Conformational polymorphism of cyclosporin A

Conformational polymorphism of cyclosporin A Daniele Altschuh', Werner Braun 2, Joerg Kallen3, Vincent Mikol 3, Claus Spitzfaden 2 , Jean-Claude Thierry', Olivier Vix1, Malcolm D Walkinshaw 3* and Kurt Wiithrich2 'Institut de Biologie Moleculaire et Cellulaire du CNRS, 15 rue Descartes, 67084 Strasbourg Cedex, France, 2lnstitut fur Molekularbiologie und Biophysik, ETH-Hbnggerberg, 8093 Zurich, Switzerland and 3Preclinical Research, Sandoz Pharma Ltd, 4002 Basel, Switzerland 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 proteinbound 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 CypAbinding 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.

Structure 15 October 1994, 2:963-972 Key words: antibody recognition, cyclophilin, cyclosporin A, ligand binding

Introduction Cyclosporin A (CsA) is an immunosuppressant drug (Fig. 1) which binds to an intracellular receptor protein, cyclophilin (Cyp). The binary complex of the drug with this protein inhibits the phosphatase calcineurin, thus blocking all subsequent steps in the immunostimulatory signal transduction pathway of T cells [1]. In view of the use of CsA (Sandimmun ) as a drug to prevent organ transplant rejection and its use in the treatment of other immune-related diseases including psoriasis [2], a study of the bioactive conformations of CsA is of considerable interest for the design of potential analogue drugs with improved action profiles. The likely target for CsA in the cell is cytosolic cyclophilin A (CypA) (Fig. 2). The highly homologous proteins cyclophilin B and cyclophilin C are also found in the endoplasmic reticulum [3]. NMR [4,5] and X-ray [6] structures of CsA-CypA complexes show CsA with all 11 amide bonds in a trans conformation. High resolution X-ray structures of complexes of cyclosporin with human cyclophilin B [7] and mouse cyclophilin C [8] have also been determined. A distinctly different conformation of CsA has been observed in the crystal structure of a complex of CsA with an anti-CsA antibody fragment (Fab) [9]. Amino acid sequences for the variable domain of this Fab fragment and for human CypA are given in Fig. 2.

There is a considerable amount of structural information concerning free cyclosporin. The NMR structure of CsA in chloroform [10,11] is very similar to that found in various single crystal forms [10,12]. Both show a compact antiparallel -sheet structure, with four intramolecular hydrogen bonds involving the four non-methylated amide-NH groups (Fig. 3) and the amide bond between N-methylleucine (MeLeu) 9 and MeLeulO in a cis conformation. The NMR structure of CsA in a complex with lithium chloride in tetrahydrofurane shows a unique all-transannular conformation [13]. CsA is only sparingly soluble in water, which precludes a detailed NMR study. However, NMR studies of CsA in methanol and aqueous methanol [14] suggest the presence of one dominant CsA conformation, consistent with that of CsA in chloroform, though at least five other conformations are also present. The three-dimensional structure of the CsA analogue [DMe-Ser3, D-Ser-(O-Gly)8]-CsA has been determined in dimethyl sulphoxide (DMSO) and water and was found to be almost identical to the cyclophilin-bound form of CsA [15]. In this paper we compare the structures and environments of CsA in the NMR structure of a CypA-CsA complex in solution [16], the X-ray structure of a monomeric crystal form of a CypA-CsA complex [17] and the X-ray crystal structure of a Fab-CsA complex [9].

*Corresponding author.

© Current Biology Ltd ISSN 0969-2126

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H,C

H

H,C C,-

HNC

0

.

CH

°

BC

C

H

C ·

H3C

H3 C

CH

CL%

X 50

bMWITVNDLI AXVD(PISQV SZEIPAWVP IMtERALS TS Y 30 40 10 20

CypA

*

*

** *

SCaHIIPGF 60 **

CH

***

bqIG'"M NXI'GSIYG EENIL 70 80

**

Kr.GCGLS AT'PNSS 90 100 110

ETICAI 120

*

WDGRVWFG K 130

Fab Light Cain

NIVE A 140

ff

/TS/K'AD 160

150

CE

DIQIIlSS LSASULG IStCRAS 10 20

UIOW DGIVKILW 40 50

IS TY 30

** *

TSZRSGWPS IRPSGGSGD YSII 60 70

avy chin

rb

Fig. 1. The CsA molecule. Chemical structure of CsA with the standard numbering of the amino acid residues. Abbreviations: Abu, L-a-aminobutyric acid; MeBmt, (4R)-4[(E)-2-butenyl]4,N-dimethyl-L-threonine; MeLeu, Nmethylleucine, MeVal, N-methylvaline; Sar, sarcosine.

