Peptidyl-prolyl cis-trans isomerases (PPlases) represent a new and rapidly expanding family of proteins that may regulate signal transduction pathways by catalyzing the cis-trans isomerization of peptide bonds. The immunosuppressive agents, cyclosporin A and FK-506 bond to and inhibit two different cytosolic PPlases (cyclophilin and the FK-506 binding protein) which are believed to be involved in the early stages of T-cell activation. Another PPlase (nina A gene product from Drosophila) has been implicated in the regulation of the visual transduction pathway. The goal of this project is to characterize these proteins and to determine the three-dimensional structures of the PPlases and PPlase/ligand complexes using NMR spectroscopy and X-ray crystallography. Cyclophilin, the FK-506 binding protein (FKBP), and the other PPlases that are required for these structural studies will be isolated and purified from natural sources or from cells that overexpress the proteins engineered in-house using molecular biological procedures. In addition to unlabeled PPlases, proteins isotopically labeled with 15N and 13C will be prepared to facilitate the NMR studies. Cyclosporin A and ascomycin (an analog of FK- 506) uniformly labeled with 13C will be isolated from cells that produce these compounds grown on isotopically labeled media. Isotopically labeled PPlase substrates will also be synthesized for the NMR studies. Using the isotopically labeled inhibitors (cyclosporin A and ascomycin) and substrates, isotope-edited proto NMR experiments will be employed in studies of cyclophilin/cyclosporin A, ascomycin/FKBP, and other PPlase/ligand complexes. These experiments, which can be rapidly performed and analyzed will be used to determine the enzyme-bound conformations of the ligands, identify the portions of the ligands involved in the interaction with the enzyme, and provide structural information on the active site. The complete three-dimensional structures of cyclophilin, FKBP, and PPlase/ligand complexes will be determined using isotopically labeled proteins and heteronuclear three-dimensional NMR spectroscopy, as well as by x-ray crystallographic methods. It is expected that the experimentally-derived three-dimensional structures of PPlases and PPlase/ligand complexes will help define the enzymatic mechanism(s) of this interesting class of proteins and aid in the design of PPlase inhibitors that are clinically useful as immunosuppressants or that block other biochemical processes of pharmacological and clinical interest that are regulated by this class of proteins.
