points" within each interface. The work will move beyond traditional alanine scanning mutagenesis in order to determine the physical basis for identified hot spots. A critical control will include determination of the effects mutations have on peptide binding by MHC using fluorescence polarization. Functional measurements of cytotoxicity and cytokine release will also be performed, allowing binding measurements to be correlated with biological activity. Interpretation of binding and functional measurements in the context of either known or modeled three-dimensional structures will permit the determinants of binding and activity to mapped to structural features of each interface. The work will culminate with attempts to identify general strategies for the rational manipulation of TCR binding and activity.