ED 80

EVLS8

P 90

PP

LPG-SLKL SCTSrm1s DYYnAHW SfEVW 20 30 40

* *

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M9i52A 52A

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A DIVI60 60

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Y0 90

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Results and discussion Backbone conformations of CypA-bound and Fab-bound CsA One crystal form of the CypA-CsA complex has an interesting oligomeric packing arrangement in which two close-packed protein pentamers form a decameric sandwich and shield the hydrophobic CsA molecules

50

*

A3109ZV 109

Fig. 2. Sequences and numbering scheme for CypA and for the variable domains of the Fab fragment. Stars represent residues which are within 4.0 A of CsA. The numbering scheme for the Fab fragment domains follows Kabat et al. [36]. Dashes in the Fab sequence indicate missing residues according to Kabat's numbering. The hypervariable loops of the Fab are the light chain residues 24-34 (L1), residues 50-56 (L2), residues 89-97 (L3) and the heavy chain residues 31-55 (H1), residues 50-65 (H2) and residues 95-102 (H3).

from bulk solvent [6]. Another monomeric crystal form of the CypA-CsA complex has also been analyzed [17]. Despite the very different crystal environments in the two structures, the average difference between corresponding CsA backbone 4) and qs angles in the decameric form and the monomeric form is less than 5°, with a maximum difference of less than 15° . The CsA

Conformational polymorphism of cyclosporin A Altschuh et al.

Fig. 3. The conformation of free CsA. Stereo drawing showing the conformation of CsA as determined by NMR in chloroform solution and by X-ray diffraction in the crystalline state [10] showing the four intramolecular hydrogen bonds and a cis amide bond between MeLeu9 and MeLeulO. Carbon, oxygen and nitrogen atoms of CsA are displayed in yellow, red and blue respectively. The figure was made using the program MOLSCRIPT [37].

Table 1. Root mean square difference in A of the individual residues of CsA after global superposition of the backbone atoms of residues 1-165 of CypA and residues 1-11 of CsA. Residues

Rmsd

forms [17]. A comparison of the conformations of a CsA derivative determined in a crystal complex with CypA angles and also showed that the differences in between the different analogous forms is on average less than 50 [18].

NMR X-ray

NMR- < NMR>

NMR denotes the ensemble of 22 energy-refined DIANA conformers representing the NMR solution structure [161; < NMR > is the mean structure of this ensemble; X-ray stands for the CsA structure determined in the monomeric crystal structure of CypA-CsA [171. The mean structure, , was calculated from the mean atomic positions after superposing the backbone atoms (N, Ca, C) of residues 1-165 of CypA and of residues 1 11 of CsA of each conformer with the corresponding atoms of conformer-1 using a least-squares fit. Numbers in the NMR< NMR > columns are the averages of the pairwise displacements for the individual residues of the 22 conformers relative to the mean structure 135]. Abbreviations for residues are defined in the legend to Fig. 1.

The NMR solution structure of the CypA-CsA complex has been calculated by distance geometry methods with the program DIANA [19] and energy refined with the program FANTOM [20]. An ensemble of 22 energy-minimized conformers has been selected according to a previously described procedure [21] to represent the NMR solution structure [16]. A mean NMR structure of the complex was calculated by superposition of the heavy atoms of the 22 conformers. The root mean square deviation (rmsd) between the mean NMR structure and the monomer form of the crystal structure of the CypA-CsA complex is 1.0 A for all CypA and CsA backbone atoms and 1.6 A when side chain heavy atoms are included. Using the global rmsd fit of all CypA and CsA backbone atoms, the backbone atoms of individual residues for the mean NMR CsA structure and CsA in the monomer CypA-CsA X-ray structure deviate within a range of 0.5-1.3 A. A similar range is observed on comparison of the 22 NMR conformers with the mean structure (see Table 1). These results show, therefore, that the orientation of the CsA ligand relative to CypA is quite similar in solution and in the crystal structure.

