X-ray Structures and Analysis of 11 Cyclosporin Derivatives ...

Article No. mb982108 J. Mol. Biol. (1998) 283, 435±449 

X-ray Structures and Analysis of 11 Cyclosporin 

Derivatives Complexed with Cyclophilin A 

Joerg Kallen, Vincent Mikol, Paul Taylor and Malcolm D.Walkinshaw* 

Structural Biochemistry Group 

The University of Edinburgh 

Michael Swann Building 

King's Buildings, Edinburgh 

EH9 3JR, UK 

*Corresponding author 

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. 

# 1998 Academic Press 

Keywords: Immunophilin; cyclosporin A; cyclophilin; ligand binding; 

molecular recognition 

Introduction 

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. 

Abbreviations used: CS, cyclosporin; CsA, cyclosporin 

A; CypA, cyclophilin A; rmsd, root mean square 

deviation; Nva, norvaline; Bmt, (4R)-4[(E)-2-butenyl]- 

4,N-dimethyl-L-threonine; Abu, L-a-aminobutyric acid; 

Sar, sarcosine; 3D, three dimensional; PPlase, peptidylprolyl 

isomerase; DMSO, dimethyl sulphoxide; PDB, 

Protein Data Bank. 

E-mail address of the corresponding author: 

M.Walkinshaw@ed.ac.uk 

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 

b-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 (Grif®th 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 vitro (Schonbrunner 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 (P¯ugl et al., 1993, 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 con®rmed 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 `èffector loop'' 

and is implicated in speci®c 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 suf®cient, for immunosuppressive 

0022±2836/98/420435±15 $30.00/0 # 1998 Academic Press

436 Cyclosporin Derivatives Complexed with CypA 

Table 1. Formulae of CsA analogues and their biological 

activities 

Figure 1. Residue labelling scheme for CsA-analogues 

(see also Table 1). Atom labelling is according to the 

IUPAC convention. CN is the methyl carbon atom of 

the N-methylated amino acids. MLE for MeLeu; MVA 

for MeVal; BMT for MeBmt; ABU for Abu; SAR for Sar; 

VAL for Val; ALA for Ala; DAL for D-Ala. All amino 

acid residues with the exception of D-Ala8 (and Sar3) 

are in the L-con®guration. 

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., 

1994a). 

This paper describes the structures of eight 

chemically distinct CsA-analogues complexed with 

CypA which show modi®cations 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. 

Results 

Side-chains for the amino acids at position 1, 2, 3 and 4 are 

shown for the 11 different CsA derivative complexes. The horizontal 

line represents the Ca-C b bond for the side-chain. The 

CypA value in column 6 gives a measure of the strength of 

binding of the derivative relative to CsA value in column 6 

gives a measure of the strength of binding of the derivative 

relative to CsA as determined using an ELISA assay 

(Quesniaux et al., 1988). The measure of the effect of suppressing 

the production of interleukin-2 relative to CsA in a whole 

cell assay is given in the column labeled IL2 (Fliri et al., 1993; 

Bollinger et al., 1990). 

CsA ˆ Abu2-CS; 116450 ˆ MeBm 2 t-CS; 33804 ˆ Val2-CS; 

27402 ˆ Thr2-CS; 224698 ˆ (5-hydroxy)Nva2-CS; 209313 ˆ 

D-MeSer3-CS; 209650 ˆ Val2-D-MeAla3-CS; 209217 ˆ Val2-D-(2- 

S-methyl) Sar3-CS; 209825 ˆ (6,7-dihydro)MeBmt-1-Val2-D-(2- 

S-methyl)Sar3-CS; 211810 ˆ (4-hydroxy)MeLeu4-CS; 211811 ˆ 

MeIle4-CS. 

a IC 50 (derivative)/IC 50 CsA. 

b 26 times less binding than CsA. 

c 4.2 times less immunosuppressive than CsA. 

d Three times better binding than CsA. 

