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J. Biol. Chem., Vol. 280, Issue 23, 21965-21971, June 10, 2005

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Structure of Human Cyclophilin A in Complex with the Novel Immunosuppressant Sanglifehrin A at 1.6 Å Resolution^*

Joerg Kallen, Richard Sedrani¶, Gerhard Zenke||, and Juergen Wagner||

From the Protein Structure Unit, ^¶Protease Platform, and ^||Transplantation Research, Novartis Institutes for BioMedical Research, Basel CH-4002, Switzerland

Received for publication, February 11, 2005 , and in revised form, March 9, 2005.

	ABSTRACT

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Sanglifehrin A (SFA) is a novel immunosuppressant isolated from Streptomyces sp. that binds strongly to the human immunophilin cyclophilin A (CypA). SFA exerts its immunosuppressive activity through a mode of action different from that of all other known immunophilin-binding substances, namely cyclosporine A (CsA), FK506, and rapamycin. We have determined the crystal structure of human CypA in complex with SFA at 1.6 Å resolution. The high resolution of the structure revealed the absolute configuration at all 17 chiral centers of SFA as well as the details of the CypA/SFA interactions. In particular, it was shown that the 22-membered macrocycle of SFA is deeply embedded in the same binding site as CsA and forms six direct hydrogen bonds with CypA. The effector domain of SFA, on the other hand, has a chemical and three-dimensional structure very different from CsA, already strongly suggesting different immunosuppressive mechanisms. Furthermore, two CypA·SFA complexes form a dimer in the crystal as well as in solution as shown by light scattering and size exclusion chromatography experiments. This observation raises the possibility that the dimer of CypA·SFA complexes is the molecular species mediating the immunosuppressive effect.

	INTRODUCTION

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More than 2 decades ago, the discovery of cyclosporine A (CsA)¹ allowed spectacular progress in the field of organ transplantation (1). Since then the search for novel immunosuppressants that interfere with T cell activation/proliferation has resulted in the isolation of several new molecules from natural sources (2). The most prominent examples are FK506 and rapamycin. Besides their important therapeutic use, these drugs have also proven to be powerful tools for dissecting signal transduction pathways at the molecular level (3). It has been shown that the biological activity of CsA, FK506, and rapamycin is mediated by intracellular binding proteins called immunophilins. CsA binds to cyclophilins (e.g. cyclophilin A (CypA)), whereas FK506 and rapamycin bind to FK506-binding proteins (e.g. FK506-binding protein-12). Immunophilin binding is required but not sufficient for the immunosuppressive activity of these drugs. Each complex interacts with a third partner, the effector protein, to achieve the full biological response. Thus, the CypA·CsA and FK506-binding protein-12-FK506 complexes inhibit the serine/threonine phosphatase activity of calcineurin, thereby blocking the production of cytokines, including interleukin-2 (4). The FK506-binding protein-12-rapamycin complex inhibits the kinase FK506-binding protein-12/rapamycin-associated protein kinase (also known as RAFT or mTOR (5)) that is involved in interleukin-2 receptor-mediated T cell proliferation (6).

We have screened microbial broth extracts to identify CypA ligands with an immunosuppressive mechanism different from CsA (i.e. searching for the "rapamycin counterpart," which binds to CypA). This search resulted in the isolation from Streptomyces sp. A92–308110 of a new class of compounds named sanglifehrins (7, 8). Among the 20 different sanglifehrins isolated so far, sanglifehrin A (SFA) is the most abundant component (Fig. 1A). The affinity of SFA for CypA in a cell-free assay is remarkably high (IC₅₀ = 6.9 nM) (i.e. approximately 60-fold higher than that of CsA) (IC₅₀ = 420 nM) (9). Furthermore, SFA displays potent immunosuppressive activity in the murine mixed lymphocyte reaction (IC₅₀ = 170 nM), an in vitro immune response assay (9). The details of the mechanism by which SFA exerts its immunosuppressive activity at the molecular level are still under intense investigation. The first results describing the effects of SFA on T cells (10–12) and dendritic cells (13, 14) were published in the last few years. These data clearly indicate that SFA acts via a mode of action that is different from that of all other known immunophilin-binding compounds, namely CsA, FK506, and rapamycin.

