Three-dimensional structuj , , immunosuppressants and

The use of the immunosuppressant drug cyclosporin A (C5A) as a biochemical tool to study the signal transduction pathway in T cells has led to the discovery of a first family of immunosuppressant-binding proteins or “immunophilins’ the cyclophiins (Cyp). Another, chemically unrelated immunosuppressant molecule, FK506, was then found to be related to a second class of immunophilins, the FK506-binding proteins (FKBPs). This paper reviews the existing structural information on these immunophiins in the context of present knowledge of the biochemical mechanisms for immunosuppression. The formation of Cyp-CsA and FKBP-FK506 complexes, and the subsequent specific interaction of these complexes with the serine/threonine phosphatase calcineurin (CN), are key steps in the cascade of events that result in the desired immunosuppression. Knowledge of the conformation of the Cyp-CsA-CN and FKBP-FK506-CN ternary complexes is of significant biomedical interest, because mimics of the composite contact surfaces of, for example, Cyp-CsA or FKBP-FK506, could provide immunosuppressant drugs with improved pharmacological profiles.Braun, W. , Kallen, J., Mikol, V. , Walkinshaw, M. D. , W#{252}thrich, K. Three-dimensional structure and actions of immunosuppressants and their immunophiins. FASEBJ. 9, 63-72 (1995) Key Wonir: 9dacponn A cyclophilin FKBP#{149} FK506#{149} ? upamyln THE IMMUNOSUPPRESSANT DRUG CYCLOSPORIN A (C5A)2 (Sandimmune) was first used in human medicine in 1978 in a kidney transplant operation to prevent organ rejection (1). Since then, more than 250,000 organ transplant operations have been carried out, with a 1-year survival rate of greater than 90%. CsA is a cyclic undecapeptide fungal metabolite (Fig. 1) that was discovered in 1973 and found to have antifungal, antiparasitic, and immunosuppressant effects (2). Since its discovery, decisive advances have been made toward understanding the biochemical rational for this drug therapy. The biochemical and structural work initiated by the discovery of CsA is now playing a central role in elucidating general mechanisms of cellular signal transduction. Cyclosporin A binds to cyclophilin Initial work on the biochemical mechanism of CsA showed that it blocks the activation of T cells by preventing the production of cytokines, including interleukin-2 (IL-2) (3). An important advance was the isolation, from human spleen and bovine thymus cytosol, of a tight-binding protein receptor for CsA, which was christened cyclophilin (Cyp) (4). FK-binding proteins The first publications, in 1987, on the biological effects of the macrolide FK506 (Fig. 1) showed that it had an immunosuppressant action similar to CsA. Within 2 years it was possible, with the use of a FK506 affinity matrix, to isolate the relevant receptor, the 107-amino acid residue FK-binding protein (FKBP), from human spleen and bovine brain (5) and from Jurkat T cells (6). It was also shown that the chemically related macrolide rapamycin binds even more strongly to FKBP than FK506, although it has a different immunosuppressive mechanism (7). The CsA-Cyp and FK506-FKBP complexes block T cell signal transduction by inhibiting calcineurin The approach for identifying the requisite partner for the immunophilin-drug complex was extended with the use of a glutathionetransferase-FKBP12 and Cyp fusion protein (8, 9), and independently with the use of a glutathionetransferaseCyp fusion protein (9). The complexes of these fusion proteins with their cognate drugs were used to probe cellular extracts. The common target was found to be a serine/threonine phosphatase, calcineurin (CN) (8). CN is a heterodimer consisting of a 59-kDa catalytic A-subunit and a 19-kDa regulatory B-subunit. That this enzyme is the relevant target has been further substantiated by experiments showing that overexpression of CN in T cells renders the cells hypersensitive to antigen and more resistant to CsA and FK506 (10, 11). There is also a good correlation between the immunosuppressive activity of various cyclosporin derivatives and the extent of CN inhibition (12). The specific target for CN in the signal transduction cascade has not yet been properly characterized, but the favored hypothesis suggests that dephosphorylation of the cytosolic component of the transcription factor NF-AT (nuclear factor for activation of T cells) is required before it can pass into the nucleus (13, 14). Immunophiins are peptidyl-prolyl cis-trans isomerases Historically, cyclophiin A was discovered independently by tracking down the protein responsible for catalyzing the cistrans isomerization of Xaa-Pro amide bonds in oligopeptides (15, 16). This catalytic activity is also found in FKBPs. Inhi‘To whom correspondence and reprint requests should be addressed. 2Abbreviations: CsA, cyclosporin A; IL-2, interleukin-2; Cyp, cyclophilin; FKBP, FK-binding protein; CN, calcineurin; NMR, nuclear magnetic resonance; NFAT, nudear factor for activation of T cells.