X-ray results 

X-ray structures of a total of 11 cyclosporines cocrystallised 

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 (Mikol & Duc, 1994). These crystals 

grew isomorphously with space group P2 1 2 1 2 1 

and cell dimensions a ˆ 36.4 AÊ , b ˆ 60.7 AÊ , 

c ˆ 72.2 AÊ . The re®ned cell dimensions for the 

different isomorphous complexes containing CsAanalogues 

varied by a maximum of 0.6 AÊ in the a- 

dimension, 1.6 AÊ in the b-dimension and 1.4 AÊ in 

the c-dimension (see Table 2). The unliganded 

CypA structure showed a shrinkage of over 2 AÊ 

in the b and c-dimensions with a ˆ 36.46 AÊ , 

b ˆ 57.32 AÊ , c ˆ 70.73 AÊ . The complex CypA/ 

209313 was crystallised in space group P2 1 2 1 2 

with unit cell dimensions a ˆ 62.9 AÊ , b ˆ 65.3 AÊ , 

c ˆ 40.8 AÊ . 

The 11 structures are re®ned to a resolution of 

between 2.2 AÊ and 1.76 AÊ . and give R-factors

Table 2. Crystallographic data for the CsA-analogue/CypA complexes 

CsA-analogue CsA 116450 33804 27402 224698 209313 209650 209217 209825 211810 211811 

Diffraction data 

a (AÊ ) 36.39 36.87 36.40 36.48 36.44 62.90 36.38 36.40 36.40 36.30 36.40 

b (AÊ ) 60.72 61.29 61.42 61.57 61.32 65.30 61.07 61.10 60.60 61.10 61.50 

c (AÊ ) 72.21 71.99 72.42 72.44 72.58 40.80 72.22 72.60 72.60 72.30 72.20 

Measured reflections 21,441 18,609 51,113 61,851 46,587 35,137 33,630 50,210 45.753 51,912 39,927 

Unique reflections 8732 7343 13,548 12,724 14,501 11,324 11,304 14,694 14,288 14,285 10,475 

R sym (%) 7.4 5.5 5.3 7.4 4.3 9.0 7.8 6.6 5.8 5.8 9.7 

Independent reflection 7428 5915 12,089 12,724 12,145 10,206 9813 13,495 12,960 11,904 9334 

Resolution (AÊ ) 2.1 2.2 1.86 1.8 1.76 2 1.9 1.8 1.8 1.8 2 

Completeness (%) 88.9 84.2 95.7 80.8 87.2 95.4 85.6 94.1 92.3 92.2 91.3 

Refinement 

No. of CsA derivative atoms used 85 86 86 86 87 87 87 88 88 86 85 

No. of solvent molecules 144 131 178 168 188 86 166 127 151 143 127 

2 

Average B-value for CypA (AÊ ) 13 13.4 15.1 20.1 14.8 28.7 13.3 15.3 16.4 14.2 15.4 

2 

Average B-value for Cs derivative (AÊ ) 15.9 20.3 16.9 24.7 16.9 24 15.5 16.5 18.5 15.8 16 

2 

Average B-value for solvent molecule (AÊ ) 37.2 37.5 32.9 44.8 36.4 45.7 35.2 36.6 40.7 36 34.6 

Range of spacings (AÊ ) 8.0±2.1 8.0±2.2 8.0±1.86 8.0±1.8 8.0±1.76 8.0±2.0 8.0±1.9 8.0±1.8 8.0±1.8 8.0±1.8 8.0±2.0 

R-value 0.167 0.163 0.177 0.186 0.175 0.194 0.186 0.185 0.165 0.163 0.169 

rmsd from ideality 

Bond length (AÊ ) 0.013 0.031 0.012 c 0.06 0.011 c 0.017 0.015 0.014 0.014 0.015 

Bond angle ( ) 2.64 2.71 1.59 c 1.88 1.57 c 2.94 2.76 2.78 2.64 2.5 2.77 

Chemical formulae for the CsA analogue codes are given in Table 1 and Figure 1. The superscript c indicates that the structure was re®ned using the stereochemical data from Engh & Huber 

(1991). All other structures used the data in the X-PLOR ®le param19X (BruÈ nger, 1993).


Figure 2. Stereo picture of the (2F o F c ) electron density for the Val-2-CS (compound 33804). This is representative 

of the quality of the electron density for all other derivatives. 

between 0.15 and 0.19 (Table 2). The root mean 

square deviation (rmsd) from ideal bond lengths 

are between 0.01 AÊ and 0.017 a and the rmsd from 

ideal angles is between 1.6 and 2.9 . Figure 2 

shows the typical quality of electron density 

obtained in these structures. The average B-value 

for the CsA-analogues vary from 24 AÊ 2 to 16 AÊ 2 

which tend to be between 5% and 25% higher than 

the average B-values of the cyclophilin atoms 

(Table 2). 