conformation in the complex with CypA appears, therefore, not to be significantly affected by the different crystal packing contacts found in the different crystal

A comparison of only the CsA ligands between the NMR and monomer CypA-CsA X-ray structures (without the backbone fit of CypA) shows even fewer structural differences. The rmsd fit of the backbone CsA

Backbone All heavy atoms Backbone MeBmtl Abu2 Sar3 MeLeu4 Va15 MeLeu6 Ala7 D-Ala8 MeLeu9 MeLeu10O MeVall11

0.77 0.94 1.02 0.97 0.88 0.90 0.99 0.81 0.64 0.66 0.69

0.94 1.01 1.08 1.50 0.94 1.00 1.03 0.80 0.87 0.67 0.75

0.93 0.96 1.28 0.61 0.66 1.13 0.90 0.55 0.59 0.53 0.61

All heavy atoms 1.12 0.98 1.35 1.46 0.92 1.39 0.95 0.63 1.17 0.98 0.76

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Structure 1994, Vol 2 No 10 atoms among the 22 NMR conformers is less than 0.3 A, while the fit of CsA in the monomer CypA-CsA X-ray structure to the NMR conformers gives an average of less than 0.5 A (Fig. 4a). Table 2 provides a comparison of the torsion angles of CsA as determined in the CypA-CsA NMR structure [16], the CypA-CsA monomeric X-ray structure [17] and the Fab-CsA X-ray structure [9]. The average deviation between the 22 and ip angles of the monomeric crystal structure and the mean NMR structure is 14 °. In particular, the angles of residues 1-4 all deviate by more than 15 ° , with the largest deviation of 460 for q' of MeLeu4. This is an angle that crucially determines the orientation of the carbonyl oxygen of residue MeLeu4, which is involved in an intramolecular hydrogen bond to the hydroxyl group of the MeBmtl side chain (Fig. 4a). In the aqueous environment used for the NMR studies it is possible that this hydrogen bond is in a dynamic equilibrium with hydrogen bonds to bulk water, which would require differences of conformation in that portion of the molecule. Differences of 170 in of MeLeu6 and 190 for q' of DAla8 between aqueous and crystal environments are not unexpected considering the conformational perturbations which are likely to be caused by the hydrophobic

MeLeu4, Va15 and MeLeu6 side chains protruding into aqueous solvent. Side chain conformations of the CypAbound CsA molecules in the crystal and in solution are also found to be quite similar, though some X angles are poorly defined in solution (Table 2). The conformation of CypA-bound CsA is markedly different from the conformation of CsA bound to the Fab fragment. The mean deviation for the 22 and angles of the CypA-CsA monomer and Fab-CsA structures is 290. Maximum deviations occur for of MeBmtl (83.40), 4' of Sar3 (99.1 °) and 4tof MeLeu6 (86.10). These differences affect the positioning of the side chains of residues 1 and 6, which have clearly different orientations in the two structures (Fig. 4b). The rmsd fit between the Fab-bound and CypA-bound CsA structures is 1.25 A for the 11 C a atoms, and 3.0 A for all non-hydrogen atoms. The Fab-bound structure and the CypA-bound structure of CsA have similar Xl angles of MeLeu4 and MeLeu6. Protein-CsA interactions Hydrogen bonds between CsA and the protein-binding surfaces of Fab and CypA are given in Table 3 and

Fig. 4. Comparison of bound CsA conformations. (a) Superposition for best fit of the 11 CsA carbon atoms of the NMR (green) and X-ray (carbon, oxygen and nitrogen atoms are displayed in yellow, red and blue respectively) structures of CsA in the CsA-CypA complex. (b) Superposition for best fit of the 11 CsA C atoms of CsA in the crystal structures of Fab-CsA (green) and CypA-CsA complexes (carbon, oxygen and nitrogen atoms are displayed in yellow, red and blue respectively).

