The goals of the proposed research are to elucidate mechanisms of T-cell signal transduction through the cell membrane by the TCR/CD3 complex, and from the cytoplasm to the nucleus by the nuclear factor of activated T cells, NFAT, as regulated by the phosphatase calcineurin and by other interacting proteins. These mechanisms are crucial for T-cell activation, T-cell function, immune suppression and general T-cell biology. The research will pursue two specific aims.
Specific Aim 1 is to elucidate the topology of the T-cell receptor complex. While structures of the ectodomain components are known, the topology of the complex and the mechanisms of signal transduction are still unclear. We propose to design systems that enable mapping of domain interfaces and determining the TCR/CD3 topology. This includes producing CD3 and TCR ectodomains anchored in membrane mimics, such as micelles and phospholipids nanodiscs. We will create e?? single chain constructs and introduce paramagnetic sites to enable domain-domain contact measurements with NMR and EPR methods. To achieve this we will link chains and tags using a sortase approach. The proposed research will lead to structures of full-length TCR/CD3 components including the transmembrane regions and eventually to complete structures of the TCR/CD3 complex. It will facilitate understanding of the mechanism of TCR/CD3 signal transduction.
Specific Aim 2 is to elucidate mechanisms that trigger nuclear translocation of NFAT. It will focus on NFAT's N-terminal regulatory domain, which contains the transactivation domain, numerous proline-rich phosphorylation sites and a nuclear localization sequence. It is primarily unstructured alone but adopts some structure when bound to the phosphatase calcineurin (Cn), to the regulatory protein Homer, to other proteins of little known function, and to transcriptional co-activator proteins in the nucleus. Despite the importance of this domain little is known about its mechanisms of function. Here we will elucidate elements of local structure and structural changes upon phosphorylation or dephosphorylation in NFAT's regulatory domain. We will determine structures of complexes with the Homer3 scaffolding protein and the transcriptional co-activator ARC105/Med15. We will study the functional relevance of these interactions. Finally, we will develop small-molecule inhibitors of the calcineurin/NFAT interaction to create immune-suppressive agents that specifically prevent NFAT dephosphorylation but will not affect calcineurin's general enzymatic activity. These new agents have the promise to avoid the long-term toxic side effects of traditional immune-suppressive drugs. The expected outcome of this research will be a better understanding of the mechanisms that regulate NFAT function triggering the immune response. In addition, new first-of-kind small molecule immune-suppressive agents will be developed.

Public Health Relevance

The planned research will elucidate mechanisms of T-cell activation. We will determine the topology of the complex consisting of the clonotypic a?T-cell receptor and the invariant CD3 components. In addition we will research the mechanisms of nuclear translocation of the nuclear factor of activated T cells (NFAT) and develop inhibitors of the NFAT/calcineurin interaction as new first-of-kind immune-suppressive agents.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Macromolecular Structure and Function B Study Section (MSFB)
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Leitner, Wolfgang W
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Harvard University
Schools of Medicine
United States
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Takeuchi, Koh; Sun, Zhen-Yu J; Li, Shuai et al. (2015) NMR resonance assignments of the catalytic domain of human serine/threonine phosphatase calcineurin in unligated and PVIVIT-peptide-bound states. Biomol NMR Assign 9:201-5
Gal, Maayan; Li, Shuai; Luna, Rafael E et al. (2014) The LxVP and PxIxIT NFAT motifs bind jointly to overlapping epitopes on calcineurin's catalytic domain distant to the regulatory domain. Structure 22:1016-27
Hyberts, Sven G; Robson, Scott A; Wagner, Gerhard (2013) Exploring signal-to-noise ratio and sensitivity in non-uniformly sampled multi-dimensional NMR spectra. J Biomol NMR 55:167-78
Gal, Maayan; Edmonds, Katherine A; Milbradt, Alexander G et al. (2011) Speeding up direct (15)N detection: hCaN 2D NMR experiment. J Biomol NMR 51:497-504
Takeuchi, Koh; Gal, Maayan; Takahashi, Hideo et al. (2011) HNCA-TOCSY-CANH experiments with alternate (13)C- (12)C labeling: a set of 3D experiment with unique supra-sequential information for mainchain resonance assignment. J Biomol NMR 49:17-26
Takeuchi, Koh; Frueh, Dominique P; Sun, Zhen-Yu J et al. (2010) CACA-TOCSY with alternate 13C-12C labeling: a 13Calpha direct detection experiment for mainchain resonance assignment, dihedral angle information, and amino acid type identification. J Biomol NMR 47:55-63
Takeuchi, Koh; Sun, Zhen-Yu J; Park, Sunghyouk et al. (2010) Autoinhibitory interaction in the multidomain adaptor protein Nck: possible roles in improving specificity and functional diversity. Biochemistry 49:5634-41
Kim, Sun Taek; Touma, Maki; Takeuchi, Koh et al. (2010) Distinctive CD3 heterodimeric ectodomain topologies maximize antigen-triggered activation of alpha beta T cell receptors. J Immunol 185:2951-9
Takeuchi, Koh; Frueh, Dominique P; Hyberts, Sven G et al. (2010) High-resolution 3D CANCA NMR experiments for complete mainchain assignments using C(alpha) direct detection. J Am Chem Soc 132:2945-51
Takeuchi, Koh; Heffron, Gregory; Sun, Zhen-Yu J et al. (2010) Nitrogen-detected CAN and CON experiments as alternative experiments for main chain NMR resonance assignments. J Biomol NMR 47:271-82

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