Comparison of cyclophilin structures 

The conformations of the cyclophilin molecules 

in all the CypA/CsA-analogue structures are in 

general very similar. A least squares ®t on to the 

596 C,N,C( and O main-chain atoms of residues 4 

to 67 and 76 to 160 of the CypA/CsA complex was 

carried out for all CsA-analogue structures (Table 3 

and Figure 3). The maximum value of 0.218 AÊ is 

for the non-isomorphous derivative 209313. For the 

ten isomorphous crystal structures, rmsd values 

vary between 0.18 AÊ and 0.10 AÊ . Apart from the 

rather ¯exible N and C-terminal residues, few 

structures have main-chain atoms which deviate 

by more than 1 AÊ from the native. The largest 

differences (excluding residues 1 to 3) are summarised 

in Table 3. The only other region which shows 

signi®cant differences between structures is in the 

``70s loop'' (residues 67 to 76 ) which shows atom 

displacements of up to 1.7 AÊ for main-chain atoms 

which can be explained by the interactions 

between modi®ed CsA residues and this 70s loop. 

A comparison of liganded and unliganded cyclophilin 

structures shows that CypA adopts a very 

well conserved conformation in many different 

environments (Braun et al., 1995). The decameric 

(P¯ugl et al., 1993) and monomeric forms (Mikol 

et al., 1993) of the CypA/CsA complex, for 

example, have an rmsd of less than 0.5 AÊ for all 

backbone atoms. The rmsd values between the 

mean NMR structure and the monomer form of 

the crystal structure of the CypA/CsA complex are 

1.0 AÊ for all CypA and CsA backbone atoms, and 

1.6 AÊ when the side-chain atoms are included. 

A comparison between CypA in the crystal structures 

of the monomeric form of the CypA/CsA 

complex and unliganded CypA (Ke, 1992) for the 

13-residue binding site gives rmsd values of 0.20 AÊ 

and 0.74 AÊ for the backbone atoms, and for all 

non-hydrogen atoms, respectively. Even on binding 

of a very different peptide ligand, N-acetyl- 

Ala-Ala-Pro-Ala-N-amidomethylcoumarin (Kallen 

& Walkinshaw, 1992), CypA does not signi®cantly 

alter its conformation; there is an rmsd ®t of 

0.48 AÊ for all atoms in the 13-residue binding site 

when ®tted against the structure of the monomeric 

crystal form.

Cyclosporin Derivatives Complexed with CypA 439 

Table 3. rmsd ®t of CypA structures 

116450 0.145 0.222 MeBm1/CH(1.23), Val5/CG1(2.61), MeLeu6/CD1(1.78), all distances

Table 4. CsA . . . CypA interaction distances

All short contacts less than 3.7 AÊ between the CsA-analogue (designated by the numerical code number) and CypA are listed. The ®ve protein-ligand hydrogen bonds are shaded.


Figure 4. Conserved intermolecular 

hydrogen bonds between CsAanalogues 

and CypA. The conformation 

of CsA in the CypA/CsA 

monomeric X-ray crystal structure 

is shown. Oxygen and nitrogen 

atoms of CsA are displayed in red 

and blue, respectively. All the 

CypA residues (dark grey bonds) 

which form hydrogen bonds (broken 

lines) and close van der Waals 

contacts (thin continuous lines) 

with the bound CsA (pale grey 

bonds) are shown in their positions 

in the crystal structure. Additional 

hydrogen bonds are mediated by 

water molecules which are also 

shown. Distances for these interactions 

for the different analogues 

are given in Table 4. 

water molecules were then de®ned such that each 

member of a cluster lies within a given radius of 

the centroid of the cluster. With a radius of 0.5 AÊ , 

there are 22 unique clusters containing a water 

molecule from each of the 11 structures. Water 

molecules are shown in Figure 3. Those water molecules 

belonging to the same cluster have been 

labeled identically in the deposited co-ordinate 

®les. All of the 22 most conserved water positions 

form at least three hydrogen bonds. and two of the 

water molecules (W30 and W27) manage to form 

four hydrogen bonds to cyclophilin. The 22 con- 

Figure 5. Stereo picture of CsA/CypA with the most conserved water molecules. CsA is coloured magenta, CypA 

is coloured cyan and the 11 water molecules which occur in 10 or 11 of the 11 structures discussed in this paper are 

shown as labelled yellow spheres. The label corresponds to the number in Table 5 and also to the label of the water 

molecule in the deposited PDB ®le. The position of the sphere is the average position of the water molecules from 

the different structures which all re®ne to within 0.2 AÊ of each other.


