Vincent Mikol

Three-dimensional structure and actions of immunosuppressants and their immunophilins.

The use of the immunosuppressant drug cyclosporin A (CsA) 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 cyclophilins (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 immunophilins 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., Wüthrich, K. Three‐dimensional structure and actions of immunosuppressants and their immunophilins. FASEB J. 9, 63‐72 (1995)

Conformational polymorphism of cyclosporin A

BACKGROUND Cyclosporin A (CsA) is a cyclic undecapeptide fungal metabolite with immunosuppressive properties, widely used in transplant surgery. It forms a tight complex with the ubiquitous 18 kDa cytosolic protein cyclophilin A (CypA). The conformation of CsA in this complex, as studied by NMR or X-ray crystallography, is very different from that of free CsA. Another, different conformation of CsA has been found in a complex with an antibody fragment (Fab). RESULTS A detailed comparison of the conformations of experimentally determined structures of protein-bound CsA is presented. The X-ray and NMR structures of CsA-CypA complexes are similar. The Fab-bound conformation of CsA, as determined by X-ray crystallography, is significantly different from the cyclophilin-bound conformation. The protein-CsA interactions in both the Fab and CypA complexes involve five hydrogen bonds, and the buried CsA surface areas are 395 A2 and 300 A2, respectively. However, the CsA-protein interactions involve rather different side chain contacts in the two complexes. CONCLUSIONS The structural results presented here are consistent with CypA recognizing and binding a population of CsA molecules which are in the required CypA-binding conformation. In contrast, the X-ray structures of the Fab complex with CsA suggest that in this case there is mutual adaptation of both receptor and ligand during complex formation.

Crystallisation and preliminary X-ray diffraction studies of cyclophilin-tetrapeptide and cyclophilin-cyclosporin complexes

Recombinant human cyclophilin has been co‐crystallised with a number of peptides to give crystals suitable for X‐ray analysis. The crystal complexes for which heavy‐atom derivatives have been prepared and X‐ray data collected are: cyclophilin with N‐acetyl‐Ala‐Ala‐Pro‐Ala‐amidomethylcoumarin (I) which crystallises in space group P212121 with a = 108.2, b = 123.0, c = 35.8 Å, and cyclophilin with cyclosporin (II) which crystallises as tetragonal plates in space group P41212 or P43212 with a = b = 94.98, c = 278.55 Å.

CONFORMATIONAL POLYMORPHISM IN PEPTIDIC AND NONPEPTIDIC DRUG MOLECULES

Macrolide ligands that bind FK506 binding proteins and cyclosporins that bind cyclophilins are chemically dissimilar but can share a number of structural and biological properties. Both families of ligands have very different conformations in the free state compared to those adopted when complexed with their binding protein. These transformations involve twisting from cis to trans about specific amide bonds, which result in significant changes in the hydrogen‐bonding capabilities of the molecular surfaces. The three‐dimensional structure of a new cyclosporin‐like ligand (SDZ214‐103) is described in the free crystalline state and bound to cyclophilin, and is shown to have a very different conformation from cyclosporin A in the free crystal, but a very similar conformation when bound to cyclophilin.