Visualizing ligand-induced signal propagation in the TCR-signaling complex

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In this proposal funds are requested for a for-cost extension for an NIH funded project at NYU School of Medicine to recover losses and delay in research time due to Hurricane Sandy under the Disaster Relief appropriations Act. This request is with the purpose to complete our proposed aims and objectives proposed in parent grant GM085586. This project described in the parent grant aims to understand how dynamic alterations and/or conformational changes at the T-cell receptor-peptide-MHC (TCR-pMHC) interface contribute to T-cell activation and to identify the mechanism that is responsible for communicating ligand engagement of TCR to CD3 signaling subunits. The central hypothesis of this application is that conformational changes and/or flexibility induced in the TCR upon ligand binding can be transmitted across the membrane to expose cytoplasmic domains in the CD3ε/ζ chains, and that differences in conformational changes and/or flexibility are responsible for quantitative and/or qualitative differences in T-cell signaling. To test our hypothesis we have combined X-ray crystallography and nuclear magnetic resonance (NMR) experiments and non-synthetic amino acid incorporation combined with cross-linking experiments and fluorescence resonance energy transfer (FRET) to analyze the changes in the overall structural organization and conformation of TCR when binding to different pMHC. We expect that these studies will provide insight into the molecular mechanism of how ligand induced conformational changes at the pMHC-TCR interface translocate to the CD3 signaling complex to influence T-cell activation outcomes with a sensitivity and resolution that have not been possible before. An increased understanding of the structural biophysics of protein-protein interactions and of the propensity of structures to undergo conformational change will be of critical importance, particularly in the case of receptors involved in cell signaling. Furthermore, such biophysical studies will provide fundamental insights into protein structure and dynamics, explain how these features are used for specific signaling purposes and how the proteins function in distinct cellular environments. These results are expected to be of interest for the scientific community interested in receptor signaling. In addition, this basic knowledge will eventually allow us to design polypeptides or other agents that can be used to monitor and manipulate cell signaling events to guide the design of therapeutics and vaccines for cancer and autoimmune disease which afflict thousands of people.