Table 5. Intermolecular distances for the 11 most conserved 

water molecules 

W11-2 Ala38 O 2.78 

W11-2 Glu43 N 3.28 

W11-2 Lys44 N 2.88 

W11-2 Phe46 O 2.73 

W11-3 Asn102 OD1 2.88 

W11-3 Val127 N 3.23 

W11-3 Asp85 OD2 3.05 

W11-3 Asn108 OD1 2.80 

W11-4 Ser51 OG 2.90 

W11-4 Cys52 O 2.60 

W11-4 Gly65 N 2.78 

W10-8 Phe53 O 2.86 

W10-8 Ile156 N 2.85 

W10-8 W01-25 O1 2.93 

W10-8 W01-604 O1 2.82 

W10-8 Thr152 OG1 3.01 

W10-9 Leu90 N 3.18 

W10-9 W11-10 O1 2.88 

W10-9 Asn87 OD1 2.83 

W10-9 Val128 O 2.97 

W11-10 Phe88 CA 3.22 

W11-10 W01-521 O1 2.86 

W11-10 Val128 N 2.91 

W11-10 Leu90 O 2.75 



W11-13 Asn106 N 3.22 

W11-13 Asp85 OD2 2.73 

W11-13 Thr107 N 2.77 

W11-13 Gly104 O 2.75 

W10-19 W05-121 O1 2.68 

W10-19 W01-194 O1 2.52 

W10-19 W02-46 O1 2.86 

W10-19 W01-698 O1 2.64 

W10-19 W01-474 O1 2.87 

W10-19 Asp13 OD1 2.82 

W10-19 His92 O 2.90 

W11-23 Glu84 O 2.83 

W11-23 Glu86 N 2.97 

W11-23 Thr32 OG1 2.81 

W11-23 Asn108 ND2 3.03 

W10-25 Ala117 O 2.85 

W10-25 Gly94 O 3.06 

W10-25 His92 C 3.09 

W10-25 Cys115 SG 3.30 

W10-25 His92 O 3.23 

W10-25 His92 CA 3.28 

W10-25 His92 CB 3.22 

W10-43 W01-153 O1 2.26 

W10-43 Gly124 N 3.11 

W10-43 Leu90 O 3.20 

W10-43 W11-10 O1 3.10 

W10-43 His126 O 2.81 

Water molecules in the 11 re®ned structures were analysed 

and classi®ed in clusters as described in the text. Those water 

molecules re®ning to a position less than 0.2 AÊ from each other 

form a cluster. All distances of less than 3.2 AÊ from the averaged 

position of the 10 or 11 water molecules and atoms of the 

native CypA/CsA complex are given. Water molecules are 

labelled Wx y where x is the number of water molecules in the 

cluster (either 10 or 11) and y is the label of the water molecule 

shown in Figure 6; it was used to label the water molecule in 

the deposited PDB ®le. The positions of these water molecules 

are shown in Figure 4. 

served water molecules make a total of 53 hydrogen 

bonds directly with the protein, of which 15 

are hydrogen bonds to side-chain atoms, 20 are 

hydrogen bonds to backbone carbonyl atoms and 

17 are hydrogen bonds to main-chain amide nitrogen 

atoms. In general, protein structures show a 

greater frequency of hydration of main-chain CÔ 

groups compared with NH groups by a ratio of 3:1 

(Baker & Hubbard, 1984). The unusually high proportion 

of hydrogen bonds to amide nitrogen 

atoms in the 22 conserved water sites in the CypA 

structures is explained by the fact that these water 

molecules tend to be buried and are likely to play 

a structural role (Williams et al., 1994; Baker & 

Hubbard, 1984). 

A more stringent criterion for a water cluster 

was made in which the cluster radius was reduced 

to 0.2 AÊ . Only 11 water molecules were found in 

ten or all 11 of the X-ray structures and these most 

strongly conserved water molecules are shown in 

Figure 5 with intermolecular distances given in 

Table 5. The average temperature factor for the 

water molecules in the different structures is 

approximately twice the amplitude of the CsA 

ligand (see Table 2). 