Conformational polymorphism of cyclosporin A Altschuh et al.

Table 2. Torsion angles (°) of CsA as determined in the CypA-CsA monomeric X-ray structure [171, the CypA CsA NMR structure [16], and the Fab-CsA X-ray structure 19].

CypXa MeBmtl Abu2 Sar3 MeLeu4 Va15 MeLeu6 Ala7 D-Ala8 MeLeu9 MeLeu10O MeVa11

'

4

Residues

-109 -116 128 -108 -76 -96 --63 100 -123 -108 -123

NMRb -132(5) -141(4) 160(8) - 154(4) --63(13) -110(4) - 65(3) 99(14) -122(2) -105(3) -125(8)

Fab

CypX

-113 -127 88 -95 -109 -97 -124 102 -123 -86 -126

171 93 -72 103 122 168 156 -141 72 170 83

NMR -166(4) 76(4) -45(3) 85(16) 136(4) -173(1) 154(9) --161(2) 69(5) 156(4) 85(8)

X1 Fab --105 139 -169 125 135 -77 144 --137 63 127 65

CypX

NMR

-51 -48

-47(13) -30(40)

-108 170 - 57

-67 -68 -168

X2 Fab

CypX

NMR

Fab

-66 -58

178

-122(51) -

147

101(56) -177(2) - 41(3)

-107 147 -62

- 50

- 54(19) -46(3) -168(12)

-143 -87 --38

-

--170 -

- 2(63) 166(1) -

172 --175 -

--148(62) -173(3) -

43 151 -177 -14

aCypX stands for the CsA structure determined in the monomeric crystal structure of CypA-CsA 117].bThe numbers in the column NMR denote the mean and the standard deviations of the dihedral angles for the 22 energy-refined DIANA conformers. Abbreviations for residues are as defined in the legend to Fig. 1.

shown in Fig. 5. The CsA-protein interactions are different in detail, but similar in a number of general ways. Both CypA and Fab bind CsA with five intermolecular hydrogen bonds and in both systems the ligand has a similar number of atoms in close contact with the protein (83 atoms in CypA-CsA and 85 atoms in the Fab-CsA complex are closer than 4 A). At least three well defined water molecules mediate CsA-protein interactions in each of the crystal structures. Despite the broad similarities in the CsA-protein contacts, the Fab-CsA affinity constant (Ka) is slightly higher than that for the CypA-CsA complex. Affinity measurements give values of Ka between 2.7x10 7 M- 1 and 5x10 9 M-l depending on the experimental procedure' for the CypA-CsA complex [22,23] and a value of Ka=3.7x10 9 M- 1 for the Fab-CsA complex [24]. The detailed mode of binding in the two complexes is indeed very different. In the CypA-CsA complex, van der Waals contacts are made between CypA and residues 9, 10, 11, 1, 2 and 3, providing a fit resembling a coin (CsA) partly inserted into a slot machine (CypA) (Fig. 6a). In contrast, CsA lies flat on the binding surface of the Fab (Fig. 6b), which is in contact with all residues of CsA except 7 and 8. Contacts to these two residues are probably missing because the antibody was raised against a CsA derivative which was linked to the matrix at position 8 [25]. It should also be borne in mind that the covalent linkage to the matrix may influence the conformation of the CsA as recognized by the antibody. The contact surface areas for the CsA-CypA and CsA-Fab complexes were calculated using Connolly's program [26] implemented in the graphics program WITNOTP (A Widmer, unpublished program). The solvent contact surface area as calculated by this program is the van der Waals surface area of the solute molecule available to solvent. The radius of the spherical probe

Table 3. Hydrogen bonds between CsA and the proteins identified in the CypA-CsA [17] and Fab CsA [9] X-ray structures. Hydrogen bond between CypA....CsA MeBmtl O'...Gln63 NF2 Abu2 N...Asn102 0' MeLeu9 O'...Trpl21 NE1 MeLeu10O O'...Arg55 Nrl MeLeu10O O'...Arg55 NT12 MeBmtl O'...W18...His54 N£2 Abu2 O'...W131 ..Thr73 O'

d (A)a 3.2 2.9 2.9 2.8 2.9 3.1, 2.8 3.3, 3.1

Hydrogen bond between Fab...CsA MeBmtl O'...H Trp100 NEl Va15 O'...HAsn100D N Va5lSN...H.Asn100D O' MeLeu6 O'...H.Asnl00D N82 MeLeu9 O'...HTyr33 OnT MeVa11 O'...W718..H Tyr35 Osq MeBmt O'...W602...LGCly91 0'

