NMR Core

ABSTRACT – CORE C To protect mammals from a vast array of diseases, adaptive immunity relies on T cells that recognize protein- derived peptides from foreign, mutant, or otherwise aberrant sources presented on the surfaces of compromised cells. Central to this process is the αβT cell receptor (TCR), which uses mechanical force to distinguish among peptide–major histocompatibility complexes (pMHC) with remarkable sensitivity and precision. Although we know a great deal about the static conformations of TCRs, pMHCs, and their complexes, we still lack a complete understanding of how these interactions generate the diverse signaling outcomes driving T cell responses. Similarly, the preTCR, a developmental precursor to the TCR, must detect self-pMHC in the thymus to guide thymocyte maturation. Recent studies highlight that TCR-pMHC ligation and signaling are inherently dynamic and that the application of piconewton-scale forces is critical. Techniques like X-ray crystallography and cryo- EM offer high-resolution static snapshots, but solution-state NMR provides an unparalleled view at atomic resolution of structural and dynamic properties across multiple timescales. Unlike most structural techniques, NMR can exploit nearly every atom as a probe of structure and dynamics, painting a more complete picture of molecular motion and energetics. This project brings together complementary methods—including cellular assays, single-molecule (SM) force measurements, MD simulations (with and without applied force), and advanced solution NMR—to deliver a holistic view of how TCRs engage pMHC. Achieving NMR objectives has required overcoming multiple technical hurdles. TCRs are challenging to express and isotope-label in mammalian systems for NMR, and the relatively large size of TCR-pMHC complexes further complicates matters. During the prior funding period, we developed new NMR methods and labeling strategies to address these challenges. With these tools now in hand, core deployment is most facile for broad usage in this PO1. Core C shall function as a NMR Core interacting with each Project where we shall: First, facilitate exploration of the dynamic complexity of TCR and preTCR by providing expertise in isotopic labeling strategies to maximize information accessible from TCR, preTCR and MHC proteins produced in E. coli or mammalian expression systems in conjunction with Core B and all Projects. Second, Core C will correlate TCR and preTCR-pMHC interaction features and signaling by measuring dynamic and structural changes while binding pMHC ligand in concert with Projects 1, 2 and 3. These orthogonal measurements will support single molecule (SM) and MD simulations exploring force-dependent and force-free changes in TCRs with binding. Third, Core C will support Projects 2 and 3 in experiments to measure known preTCR-pMHC interactions and aid in screening for enhanced binding of known preTCRs for their ligand and for discovery of novel preTCR clonotypes.