CypA-CsA complexes with modifications in 

CsA at position 2 

The Abu-binding pocket 

In the CypA/CsA complex and indeed in all 

derivatives with Abu in position two, the ethyl 

side-chain ®ts snugly into the `Àbu-pocket'' and 

makes between 11 and 13 van der Waals contacts 

(C . . . C


Figure 6. Abu-pocket showing hydrogen bonding of conserved water molecules. Picture highlighting the conserved 

water molecules W5, W6 and W7 in the binding pocket for Abu2. These water molecules are present in nine of 

the complexes examined in this paper. Part of the CsA molecule is shown in magenta, water molecules are yellow 

and hydrogen bonds are drawn in green. W7-Ser110 ˆ 2.75 AÊ , W7-Gly74 ˆ 3.29 AÊ , W7-Asn111 ˆ 3.13 AÊ , 

W5-Asn111 ˆ 3.12 AÊ , W5-Gly109 ˆ 2.87 AÊ , W5-W6 ˆ 3.26 AÊ , W5-Ala101 ˆ 2.70 AÊ , W6-Thr107 ˆ 2.77 AÊ , W6- 

W244 ˆ 2.75 AÊ . 

59 , 53 for Abu2, Val2 and Thr2, which places 

Val2:C g0 

and Thr2:O g in the trans and gauche conformations, 

respectively. The conformation of Thr- 

2 is stabilised by a strong 2.7 AÊ intramolecular 

hydrogen bond from the g OH to the main-chain 

carbonyl oxygen atom. This conformation is clearly 

disfavoured in the Val2 structure because of steric 

interaction between the C g methyl group and the 

carbonyl oxygen atom. The alternative trans conformation 

adopted by Val2 results in a number of 

rather short van der Waals contacts to residues lining 

the Abu-binding pocket. In particular there is a 

short Val2/CG . . . Gln/NE2 contact of 3.56 AÊ . 

There is a signi®cant effect of this repulsive 

interaction on the conformation of CypA (Figure 7), 

which results in a shift in position of the 70s loop 

of CypA. A comparison with the native CypA/ 

CsA structure shows a shift in this loop which is 

greater than 1.2 AÊ for each of the main-chain 

atoms of residues 69 through 73. This compares 

with an average displacement of less than 0.2 AÊ 

for the corresponding region in all other derivative 

structures. These changes, along with the presence 

of the second C g group in the Abu-binding pocket, 

prevent the formation of the hydrogen-bonded 

water network found in the native structure 

(Figure 6). An alternative water hydrogen-bonding 

pattern is formed with the Val2C/S complex in 

which the water molecule corresponding to W6 in 

the native structure moved relative to the native 

structure by more than 0.5 AÊ . 

The threonine hydroxyl group from Thr2-CS 

group in the Abu-binding pocket is involved in 

hydrogen bonds to two water molecules, one of 

which is 0.8 AÊ from the conserved W6 position. 

Despite these additional hydrogen bonds and the 

lack of steric clash of the Thr side-chain in the 

Abu-pocket the IC50 increases by a factor of 1.4 

(Table 1). This corresponds to a small reduction in 

binding strength that may be explained by a loss 

of entropy (rotational freedom) of the Thr sidechain. 

The Val2 analogue (Figure 7) shows a sixfold 

reduction in binding to CypA and a corresponding 

threefold drop in biological activity in vitro. Again 

the entropy loss of the Val side-chain on binding 

may play a role. In addition, the distortion in the 

70s loop found in the Val2/CS complex may also 

provide a negative energy term for ligand binding. 

The precision of the atomic positions of these structures 

is about 0.2 AÊ and in many cases small 

(0.5 AÊ ) movements of protein loops may be signi®cant. 

The X-ray structure of the CypA/224698 complex 

which was designed with a modi®cation at 

position 2 to enhance speci®city for CypB-binding 

(Mikol et al., 1995) shows that the hydroxypropyl 

side chain penetrates deep into the Abu-pocket


Figure 7. Overlay of CypA/Val2-CS (analogue 33804) and CypA/CsA showing close contacts to the 70s loop. The 

native CypA/CsA structure is shown in magenta and CypA/Val2-CS has atom-type colouring. The CsA ligands are 

drawn with thick bonds.The additional branched C b of Val2 induces a signi®cant shift in the position of the 70s loop. 

making one weak additional van der Waals contact 

and a weak hydrogen bond. Binding of 224698 to 

CypA was reduced by a factor of 7. The displacement 

of water molecules by the side chain and loss 

of rotational entropy of the side chain are again 

invoked to explain the overall loss of binding. 