In addition to its interesting biological profile, SFA has a unique chemical structure. The compound consists of a 22-membered macrocycle, bearing in position 23 a nine-carbon tether terminated by a highly substituted spirobicyclic moiety. The macrolide contains an E,E-diene, a short polypropionate fragment, and a tripeptide unit composed of valine and two unusual amino acids, piperazic acid and meta-tyrosine. It is the -nitrogen of piperazic acid that is involved in the amide bond formation, which stands in contrast to all other piperazic acid containing natural products isolated so far. The unique chemical structure of sanglifehrin combined with its immunosuppressive activity generated broad interest. Chemical efforts resulted in the synthesis of several fragments and finally culminated with the total syntheses by Nicolaou et al. (15) and Paquette et al. (16). Finally, degradation and synthetic work on the SFA macrocycle, together with x-ray analysis of the 22-membered macrocycle alone bound to CypA, has been published recently (17).

Here we present the crystal structure of the complex between human CypA and SFA. The high resolution of the x-ray data allowed the determination of the absolute configuration of all chiral centers of SFA and revealed the details of the CypA/SFA interactions. Furthermore, the structure of the CypA·SFA complex was compared with the structure of the CypA·CsA complex, since both immunosuppressants bind to the active site of CypA. Also, the finding that the CypA·SFA complex is present as a dimer in the crystal as well as in solution raises the possibility that the immunosuppressive effect of SFA might be mediated through a dimer of CypA·SFA complexes.

	MATERIALS AND METHODS

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Protein Preparation, Crystallization, and Data Collection—Human CypA was purified to homogeneity and SFA was obtained as described (7). The complex CypA·SFA was prepared by adding the ligand (100 mM solution in ethanol) in a 3-fold molar excess to the protein solution (12 mg/ml cyclophilin A, 20 mM NaCl, 20 mM Hepes, 0.02% NaN₃, pH 7.0). Cocrystallization was performed using a standard vapor diffusion hanging drop set-up, with VDX crystallization plates and siliconized microscope coverslips from Hampton Research. Crystallization droplets were made by mixing on the coverslips 1.0 µl of the protein solution with 1.0 µl of reservoir solution and equilibrated against 700 µl of reservoir solution at 20 °C. Commercially available screening kits from Hampton Research were used to find preliminary crystallization conditions. In the refined conditions, crystals grow within 3 weeks at 20 °C to a size of 0.1 x 0.1 x 0.5 mm³ with a reservoir of 80 mM magnesium acetate, 50 mM sodium cacodylate, pH 6.5, and 30% (w/v) polyethylene glycol 4000. A crystal was mounted in a glass capillary for data collection at 20 °C. X-ray diffraction data (347 frames of 1° rotation) were collected with a mar345 detector at the Swiss-Norwegian beamline of the European Synchrotron Radiation Facility, using a wavelength of, = 0.873 Å. Intensity data were processed with DENZO/SCALEPACK (18) and converted to structure factors with the CCP4 suite (19).

Structure Determination, Model Building, and Refinement—The molecular replacement solution was found with X-PLOR, version 3.1 (20), using the protein coordinates of CypA (21) and diffraction data from 8 to 3 Å. For the subsequent refinement steps, all data from 15 to 1.6 Å were used. The two complexes were refined without noncrystallographic symmetry restraints. Alternate cycles of model building with the graphics program O (22) and refinement yielded a model with 256 added water molecules, but no ligands yet, with an R-factor of 23.2%. The resulting 2F_o – F_c and F_o – F_c maps were of excellent quality for the missing ligands and allowed the clear determination of the absolute configurations at all 17 chiral centers of SFA. Further refinement with REFMAC version 5.0 (23), water insertion with ARP/wARP (24), and rebuilding steps yielded the final model, consisting of two complexes CypA·SFA (including residues 2–165 of CypA) and a total of 361 water molecules, which has an R-factor of 16.3% and R_free of 18.5% (no cut-off, 15 to 1.60 Å, working set of 39,452 unique reflections, test set of 2096 reflections).

The quality of the model was assessed with PROCHECK version 3.3 (25) and REFMAC. 86.5% of the amino acids are in the most favored region, and 13.5% are in the additionally allowed region of the Ramachandran plot (no amino acids are in disallowed regions). The overall G-factor is 0.16, with r.m.s. bond lengths of 0.007 Å and r.m.s. bond angles of 1.18°. Buried solvent-accessible areas were calculated with the program AREAIMOL as implemented in the CCP4 suite (19), using a probe radius of 1.4 Å.

Dynamic Light Scattering and Size Exclusion Chromatography— Dynamic light scattering experiments were performed with a DynaPro-801 molecular sizing instrument in conjunction with the software package "Dynamics (version 3.30)," both from Protein Solutions Ltd. The complexes were prepared as 1:1 mixtures of CypA and ligand (both at 56 µM), in 100 mM NaCl, 20 mM HEPES, pH 7.4, and measurements were performed at 20 °C.

