This application, an extension of research derived from a program project grant, has as its focus the structural and functional role of the cyclophilin (CyP) family of proteins. In this rapidly evolving field, we will continue our structural and kinetic characterization of the interaction between the Cyclosporin A (CsA) related ligands as they associate with the CyP family members and calcineurin, currently considered a major target within various cell types. A central focus will be NMR methods coordinated with an assessment of kinetic and thermodynamic parameters of these interactions with stopped-flow fluorescence studies. Human CyP-18, the predominant cytosolic receptor, as well as new CyP-40c species will be provided by isolation from tissues and in recombinant form from E. Coli. We will also prepare and study site-directed mutant forms of these species. In addition, two new membrane bound CyP species, CyP-22m, richly associated with the endoplasmic reticulum and CyP-20, will be available in their native glycosylated state. Recently, it has been shown that the ligand- immunophilin complex associates with calcineurin in a Ca++-dependent reaction, thus a variety of multidimensional metallo-NMR as well as 1H, 13C and 15N techniques are possible. Specifically, 113Cd NMR methods will be used to provide an isomorphic, magnetically active probe of the Ca++ sites as they are involved in the structure and dynamics of these multimeric immunophilin complexes. Additionally, multidimensional NMR methods utilizing 1H, 13C and 19F isotopically labeled CsA derivatives will aid in the identification of specific amino acids located at sites of interaction in the multimeric complex. Independent assessment of kinetic aspects of these interactions will be accomplished by transient state fluorescence measurements using the single tryptophan in CyP which has been shown to sensitively reflect CyP's interaction with ligands such as CsA. This interaction will be assessed by stopped-flow fluorescence studies to develop a complete kinetic and thermodynamic understanding of the CsA-CyP interaction as well as provide a basis for characterizing new CyP proteins and site-directed mutant forms. These studies will also be extended to examine the interaction of calcineurin with the immunophilin complex. Preliminary information obtained by chemical crosslinking has revealed an unexpected topological relationship of the components in the CyP-calcineurin complex. These results will be extended to the substrate site and potential new cellular targets for the CyP-CsA complex may also be examined with these physical techniques. These unique opportunities for integrating different talents are expected not only to define specific amino acid loci of interaction with dimensional information but also serve as a guide for new concepts in drug synthesis and an understanding of the mechanism of action of this new class of immunosuppressive agent.
