Journal of Molecular Biology
Volume 283, Issue 2, 23 October 1998, Pages 435-449
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Regular article
X-ray structures and analysis of 11 cyclosporin derivatives complexed with cyclophilin A1

https://doi.org/10.1006/jmbi.1998.2108Get rights and content

Abstract

Eight new X-ray structures of different cyclophilin A/cyclosporin-derivative complexes are presented. These structures, combined with the existing three published cyclosporin complexes, provide a useful structural database for the analysis of protein-ligand interactions. The effect of small chemical differences on protein-ligand hydrogen-bonding, van der Waals interactions and water structure is presented.

Introduction

Cyclosporin A (CsA) and FK506 are established drugs used in the treatment of a variety of autoimmune diseases and are also useful biochemical tools (Schreiber et al., 1993). They led to the discovery of the immunophilin family of proteins and have been used to study the signal transduction pathway in T-cells (Galat & Metcalfe, 1995). CsA is a cyclic undecapeptide which has 7 of the 11 amides in the N-methylated form (Figure 1). Initial attempts at relating the molecular structures of many different CsA derivatives with biological function were based on the 3D structure of free CsA. The NMR structure of CsA in chloroform and the structures in various single crystal forms (Loosli et al., 1985) all contain a compact antiparallel β-sheet, with four intramolecular hydrogen bonds involving the four non-methylated amide-NH groups. This tightly folded structure results in a very hydrophobic outer surface.

The immunosuppressive mechanism of CsA requires formation of a tightly bound binary complex of CsA with cyclophilin A (CypA), a ubiquitous 165 amino acid long cytosolic protein (Handschumacher et al., 1984). The composite CsA/CypA surface binds and inhibits the serine/threonine phosphatase calcineurin Griffith et al 1995, Kissinger et al 1995, preventing further transduction of the immuno-activation signal. Cyclophilins also have peptidyl-prolyl isomerase (PPIase) activity and they can speed up the refolding of proteins in vitroSchonbrunner et al 1991, Kern et al 1995. This activity seems unrelated to the mechanism involved in immunosuppression.

The conformation of the inhibitory complex of CsA bound to cyclophilin is very different from the dominant conformations of free CsA in chloroform or in single crystals. In the cyclophilin-bound form all CsA peptide bonds are trans and none of the intramolecular hydrogen bonds found in the free structure are present. A single intramolecular hydrogen bond exists between the hydroxyl group on the Bmt-1 side-chain and carbonyl oxygen of MeLeu-4. This conformation has been observed in solution by NMR and in two different crystal forms (Altschuh et al., 1994). X-ray structures of a decameric Pflugl et al 1993, Pflugl et al 1994 and monomeric Mikol et al 1993, Taylor et al 1997 crystal form of the CypA/CsA complex have been determined and the CsA binding site has been confirmed as the PPIase active site. Furthermore, only residues 9, 10, 11, 1, 2 and 3 of the CsA ligand are in contact with CypA; the remaining residues (4 through 8) protrude out from the CypA surface. This hydrophobic protrusion is termed the “effector loop” and is implicated in specific interactions with calcineurin (Liu et al., 1992). The 3D structure of the CypA/CsA complex therefore helps explain the initially puzzling observation that binding of a cyclosporin derivative to cyclophilin was a requirement, but not sufficient, for immunosuppressive activity (Sigal et al., 1991). Even small chemical changes to residues of the effector loop can destroy the immunsuppressive effect without reducing the ability to bind cyclophilin Papageorgiou et al 1994, Papageorgiou et al 1994.

This paper describes the structures of eight chemically distinct CsA-analogues complexed with CypA which show modifications in both the cyclophilin-binding residues and the effector loop. A comparison of each complex with the native CypA/CsA structure shows differences in conformation, molecular rigidity and water binding. This small library of closely related ligand structures also provides a picture of the frequently unpredictable effects of small chemical changes on 3D structure and biological activity.

Section snippets

X-ray results

X-ray structures of a total of 11 cyclosporines co-crystallised with cyclophilin are presented. The labelling scheme is shown in Figure 1 and chemical structures of the cyclosporines are described in Table 1. For completeness, these include three previously published structures: native CsA (Mikol et al., 1993), 116450 ((4-methyl)MeBmt1-CS) (Mikol et al., 1994) and 224698 ((5-hydroxy)Nva2-CS) (Mikol et al., 1995). Most of the CypA/CsA-analogue complexes were grown using a cross-seeding technique

Discussion

In this reported series of CsA-analogues, both the ability to bind cyclophilin and the immunosuppressant activity vary considerably (Table 1). In general, derivatives at residues 9, 10, 11, 1 or 2 which change the cyclophilin-binding surface of CsA, diminish binding to cyclophilin. This diminished binding correlates well with a reduction of immunosuppressive activity Wenger 1985, Fliri et al 1993, Sigal et al 1991. Similarly, modifications of residues 4, 5 and 6 which comprise the protruding

Crystallisation

Recombinant human CypA was purified and concentrated to between 15 and 20 mg ml−1 (1 mM). CsA analogues were synthesised using methods described Seebach et al 1993, Wenger et al 1992, Wenger 1990 and were dissolved in DMSO to a concentration of 10 mg ml−1 (8 mM). An equimolar solution of CypA and CsA analogue was incubated at 310 K for 30 minutes and centrifuged at 14,000 rev min−1 Crystals of the CypA/CsA-analogue complexes were grown by vapour diffusion at 295 K using the hanging drop method.

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    Edited by I. A. Wilson

    2

    Present address: J. Kallen, Novartis Pharma AG, S-503.12.08, 4002 Basel, Switzerland; V. Mikol, CRVA, Rhone-Poulenc Rorer, 13 Quai J. Guesde B.P. 14, F-94403 Vitry/Seine, France.

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