3.0 2.6 3.3 2.6 3.2 2.9, 2.7 3.3, 3.3

The following criteria were used to search for hydrogen bonds: the maximum O...O(N) search distance was 3.3 A and the minimum search ° C-O...O(N) angle was 100 . aThe distance 'd' (A) is given between the oxygen or nitrogen atoms of the donor and acceptor atoms involved in the hydrogen bond. For those water (W) mediated hydrogen bridges between CsA and protein involving only one water molecule, both relevant distances are given.

atom used to represent a solvent molecule was set to 1.5 A, and all hydrogen atoms of CsA and the protein were included in the calculation in idealized calculated positions. Crystallographic water molecules were excluded. Contact surface areas of free CsA in the conformation observed in the Fab and CypA complexes are 1070 A2 and 885 A2, respectively. The total loss of contact surface area on formation of the CypA-CsA complex is 630 A2, of which 300 A2 is attributable to CsA. The total buried contact area in the Fab-CsA complex is 835 A2, of which 395 A2 is attributable to CsA.

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Fig. 5. Hydrogen bond interactions of bound CsA. (a) Stereo picture showing the conformation of CsA in the CypA-CsA monomeric X-ray structure [17]. Carbon, oxygen and nitrogen atoms of CsA are displayed in yellow, red and blue respectively. Hydrogen bonds are drawn as dashed lines. All the CypA residues which form hydrogen bonds with the bound CsA are shown with black atoms. (b) Stereo picture showing the conformation of CsA in the Fab-CsA X-ray structure [9]. Carbon, oxygen and nitrogen atoms of CsA are displayed in yellow, red and blue respectively. Hydrogen bonds are shown as dashed lines. All Fab residues which form hydrogen bonds with the bound CsA are shown with black atoms. Fig. 7 shows those regions on the surface of both CsA and protein which are within 1.5 A of one another in the complexes and which define areas of close steric complementarity between protein and ligand where there is no room for solvent. These surfaces were generated using a triangulation of the Connolly contact surface as implemented in the program VIEW [27]. The significantly larger buried surface area of CsA in the Fab complex (395 A2) compared with CsA in the CypA complex (300 A2), (Figs 6 and 7), is consistent with the measured binding affinity, which is higher than for the CypA-CsA complex. The buried surface areas for each CsA residue in the CypA and Fab complex, as calculated using the program ACCESS [28], are shown in Fig. 8. The main differences are found for residues 4 and 6, which have 140 A2 and 50 A2 of buried surface in the Fab complex, respectively, but have no contacts to CypA. In both complexes, N-methylvaline (MeVal) 11 is seen to be a key recognition feature, and it is

completely buried in the CypA complex, as clearly seen in Fig. 6. Protein-CsA complex formation may require different degrees of conformational adaptation The conformation of the CypA around its active site is well conserved in crystal complexes with CsA [17], [6], and a CsA derivative [18]. A comparison between the CypA structure of the monomer form of the CypA-CsA complex and unliganded CypA [29] for the 13-residue binding site gives an rmsd of 0.2 A and 0.74 A for backbone atoms and all non-hydrogen atoms respectively. The largest difference is observed for the guanidinium group of Arg55, where both peripheral NxI atoms have moved, by 1.9 A and 1.4 A respectively. Even on binding of a very different peptide ligand (N-acetyl-alanyl-alanylprolyl-alanyl-N-amidomethyl coumarin) [30], CypA does not significantly alter its conformation; there is an rmsd fit of 0.48 A for all atoms in the 13-residue binding

Conformational polymorphism of cyclosporin A Altschuh et al. NMR studies of CsA derivatives in various non-polar organic solvents [10,13] all show one dominant 'foldedin' conformation (Fig. 3), while in more polar solvents like DMSO and methanol, CsA shows more than five different conformations in solution [14]. CsA is too insoluble in water to allow conformational studies using NMR [31]. However, one water-soluble CsA derivative has been shown to adopt a conformation which is similar to the cyclophilin-bound form [15]. The implication here is that at least one family of CsA conformers in aqueous solution is in the CypA-bound conformation. Coupled with our knowledge of the rather rigid CsAbinding surface of CypA, a picture of CypA-CsA recognition emerges in which CypA selects and binds a sub-population of CsA which is already in the correct conformation in solution.