Size exclusion chromatography to detect monomer/dimer formation was done by injecting samples of 40-µl total volume containing 29 µM CypA and 126 µM ligand (or no ligand, as a reference) at pH 7.0 in 20 mM ammonium bicarbonate buffer. The 5 x 100-mm column was packed with BioGel P30 (45–90 µm) from Bio-Rad, and the flow rate was 0.06 ml/min. The collected fractions with CypA (detected at 210, 254, and 280 nm) were then subjected to liquid chromatography/mass spectroscopy analysis for verification of complex formation and molecular weight of ligand.

	RESULTS

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The SFA-binding Site—We have determined the three-dimensional structure of CypA bound to SFA by x-ray crystallography. The 1.6 Å resolution data were phased by molecular replacement using CypA (21) as a search model, yielding a dimer of CypA·SFA complexes as solution. The atomic structure includes residues 2–165 of two CypA molecules, two SFA ligands and a total of 361 water molecules. The results of the crystallographic refinement are summarized in Table I. The high resolution of the x-ray data allowed the determination of the absolute configuration of all 17 asymmetric centers of the macrocyclic and spirobicyclic fragments of SFA (Fig. 1, A and B).

View larger version (31K): [in this window] [in a new window]   FIG. 1. Chemical structure and conformation of sanglifehrin A bound to CypA. A, chemical formula, atom numbering, and absolute configuration of the 17 chiral centers of SFA as determined by the electron density at 1.6 Å resolution. B, wall-eyed stereo view of the final 2Fo – Fc electron density (contoured at 1, resolution range 15 to 1.60 Å) for SFA. Cyan, carbon; red, oxygen; blue, nitrogen.

View larger version (70K): [in this window] [in a new window]   FIG. 2. Sanglifehrin A fits nicely into the active site of CypA. Shown is a wall-eyed stereo view of SFA (ball-and-stick model; cyan, carbon; red, oxygen; blue, nitrogen) bound to the active site of CypA (stick model; yellow, carbon; red, oxygen; blue, nitrogen; green, sulfur), for which the van der Waals surface is depicted in white. Water molecules are shown as green spheres. The piperazic acid moiety of SFA is deeply buried in a hydrophobic pocket formed by the amino acids Phe60, Met61, Phe113, and Leu122 of CypA. The presence of the linker between the macrocycle and spirobicycle of SFA imposes a side-chain reorientation on Trp121, compared with unliganded CypA.

View larger version (46K): [in this window] [in a new window]   FIG. 3. Comparison of the CypA·SFA and CypA·CsA complexes. A, detailed view of the interactions between the macrocycle of SFA (ball-and-stick model, cyan) and CypA (stick model, yellow). There are six direct intermolecular hydrogen bonds (cut-off 3.2 Å) formed by the tripeptide moiety Pip-meta-Tyr-Val of SFA. The distances indicated are averaged over the two independently refined complexes of the dimer. B, detailed view of the interactions between CsA (ball-and-stick model, magenta) and CypA (stick model, white). The projected view is identical to the one in A. There are five direct intermolecular hydrogen bonds (cut-off 3.2 Å) made by CsA (the hydrogen bond between CO-MeLeu10 and NE1-Trp121 is only indicated in the scheme at the right). The main side-chain differences between the complexes CypA·SFA and CypA·CsA occur for Trp121 (not shown) and Arg55.

Overall, the x-ray crystal structure of SFA bound to CypA confirms that the affinity of the ligand for CypA is primarily mediated by the 22-membered macrocycle, whereas the spirobicyclic unit remains essential for the immunosuppressive activity.

Comparison of the CypA·SFA and CypA·CsA Complexes: A Case of Induced Fit and Binding with Opposite Peptide Backbone Directions—A least squares superposition of CypA·SFA and CypA·CsA shows that the protein backbones are similar in both complexes. The r.m.s. deviation of C positions for CypA in complex with SFA (monomer A in the present structure) and CsA (accession code 1CWA), using residues 2–165, is 0.30 Å. The largest difference in the position of backbone atoms occurs for the 68–73 loop, with maximal deviations of 1.7 and 1.4 Å for the Cs of Asn⁷¹ and Gly⁷², respectively. In addition, there are two important side-chain differences in the ligand binding pockets that occur for Arg⁵⁵ (Fig. 3, A and B) and Trp¹²¹. These latter differences are essential for binding the two respective ligands. The side chain of Arg⁵⁵, mediating a hydrogen bond with the carbonyl oxygen of valine (SFA), rotates into a new position in the CypA·CsA complex in order to form a hydrogen bond with the carbonyl oxygen of MeLeu¹² (CsA) and to avoid a steric clash with Val⁵ (CsA). In return, the latter position of Arg⁵⁵ from the Cyp·CsA complex would lead to a steric clash with the ester and diene groups of SFA. The side chain of Trp¹²¹ rotates into a new position in the CypA·CsA complex in order to form a hydrogen bond with the carbonyl oxygen of MeLeu⁹ (CsA). In return, the latter position of Trp¹²¹ from the CypA·CsA complex would lead to a steric clash with the linker that extends between the macrocycle and spirobicycle of SFA. These two fundamental side-chain adaptations can be regarded as examples of induced fit in order to bind two chemically very different ligands.

