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European Journal of Medicinal Chemistry 46 (2011) 5556e5561

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European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Original article

Oligopeptide cyclophilin inhibitors: A reassessment

Michael Schumann, Günther Jahreis, Viktoria Kahlert, Christian Lücke, Gunter Fischer*

Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, D-06120 Halle/Saale, Germany

a r t i c l e i n f o

Article history:Received 1 September 2011Received in revised form12 September 2011Accepted 14 September 2011Available online 21 September 2011

Keywords:CyclophilinCyclosporin AProlyl bond cis/trans isomerizationInhibitioncis/trans ratioIsothermal titration calorimetry

Abbreviations: CypA, cyclophilin A; CsA, cyclospocis/trans isomerase; ITC, isothermal titration calorime* Corresponding author. Tel.: þ49 345 5522800; fa

E-mail address: fischer@enzyme-halle.mpg.de (G.

0223-5234/$ e see front matter � 2011 Elsevier Masdoi:10.1016/j.ejmech.2011.09.019

a b s t r a c t

Potent cyclophilin A (CypA) inhibitors such as non-immunosuppressive cyclosporin A (CsA) derivativeshave been already used in clinical trials in patients with viral infections. CypA is a peptidyl prolyl cis/transisomerase (PPIase) that catalyzes slow prolyl bond cis/trans interconversions of the backbone of substratepeptides and proteins. In this study we investigate whether the notoriously low affinity inhibitoryinteraction of linear proline-containing peptides with the active site of CypA can be increased througha combination of a high cis/trans ratio and a negatively charged C-terminus as has been recently reportedfor Trp-Gly-Pro. Surprisingly, isothermal titration calorimetry did not reveal formation of an inhibitoryCypA/Trp-Gly-Pro complex previously described within a complex stability range similar to CsA,a nanomolar CypA inhibitor. Moreover, despite of cis content of 41% at pH 7.5 Trp-Gly-Pro cannot inhibitCypA-catalyzed standard substrate isomerization up to high micromolar concentrations. However, in thecontext of the CsA framework a net charge of e7 clustered at the amino acid side chain of position 1resulted in slightly improved CypA inhibition.

� 2011 Elsevier Masson SAS. All rights reserved.

1. Introduction

Cyclophilins represent a subfamily of peptidyl prolyl cis/transisomerases (PPIases, EC. 5.2.1.8) that can bind and catalyticallyinterconvert prolyl cis/trans isomers in all folding states ofa proline-containing polypeptide chain. The functional importanceof the prototypic enzyme cyclophilin A (CypA) for a variety ofbiological processes such as viral infections [1e4], chronic inflam-mation [5e8] and malignancies [9e13] has stimulated a consider-able interest in the development of CypA inhibitors as mechanistictools and potential drugs for various diseases.

The immunosuppressive cyclic undecapeptide cyclosporin A(CsA) and non-immunosuppressive CsA derivatives represent low-nanomolar inhibitors of the PPIase activity of CypA [14,15]. Besideinhibition of CypA catalytic activity, CypAeCsA complex formationalso precedes calcineurin inhibition and subsequent suppression ofIL-2 expression which may explain how CsA mediated immuno-suppression can arise [16]. Non-peptidic small molecule inhibitorshave been designed but require considerable improvement toexhibit CsA-like inhibitory potencies [17]. While the active cleft ofCypA orients CsA and oligopeptide substrates in the opposite

rin A; PPIase, peptidyl prolyltry.x: þ49 345 5511972.Fischer).

son SAS. All rights reserved.

direction of the contacting sites in their respective complexestopologies are defined by the superposition of the proline ring ofthe substrate and the MeVal-11 side chain of CsA [18]. The markedincrease of about >104 fold in the CypA affinity for CsA whencompared to a linear oligopeptide substrate can be rationalized bythe transition state-like structural constraints imposed by themacrocycle [19]. In an extensive search of heptapeptide sequencesderived from the HIV-1 Gag polyprotein capsid domain containing-Xaa-Gly-Pro- moieties only very weak CypA binders could beidentified [20]. On the other hand, the internally positioned -Gly-Pro- sequence motif was found as an essential feature for CypAbinders identified by phage display screening and by planar peptidearrays [21]. Consequently, the question arises whether linearpeptide inhibitors of low molecular masses, whose particularamino acid sequences and internal charge distributions allow fora transition state complementary geometry, might exist. Recently,based on the identification of critical residues of CypA for theCypAeCsA interaction by calculating the electronic structure ofboth molecules [22], a virtual oligopeptide library database hasbeen ranked for CypA affinity by using a molecular docking algo-rithm [23]. Interestingly, the calculations have resulted in a fewcharged oligopeptides that exhibited interaction energies superiorto those of the CypAeCsA interaction. Based on these calculationsthe authors have carried out an experimental approach with PPIaseassays, surface plasmon resonance analyses and viral replicationdata. The inhibitory tripeptide Trp-Gly-Pro eventually emerged,exhibiting a CypA binding and inhibiting affinity nearly as strong as