CsA-analogues with valine at position 2 and 

methyl or S-methyl at position 3 

Only four of the CsA analogues, namely 33804, 

209650, 209217 and 209825, have a valine at position 

2. All have nearly identical conformations of 

the valine side-chain as that shown in Figure 7. 

The effect of the additional methyl group of the 

valine is to push the main-chain atoms of residues 

69 to 73 (the 70s loop) by over 1 AÊ from the native 

Cyp/CsA positions. Despite the additional methyl 

group, only two non-bonded contacts of less than 

3.7 AÊ are made: Gln:NE2 and Thr73:O (Table 4), 

suggesting that the Abu-binding pocket is not 

designed for accommodating b-branched amino 

acids. 

The three Val2-CS analogues with methyl or 

S-methyl side-chains at position 3 all show a 

signi®cantly increased relative binding strength for 

cyclophilin. This effect can not be explained by the 

formation of additional van der Waals contacts or 

hydrogen bonds between ligand and protein. The 

S-Me side-chains of 209217 and 209825 hardly 

contribute to additional van der Waals interaction: 

the terminal methyl group makes one contact 

(3/CG . . . Thr73/O ˆ 3.5 AÊ ) while the bulky sulphur 

atom does not interact at all with cyclophilin 

(Table 4). Similarly, the methyl group of D-MeAla3- 

CS does not make any contacts of less than 3.7 AÊ 

with the protein. 

D-MeSer3-CS is the only complex in this series 

which was grown in a different crystal form 

(Table 2). The rms ®t for the 596 backbone atoms 

onto the native CypA/CsA structure is 0.22 AÊ 

compared to the average of 0.13 AÊ for the other 

nine structures. The D-MeSer3 side-chain of 209313 

is within 3.7 AÊ of Thr73, but the hydroxyl oxygen 

atom points out into the solvent and no proteinligand 

hydrogen bond is made. 

The effect on CypA binding by CsA residues 

modified at position 3 

The CsA derivatives studied in this paper have 

methyl, hydroxymethyl, and S-methyl groups substituting 

the pro-R hydrogen of the sarcosine residue 

at position 3. All show an approximately twofold 

stronger binding to CypA compared with 

CsA. Derivatives 209650, 209217 and 209825 also 

have an Abu2Val mutation, which was shown to 

reduce binding to CypA by a factor of about 6. The 

reason for the enhanced binding of derivatives 

with both hydrophobic or hydrophilic substituents 

at position 3 is not clear. There is no obvious pocket 

in the protein to accept the residue-3 side-chains 

and only one or two additional van der Waals contacts 

are made between ligand and protein, even 

for the large S-methyl group (Table 4). The X-ray 

structure of the 209313/CypA complex shows that 

the hydroxy group of the D-MeSer3-CS points out 

into the solvent and forms no direct or water 

bridged hydrogen bonds to the protein. 

A possible explanation for the anomalous 

increase in binding strength comes from the observation 

using NMR spectroscopy that unliganded 

D-MeSer3-CS in aqueous solution adopts an exclusively 

the CypA-bound conformation (Wenger 

et al., 1994). NMR studies of native CsA have 

shown mixtures of signi®cantly different conformers 

in solution (Braun et al., 1995). The consistently 

better CypA-binding properties of these


derivatives may then be explained by a higher concentration 

in solution of the required binding conformation 

compared with native CsA. 

CypA/CsA complexes with modifications in 

CsA at position 4 

The side-chain modi®cations at position 4 for 

CsA derivatives 211810 ((4-OH)MeLeu4-CS) and 

211811 (MeIle4-CS) do not make any contact with 

CypA. The CD1 methyl group of MeLeu4 in 

211811 is twofold disordered with w 2 values of 

60 and 180 while there is only one conformation 

about the Ca-C b bond with w 1 ˆ 50 . The 

X-ray structure of the complex of CypA with 

211810 (( 4-OH)MeLeu4-CS) is virtually identical to 

that of the CypA/CsA complex. It was not possible 

on the basis of electron density maps or intermolecular 

crystal contacts to distinguish between the 

OH group and the C g methyl groups, but in any 

case the g-OH on residue 4 makes no intramolecular 

contacts and points out into the solvent region. 