Fig. 6. The binding surface of CsA. (a) Binding to CypA. (b) Binding to the Fab. The surfaces were generated using a triangulation of the Connolly surface as implemented in the program VIEW [27]. The probe radius was 1.5 A and all hydrogen atoms (not shown) were included in the surface calculation in idealized positions. Water molecules were not included in the surface calculation. Regions of protein surface are shown when the CsA surface is within 1.5 A of the protein surface. The contact surface of the protein is coloured on a charge-coded spectrum from blue (positive) to red (negative). CsA is shown as a ball-and-stick model with carbon atoms yellow, nitrogen atoms blue and oxygen atoms red. The protein backbone (N, C, C) trace of the protein is displayed as a stick model. Those residues which have at least one atom (including hydrogen) within 3.0 A of any atoms of CsA, are also displayed with carbon atoms white, nitrogen atoms blue and oxygen atoms red. Water molecules are shown as space filling models with red oxygen atoms and blue hydrogen atoms. site when fitted to the monomer form of the CypA-CsA crystal structure. The NMR structure of the CypA-CsA complex [16] also shows that the 13- residue CsAbinding site of CypA is conformationally conserved in solution. Thus the available structural information indicates that CypA has a rather inflexible CsA-binding surface, in a state that is already close to the ideal CsAbinding conformation.

The X-ray structure of the unliganded Fab fragment has been solved at 2.6 A resolution and has a disordered H3 loop which is not clearly defined in the electron density (O Vix, D Altschuh and J-C Thierry, unpublished data). In contrast, in the CsA-Fab complex the H3 loop is well ordered and four of the five intermolecular bonds with CsA are made to residues of this loop (Table 3). The conformational ordering of the CsA-bound Fab suggests that CsA has induced or selected a particular conformation of the binding site of the Fab. As mentioned previously, the conformation of CsA bound to Fab is significantly different from the CypA-bound conformation. Two hydrogen bonds (between the backbone N and O atoms of H-Asn100D in the H3 loop and the O and N atoms of MeVall 1 of CsA) form a short stretch of antiparallel sheet, which may be a driving force determining the Fab-bound CsA conformation. Thus for the CsA-Fab interaction, both ligand and receptor seem to change conformation, offering a mutually adaptive recognition mechanism.

Biological implications The immunosuppressive drug, cyclosporin A (CsA) is used to prevent organ rejection after transplant surgery. It blocks the signal transduction pathway in T cells, preventing T-cell activation and proliferation. The likely molecular target for CsA is the cytosolic protein cyclophilin A (CypA). The heterodimeric molecular complex of CypA-CsA inhibits the cytosolic phosphatase, calcineurin, thereby preventing the subsequent transmission of the signal into the nucleus for cytokine expression. Neither CypA nor CsA alone inhibit calcineurin and it is therefore likely that a composite CypA-CsA surface is required for recognition and binding to calcineurin. Mutational studies on CypA have identified some residues which play a role in this interaction [32]. Knowledge of the conformation of CsA in the calcineurin-CsA-CypA ternary complex is of significant biomedical interest, since rigid

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Fig. 7. Stereoviews of the molecular surface areas involved in intermolecular contacts in complexes with CsA. The surfaces were generated as for Fig. 6. (a) The CsA-CypA complex. The transparent contact surface of CsA is coloured pale green and the closely complementary solid CypA surface can be seen through it. Interpenetrating surface areas correspond to regions of close contacts. For instance the terminal ¶jNHs of Arg55 can be distinguished as the two blue lobes pointing through the CsA surface to the carbonyl oxygen of MeLeul 0. (b) Same as (a) but with CsA translated horizontally and the CsA surface shown in solid colour with the inside surface coloured orange and the outer surface coloured green. (c) The CsA-Fab complex. The transparent contact surface of CsA is coloured pale green and the closely complementary solid CypA surface can be seen through it. (d) Same as (c) but with CsA translated horizontally and the CsA surface shown in solid colour with the inside surface coloured orange and the outer surface coloured green.