M. Schumann et al. / European Journal of Medicinal Chemistry 46 (2011) 5556e5561 5557

those found for CsA. Even more, marked antiviral effects in a HIV-1replication assay have been noted [23]. From the cellular viewpoint,these findings would be particularly intriguing since they implicateprotein degradation products, especially those resulting fromcatalysis by proline-specific proteases, in the regulation of endog-enous CypA activity.

Structurally, the major differences found between previouslytested peptides and Trp-Gly-Pro were found in the charge distri-bution next to the critical proline and the presence of a Trp residue.To explain the possible origin for the said CsA-like biochemicaleffects, the interplay between a negative charge at the backboneand a potentially Trp-mediated increase of the cis/trans ratio wasconsidered to play a dominating role.

Here we investigate anionic peptides in conjunction with anNMR-based analysis of prolyl cis/trans isomers to evaluate theirCypA inhibitory potency.We show below that various PPIase assaysand isothermal titration calorimetry revealed a drastic loss of CypA-peptide complex stability for peptides bearing a negative chargenext to proline on the backbone. Taken together, our results do notonly contradict CypA inhibition but furthermore indicate a lack ofaffinity of short linear oligopeptides, such as Trp-Gly-Pro, for CypA.

2. Results and discussion

2.1. Isothermal titration calorimetry

Direct comparison of surface plasmon resonance, which hasbeen used as an activity independent binding assay by Pang et al.[23], and isothermal titration calorimetry (ITC) has been already

Fig. 1. Representative isothermal titration calorimetry of CsA (A) and Trp-Gly-Pro (B) with Cpeak corresponds to the injection of 15 ml of 100 mM cyclophilin into the titration cell contatitrated with 100 mM peptide solution. Capacity of the titration cell was 1.4 ml. Referenceseparately obtained and subtracted from the thermogram of the sample titrations. Cumulatipart). The solid line of the CsA titration represents the least square fit of the compiled dataa complex stoichiometry N ¼ 1.023 � 0.08 and a reaction enthalpy DH ¼ 10.6 � 0.1 kcal mcalorimetry.

utilized to analyze CypA inhibitor interactions [24]. Our ITCexperiments indicated an exothermally formed CypAeCsA complex(DH ¼ 10.6 kcal mol�1) with a Kd value of 7.6 nM together witha complex stoichiometry of 1.02 in good agreement with publisheddata [24,25]. However, under the same conditions, titration of Trp-Gly-Prowith CypA did not indicate any binding up to 25 mMpeptidein the titration cell (Fig. 1).

It can be hypothesized that the reportedly high affinity of Trp-Gly-Pro resulted from the combination of a high cis prolyl isomercontent and a negatively charged proline. Indeed, 1H NMR inves-tigations on linear oligopeptide substrates revealed that the KMvalue of a cis isomer is significantly lower than that of the respectivetrans isomer [26,27]. It is also known that proline-containing linearpeptides undergo relatively slow cis/trans equilibration afterdissolution in aqueous buffer and that this might lead to a lag andlatency issue for the cis isomer content. It follows that very slowisomerization kinetics might be able to disturb our ITC experiment[28]. Thus, we characterized the prolyl isomer composition of Trp-Gly-Pro in buffer solution at pH 7.5 by NMR spectroscopy.

2.2. Isomer-specific NMR signals

To identify isomer-specific 1H and 13C resonances of Trp-Gly-Prothe signals were completely assigned by means of homo- andheteronuclear 2D NMR spectra (Supplementary Tables S1 and S2).The trans and cis prolyl bond isomers were identified based on theCb and Cg chemical shift values [29], and additionally confirmed bysequential ROEs between Gly2 Ha and either Pro3 Ha or Hd. Due tostrong resonance overlap, the cis/trans ratio was obtained from the

ypA in 10 mM HEPES buffer pH 7.5, 100 mM NaCl at 20 �C. For the CsA experiment eachining 10 mM CsA. For the tripeptide the titration cell contained 10 mM CypA which wastitration of buffer versus buffer, CypA versus buffer and peptide versus buffer were

ve heat of reaction is displayed as a function of the mole ratio of inhibitor/CypA (lowerpoints. (A) CsA binding to CypA gave an association constant Ka ¼ (1.3 � 0.6) � 108 M,ol�1 (B) CypA did not show any affinity for the tripeptide in the isothermal titration