As in the case of derivatives at position 3 it is 

dif®cult to explain the small increase in CypAbinding 

strength for 211811 (Table 1). The sidechain 

of derivatives at position 4 are even more 

remote from the surface of CypA and again the 

only reasonable explanation is that such chemical 

modi®cations help stabilise the CypA-bound conformation 

in solution. There are, however, no 

NMR experiments to support this in the case of 

these derivatives. Position 4 is a very sensitive recognition 

site for the CypA/CsA complex by calcineurin. 

A branched C b atom is enough to 

essentially destroy the calcineurin inhibitory (and 

therefore immunosuppressive) effect. The extent to 

which this site can be varied has been explored in 

a series of synthetic studies (Papageorgiou et al., 

1996) and the binding properties of a number of 

position 4 derivatives have been compared. The 

ability to decouple the cyclophilin inhibitory property 

from its immunosuppressive activity by such 

modi®cations is of potential value in developing 

non-immunosuppressant derivatives which prevent 

cyclophilin being incorporated into the HIV 

protein coat. It has been shown that 211811 is a 

potentially useful HIV inhibitor which blocks viral 

replication (Papageorgiou et al., 1996; Rosenwirth 

et al., 1994). 

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, modi®cations 

of residues 4, 5 and 6 which comprise the 

protruding effector loop can strongly affect immunosuppressant 

activity without substantially affecting 

cyclophilin binding (Sigal et al., 1991; 

Papageorgiou et al., 1994a,b). It is, however, not so 

straightforward to correlate all of the small structural 

changes observed in the different X-ray analyses 

with the measured CypA/CsA IC50 values. 

Various attempts have been made to analyse 

protein-ligand binding by using a number of discrete 

linearly related energy terms (Ca¯isch & 

Karplus, 1995; Morton & Matthews, 1995; Morton 

et al., 1995). An analysis of the X-ray structures of a 

set of seven peptide ligands bound to HIV-protease 

has led to an empirical set of energy terms which 

accurately describe the binding of this training set 

(Verkhivker et al., 1995). A simple and computationally 

very ef®cient scoring function which gives 

a good estimate of ligand binding energies has 

been developed (Bohm, 1994). The function correlates 

well with observed dissociation constants and 

is implemented in the program Ludi (Bohm, 1992, 

1996). The free energy of a neutral hydrogen bond 

contributes 1.1 kcal mol 1 and the free energy 

contribution from the interaction between lipophilic 

surfaces contributes 0.04 kcal mol 1 AÊ 2 . Each 

rotatable bond in the ligand which is frozen on 

binding to the protein costs 0.33 kcal mol 1 (Bohm, 

1994). 

Application of Bohm's scoring function to the 

CypA/CsA complex provides an insight into 

the relative importance of the different terms in the 

recognition and binding process. The binding of 

CsA to CypA results in a loss of contact surface 

area of CsA of 300 AÊ 2 (Altschuh et al., 1994). This 

gives a lipophilic contribution to binding of 

4.2 kcal. The ®ve direct CsA-CypA hydrogen 

bonds contribute a total of 5.6 kcal mol 1 . An 

entropy term due to loss of rotational and translational 

motion of the whole ligand reduces the binding 

energy by 1.3 kcal mol 1 . There are 11 freely 

rotatable side-chain bonds in CsA which are frozen 

by the interaction with CypA and reduce the binding 

by 3.6 kcal mol 1 . The total binding energy, 

using this simple procedure sums to (11 0.33) 

‡ 1.3 (300 0.04) (5 1.1) ˆ 12.6 kcal mol 1 

and corresponds to a K d value of 5.9 10 10 M. 

The experimental dissociation constant for the 

CsA/CypA complex is in the range 10 8 to 10 9 M 

(Galat, 1993), which corresponds to binding energies 

between 10.9 kcal mol 1 and 12.3 kcal 

mol 1 . The slight over estimate for binding energy 

may be accounted for by the estimate of the number 

of frozen rotatable bonds, as binding will also 

to some extent lock the backbone conformation of 

the CsA main-chain. The loss of conformational 

freedom is particularly dif®cult to estimate for the 

binding of CsA as it is known known to adopt a 

variety of conformations in solution (Ko & Dalvit, 

1992). Loss of rotational freedom of an additional 

11 backbone torsion angles (a justi®able compromise 

for the partially constrained cyclic CsA molecule) 

over compensates and reduces the 

calculated binding energy to 9.1 kcal mol 1 with a 

corresponding K d value of 2.1 10 7 M.