View larger version (25K): [in this window] [in a new window]   FIG. 4. SFA and CsA bind to CypA with opposite peptide directionalities. Wall-eyed stereo view of the superposition between the tripeptide moiety of SFA (cyan, carbon; red, oxygen; blue, nitrogen) and the corresponding region of CsA (magenta, carbon; red, oxygen; blue, nitrogen). The peptide directionality is from left to right for CsA and from right to left for SFA. The main-chain carbonyl oxygens of meta-Tyr and Val for SFA superimpose with the carbonyl oxygens of MeVal11 and MeBmt1 for CsA, respectively (cf. black circles). The main-chain nitrogens of meta-Tyr (SFA) and Abu2 (CsA), although not coincident (cf. black arrows), are both located such that they can accept a hydrogen bond from O-Asn102 (Fig. 3). The piperazine ring and the valine side chain of SFA superimpose with the CsA side chains of MeVal11 and Abu1, respectively.

View larger version (38K): [in this window] [in a new window]   FIG. 5. Crystal structure of the CypA·SFA dimeric complex. A, overview of the dimer (dimer axis vertical, gray line) of CypA·SFA complexes (yellow/magenta and gray/gray). SFA ligands are shown as ball-and-stick models. The spirobicyclic (top) and -ketobutyrate (bottom) moieties are exposed to the solvent. B, close-up of the dimer interface (dimer axis horizontal, gray line) between the two CypA·SFA complexes (yellow/cyan and white/magenta). SFA ligands are shown as CPK models; the local dyad is approximately parallel to the direction defined by the conjugated double bonds in the macrocycle of SFA. Selected CypA residues and SFA carbon atoms (italic type) are labeled in black and white for the bottom and top complex, respectively. For SFA atom numbering, see Fig. 1A.

Whereas both immunosuppressants SFA and CsA, despite their completely different chemical structures, fit nicely into the active site of CypA, their effector domains are clearly very different, already strongly suggesting that the respective immunosuppressive mechanisms are different. Indeed, a superposition with the ternary complex CypA·CsA·calcineurin (31, 32) shows that CypA·SFA cannot interact with calcineurin.

Structure of the Local Dimer of CypA·SFA Complexes: The Dimer Hypothesis—The two CypA·SFA complexes are related by an approximate local dyad axis (rotation angle = 176°, calculated from the least squares superposition of protomers A and B using residues 2–165), which is located between the two SFA ligands and oriented almost parallel to the longest dimension of SFA (Fig. 5, A and B). The slight disturbance of the C2 symmetry can be explained by crystal packing (see below). With the exception of the spirobicyclic and -ketobutyrate moieties (which extend toward the outside of the dimeric complex), all of the remaining parts of the SFA ligands are deeply buried in the dimer. Upon dimer formation, a total of 1504 Ų of the solvent-accessible surface from the two monomers is buried (721 Ų from the protein molecules and 783 Ų from the SFA ligands). Interestingly, these values are even larger than the ones for monomer complex formation itself (see above). The two SFA ligands make extensive vdW contacts with each other along the local dyad. In particular, the meta-tyrosine side chain is in close proximity to the E,E-diene fragment of the neighboring SFA molecule, and the C26–C28 fragments form vdW contacts with each other. In addition, a number of water-mediated hydrogen bonds are formed between the two SFA ligands (e.g. between the C41 carbonyl oxygen and the C31 hydroxy group), between a SFA ligand and the neighboring protein molecule of the dimer (e.g. between the C17 hydroxy group and the Asn¹⁰² side chain), and between the two protein molecules. Finally, a direct hydrogen bond links NE1-TrpA¹²¹ and CO-ArgB¹⁴⁸ (distance 3.1 Å).