Conformational polymorphism of cyclosporin A Altschuh et al. bond angles were 0.013 A and 2.64 ° , respectively, using the PARAM19X.PRO file from X-PLOR [34]. The average B-factors for CypA and for CsA are 13.0 A2 and 15.9 A2 , respectively. The crystal structure of the Fab-CsA complex has been refined at 2.65 A resolution to a crystallographic R-factor of 16.3% with 173 solvent molecules [9]. The rmsd from ideal bond lengths and bond angles were 0.015 A and 3.5° , respectively, using the PARAM19X.PRO file from X-PLOR [34J. The average B-factor for the Fab and for CsA are 11.3 A2 and 16.9 A2, respectively.

-

I-

1

2

3

4 5 6 7 8 Residue number

9

10 11

Fig. 8. Comparison of the buried area for each of the residues of CsA in the CypA-CsA and Fab-CsA crystal structures. Buried surface areas of each of the CsA residues calculated with the program ACCESS [28] using the coordinates from the monomeric CsA-CypA X-ray structure and the CsA-Fab X-ray structure. analogues of CsA, constrained to be in the right conformation for binding, or which mimic the CsA-CypA composite surface, could provide improved immunosuppressant drugs. In this paper we present a comparison of various structures of CsA. We have seen that CsA binds to CypA with a conserved conformation both in solution and in various different crystal packing arrangements. There are small but real differences in the effector loop of CsA, particularly in the side chain conformations of N-methylleucine groups at positions 4 and 6. These results indicate that although CsA is a flexible molecule which can adopt widely different conformations depending on the nature of the solvent or protein environment, the CypA-bound conformation of CsA is much more restricted. The possibility that CsA undergoes a conformational change on binding of the CypA-CsA complex to calcineurin has been raised [33], but our results suggest that major changes are unlikely. We could expect some sidechain rotations of CsA on binding to calcineurin, but the backbone conformation would be anchored by the cyclophilin binding site and the backbone atoms of the effector loop are unlikely to move significantly away from the NMR and X-ray determined structures. This makes rigid analogues of CsA in the CypA-bound conformation an appealing target for structure-based drug design.

Materials and methods Atomic coordinates used in this paper were taken from the following protein X-ray and NMR structures. The crystal structure of the monomeric CypA-CsA complex has been refined at 2.1 A resolution to a crystallographic R-factor of 16.7% with 144 solvent molecules [17]. The root mean square deviation (rmsd) from ideal bond lengths and

The three-dimensional NMR solution structure of the Cyp-CsA complex was determined using heteronuclear threedimensional and four-dimensional experiments in different combinations of the unlabeled, 15 N-labelled, 13C-labelled and [15 N, 3 C]-doubly-labelled CypA and CsA samples [16]. A final data set of 1810 intra-CypA, 107 intra-CsA and 63 intermolecular NOE upper distance constraints was used in distance geometry calculations with the program DIANA [19]. A linker peptide of 34 dummy glycyl residues with vanishing van der Waals radii of the dummy atoms was connected to the amino terminus of CsA and the carboxyl terminus of CypA to perform distance calculations in dihedral angle space. An ensemble of 22 conformers, selected by an rmsd x plot [21] and energy-minimized with the program FANTOM [20] represents the NMR solution structure. The average rmsd value of the 22 conformers of the complex relative to the mean structure is 1.1 A for backbone and 1.7 A for all non-hydrogen atoms of all residues of CypA and CsA. Restricting the optimal superposition to the binding site residues of CypA and CsA, the average rmsd values to the mean structure are 0.7 A for backbone and 1.2 A for all non-hydrogen atoms. The NMR distance constraints and the atom coordinates have been deposited with the Brookhaven Protein Data Bank (accession entry code 3CYS).

References

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