Flexibility of ligand and protein may play a signi®cant 

role in general recognition and binding 

process. An analysis of a series of X-ray structures 

of lysozyme/ligand complexes suggested that the 

more rigid part of the protein template confers 

speci®city of binding and only one shape or conformer 

of the ligand is accepted (Morton & 

Matthews, 1995; Morton et al., 1995). However, 

neither the ligand nor the CypA template show 

¯exibility in the family of X-ray structures discussed 

here. The only signi®cant change in CypA 

structure occurs with complexes of compounds 

33804, 209650, 209217 and 209825, which have a 

valine side-chain at position two. This increased 

bulk in the Abu-pocket pushes main-chain atoms 

in the 70 s loop by just over 1 AÊ from the native 

CypA/CsA position. There may be some inherent 

¯exibility in this loop as it shows multiple conformations 

in NMR structures of the CypA/CsA complex 

(Theriault et al., 1993; Spitzfaden et al., 1994). 

All other X-ray structures of CypA with a wide 

range of different ligands, including peptides and 

in different crystal environments, show a very well 

conserved protein conformation (Braun et al., 

1995). CypA provides an example of an unyielding 

recognition template which may be important for 

its role as a PPIase or chaperone protein in the cell. 

Small prolyl peptides complexed with CypA are 

always bound in the cis conformation, while longer 

peptides with an accessible Gly-Pro sequence are 

bound in the trans conformation with proline occupying 

the position of MeVal11 in the hydrophobic 

pocket (Taylor et al., 1997). All the structural results 

for the CsA complexes suggest that CsA must be 

in the correct conformation in solution before it can 

bind to CypA. This could be analogous to the recognition 

or escort role played by CypA in the cell 

where it only binds selected prolyl peptides. 

Materials and Methods 

Crystallisation 

Recombinant human CypA was puri®ed 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. The 

1 ml precipitating solution in the well consisted of 

100 mM Tris-HCl (pH 8.0), 19% (w/v) PEG 8000, 6% (v/ 

v) DMSO, 0.04% (w/v) NaN 3 . The initial 6 ml drops consisted 

50 mM Tris-HCl (pH 8.0), 9.5% PEG 8000, 3% 

DMSO, 0.02% NaN 3 , 0.5 mM CsA-analogue and 0.5 mM 

CypA. In most cases, crystals of the complex were prepared 

by cross-seeding (Mikol & Duc, 1994). After equilibrating 

for four days, individual seeds of about 10 mm 

from a crushed CypA/CsA crystal were transferred into 

the hanging drop. Crystals obtained from cross-seeding 

were used to seed at least one subsequent crystallisation 

in order to dilute out the effect of the CsA in the seed. 

X-ray diffraction measurements 

Crystals were mounted in Debye-Scherrer glass capillary 

tubes. X-ray intensity data were collected using a 

FAST television area-detector diffractometer mounted on 

a FR571 rotating-anode generator operating at 40 kV and 

80 mA. The program MADNES was used to process the 

data (Messerschmidt & P¯ugrath, 1987). 

X-ray structure refinement 

All structures were re®ned with the program X-PLOR 

(BruÈ nger, 1993) using alternating rounds of positional 

re®nement followed by temperature factor re®nement. 

Structures were re®ned using the Param19x parameters 

(BruÈ nger, 1993) except 33804, 27402 and 224698 which 

were re®ned using different stereochemical data from 

Engh & Huber (1991). The programs O (Jones et al., 

1991) and WITNOTP (Widmer, 1997) were used to visualise 

the structures and ®t electron density maps. 

Water molecules were selected from peaks on difference 

electron density Fourier maps which had peak 

heights greater than 3s and which were between 

2.1 AÊ and 3.4 AÊ from atoms in the current model. 

Buried surface areas were calculated using Connolly's 

algorithm implemented in the program WITNOTP 

(Widmer, 1997). All hydrogen atoms were added in calculated 

positions on the CsA derivatives and on CypA. 

The buried surface is the contact surface area of CypA 

not accessible to a probe atom of radius 1.5 AÊ when the 

CsA ligand is bound. 

Atomic co-ordinates of all new structures described 

here have been deposited in the Brookhaven Database 

with the following accession numbers: 33804, 1cwf; 

27402, 1bck; 209313, 1cwh; 209650, 1cwi; 209217, 1cwj; 

209825, 1cwk; 211810, 1cwl; 211811, 1cwm. 

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Edited by I. A. Wilson 

(Received 22 December 1997; received in revised form 18 June 1998; accepted 16 July 1998)

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