The two CypA·SFA complexes in the asymmetric unit were refined without noncrystallographic symmetry restraints. They show an r.m.s deviation of 0.26 Å for the C positions of residues 2–165 after least squares superposition. The main side-chain differences in proximity of the SFA binding site occur for Trp¹²¹, Lys¹²⁵, and Arg¹⁴⁸. These differences can be explained by differences in crystal contacts: for protomer A, ArgA¹⁴⁸ is involved in a salt bridge with GluB'¹³⁴ of a neighboring protomer B' in the lattice and thus adopts a different side-chain conformation than ArgB¹⁴⁸. As a consequence, TrpB¹²¹, which stacks on ArgA¹⁴⁸, adopts a different side-chain conformation than TrpA¹²¹. Finally, LysA¹²⁵, which stacks on TrpA¹²¹, adopts a different side-chain conformation than LysB¹²⁵. Since the side-chain conformations of TrpA¹²¹ and LysA¹²⁵ are not influenced by crystal packing, they probably represent the state for the dimer in solution.

The fact that the crystallographically observed CypA·SFA dimer is stabilized by numerous interactions raised the possibility that this dimer also exists in solution. Toward this end, monomer/dimer formation was followed by size exclusion chromatography as well as dynamic light scattering. Using the latter method, CypA alone has an apparent molecular mass of 14 kDa, the actual mass being 18 kDa. In contrast, the apparent molecular mass of the CypA·SFA complex was 31 kDa, clearly confirming the existence of a dimer in solution. In a further experiment, a SFA derivative was tested that retains a high affinity for CypA but for which dimer formation is predicted to be impaired. The apparent molecular mass of (26S,27S)-dihydroxysanglifehrin A (33) in complex with CypA is 17 kDa, confirming the prediction, based on the crystal structure, that the two additional hydroxy groups would lead to steric clashes with themselves and thus block dimerization (in the intact dimer, the atoms C26 and C27 are at distances of 3.8 and 4.0 Å with their corresponding partners) (Fig. 5B). Also, size exclusion chromatography (followed by liquid chromatography/mass spectroscopy for verification of complex formation and molecular mass of ligand) showed that CypA complexed with the latter derivative displayed a retention time (6.5–7 min), which is very similar to unliganded CypA and CypA·CsA and thus corresponds to a monomer. CypA bound to the 22-membered SFA macrocycle (17) alone showed the same result, again indicating a monomer. The complex CypA·SFA, on the other hand, had a clearly shorter retention time of 5 min, which indicates probable dimer formation.

The spirobicycle linker, which is missing in the case of the 22-membered SFA macrocycle alone, mediates important vdW contacts within the dimer and also induces a side-chain conformation of Trp¹²¹ (Fig. 2), which enables interactions with the other monomer. The latter influence on Trp¹²¹ could be denoted as the "Trp switch," which yields the intersubunit hydrogen bond between NE1-TrpA¹²¹ and O-ArgB¹⁴⁸. Importantly, both derivatives of SFA (dihydroxy-SFA and the macrocycle) lack immunosuppressive activity in the murine mixed lymphocyte reaction, although their affinity for CypA is unaffected (Table II). 42-N-methyl-SFA, on the other hand, is an example of a derivative retaining immunosuppressive activity (Table II) and still forming a dimer in solution. The apparent molecular mass of the CypA·42-N-methyl SFA complex using the dynamic light scattering method was 26 kDa. Taken together, these results suggest that the ability of SFA derivatives to form a dimer similar to the one observed by crystallography might be related to their immunosuppressive activity as measured in the murine mixed lymphocyte reaction assay.

	DISCUSSION

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	FOOTNOTES

 
^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 1YND) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

To whom correspondence should be addressed: Protein Structure Unit, Novartis Institutes of BioMedical Research, CH-4002 Basel, Switzerland. Tel.: 4161-324-5579; Fax: 4161-324-2686; E-mail: joerg.kallen{at}pharma.novartis.com.

¹ The abbreviations used are: CsA, cyclosporine A; CypA, cyclophilin A; SFA, sanglifehrin A; r.m.s, root mean square; Pip, piperazic acid; vdW, van der Waals; Bmt, (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine; Abu, (L)--aminobutyric acid.

	ACKNOWLEDGMENTS

 
We thank J. Causevic and M. Zurini for protein purification and light scattering experiments as well as G. Hegy for size exclusion chromatography and liquid chromatography/mass spectroscopy measurements. We thank C. Maurer, C. Vedrine, and V. Quesniaux for CsA/CypA ELISA measurements. Experimental assistance from the staff of the Swiss-Norwegian Beam Lines at the European Synchrotron Radiation Facility, Grenoble, is acknowledged. We thank M. Walkinshaw for initial interest and discussions in the project.

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