Structural and chemical genetic insights into protein-protein interactions of phosphate-dependent cell signaling: The cases of calcineurin and protein kinase C iota

This thesis describes structural and chemical genetic insights into protein-protein interactions of phosphate-dependent cell signaling.; Section I focuses on the interaction between the phosphatase calcineurin and transcription factors of the NFAT family. We discovered novel small-molecule inhibitors of NFAT-calcineurin association (INCAs) that selectively disrupt this interaction. We introduce general methods for the rational development of competitive high-throughput screening assays for small-molecule inhibitors of protein-protein interactions by fluorescence polarization. INCAs do not compromise the enzymatic activity of calcineurin towards phosphorylated RII peptide, a substrate that does not require this NFAT docking site. In T cells, INCAs interfere selectively with the calcineurin-NFAT interaction. This substrate-selective enzyme inhibition represents a conceptual and practical advance over inhibition with drugs like cyclosporin A or FK506. Furthermore, we solved the 2.8-A crystal structure of the catalytic domain of human calcineurin A. Structural, mutational, and exhaustive computational INCA docking experiments lead us to a prediction of the VIVIT binding site near the catalytic domain's C-terminal beta sheet, but remote from its active site. We conclude with progress towards the complete backbone chemical shift assignment of the catalytic domain of calcineurin by multidimensional NMR spectroscopy.; Section II describes the three-dimensional structure of the V1 domain of human protein kinase C iota (PKC &igr;) by NMR spectroscopy. Dysregulation of PKC &igr; makes leukemia and breast cancer cells highly resistant to chemotherapeutic agents. The N-terminal V1 domain of PKC &igr; participates in cellular targeting and regulation, thus rendering it an attractive object for pharmacologic intervention. We describe the biosynthetic production and biochemical purification of recombinant PKC &igr; V1. Subsequently, the chemical shift assignment and the solution structure of the V1 domain were obtained using NMR spectroscopy. The protein assumes an ubiquitin-like beta grasp fold. We close with an outlook on work that investigates the interaction of PKC &igr; V1 with Par-6.; The Appendix addresses an interesting, but often overlooked, issue concerning the correspondence between spin-dynamic and pulse program phase behavior of NMR spectrometers. The results are important for spin state selective experiments, such as TROSY-type experiments, which I have implemented and relied on for the NMR spectroscopy of calcineurin.