The objectives of the study are to understand the molecular mechanisms of catalysis, regulation, and inhibition of the serine/threonine protein phosphatases calcineurin and protein phosphatase 1 (PP1). Since little is known regarding the mechanism of phosphate ester hydrolysis of these enzymes, the long term goals of this study will be to arrive at a model for the catalytic mechanism which can be tested by biochemical and spectroscopic approaches. The broad objectives are: 1) to characterize the binuclear metal centers of calcineurin and PP1 and determine their role in catalysis; 2) to understand the structural basis for divalent metal ion activation of these phosphatases; 3) to examine the role(s) of specific amino acid residues surrounding the binuclear metal centers; 4) to investigate the inactivation of calcineurin by mechanism-based inhibitors; and 5) to understand the structural basis for calcineurin inhibition by cyclosporin A (CsA)-cyclophilin complexes. The above objectives will be accomplished by using a variety of techniques including EPR, Mossbauer, NMR, ENDOR, and ESEEM spectroscopies. In addition, metal analyses and determinations of enzyme activity of calcineurin and PP1 in various states will be carried out in order to determine whether a correlation exists between enzyme activity and redox state of the bound metal ions. These spectroscopic techniques should also be able to resolve discrepancies between current active site models of calcineurin and PP1, and will provide additional handles for uncovering aspects regarding the mechanism of catalysis. X-ray crystallography will be applied to understand the similarities and differences of calcineurin inhibition by CsA-cyclophilin and FK506-FKBP complexes. Since calcineurin is the target of the immunosuppressant drugs cyclosporin A and FK506, a knowledge of the mechanism of CsA-cyclophilin inhibition and details relating to the mechanism of catalysis may assist in the future design of novel immunosuppressive agents with decreased toxicity. The continued study of calcineurin inactivation by mechanism-based inactivators will provide information about the role of active site residues and can assist in the design of additional structural elements for increased inhibitor potency.