M. Schumann et al. / European Journal of Medicinal Chemistry 46 (2011) 5556e55615558

2D NMR data of Trp-Gly-Pro. Interestingly, under the solventconditions used in the PPIase assays, this tripeptide showed a highcis content of 41% as based on integration of the CaH signals in the1H/13C-HSQC spectrum (Fig. 2). This finding opens up the possibilityof assuming the cis isomers of linear peptides bearing a negativecharge as highly inhibitory toward CypA. We note that in the NMRexperiments the cis/trans isomerization rate was shown to be fastenough to equilibrate the isomers within 30 min after dissolutionindicating that the ITC experiment was not corrupted by theisomerization kinetics.

Fig. 3. Inhibition of the PPIase activity of cyclophilin A (CypA) by Trp-Gly-Pro (trian-gles), Succinyl-Ala-Ala-Pro-Phe (squares), VK-053 (diamonds), and cyclosporin A (CsA)(circles) in the protease-coupled assay. The PPIase activity of 0.5 nM CypA wasmeasured in 10 mM HEPES buffer pH 7.5, 100 mM NaCl at 10 �C using Succinyl-Ala-Ala-Pro-Phe-4-nitroanilide as substrate. Each data point represents the average of threemeasurements. The error bars represent standard deviation. PPIase activity of CypAwas inhibited by VK-053 and CsA. Drawn lines were calculated from the hyperbolicequation using an IC50 value of 4.9 � 0.4 nM (VK-053) and 9.3 � 1.5 nM, (CsA),respectively.

2.3. Inhibition of PPIase activity

We have carried out CypA inhibition experiments where Trp-Gly-Pro was allowed to preequilibrate in HEPES buffer pH 7.5,100 mM NaCl prior to testing.

Surprisingly, no inhibition of CypA could be seen in thechymotrypsin-coupled standard PPIase assay even at high concen-trations of Trp-Gly-Pro. According to the reported IC50 value of33.1 nM [23], a negligible residual activity should result at 100 mMTrp-Gly-Pro in this assay contrasting the almost uninhibited isom-erization catalysis depicted in Fig. 3. However, under similarconditions our experiments revealed an IC50 value of 9.5 � 1.5 nM(Fig. 3) for CsAwhich iswithin the rangeof inhibition data publishedelsewhere [25].

We then examined the effects of increasing concentrations ofanother anionic peptide, Succinyl-Ala-Ala-Pro-Phe on the PPIaseactivity of CypA. This peptide is an intrinsic constituent in thechymotrypsin-coupled standard PPIase assay [24] because it isincreasingly present in the assaymixture bycleavage of Succinyl-Ala-Ala-Pro-Phe-4-nitroanilide as the isomerization reaction proceeds.While tetrapeptide 4-nitroanilides bind CypA with KM values in thehighmicromolar range [26,27], their peptidic fragment of proteolysisfailed to compete in the catalyzed substrate isomerization up to100 mM concentrations (Fig. 3). Lack of active site binding affinity ofthe anionic peptide is in accord with the strict first-order kineticsobserved in the time courses of protease-coupled PPIase assays [31].This result is reminiscent to the PPIase Pin1which is mechanisticallyrelated to CypA. Here, prevention of a negatively charged peptidebackbone alongwith chain elongation increasedbinding to the activesite of Pin1 more than ten-fold [32].

Fig. 2. Section from the 1H/13C-HSQC spectrum of Trp-Gly-Pro (in 50 mM phosphatebuffer, pH 7.5, 25 �C) displaying only the CaH signals of the trans and cis prolyl bondconformers. The 1D 1H trace is shown on the top.

In the light of the above findings it is important to analyzewhether the net negative charge of an inhibitor is generallyrepulsive for its interaction with CypA. Given that the very lowCypA affinity of linear oligopeptides makes them unsuitable fordetecting small effects, the CsA framework would provide a sensi-tive probe of binding energy changes.

The CsA derivative VK-053 has been synthesized and charac-terized in a PPIase assay by comparisonwith CsA. Seven carboxylateresidues covalently added to the side chain of MeBmt amino acid atthe 1-position of CsA (Supplementary Scheme S1) suffice toimplement a moderate increase in the inhibitory potency(4.9 � 0.4 nM) when compared to CsA (Fig. 3). We note that evenclustering the negative charge adjacent to the binding site ofa transition state-like CypA inhibitor did not impair its intrinsicallyhigh affinity.

Fig. 4. Inhibition of the PPIase activity of cyclophilin A (CypA) by Trp-Gly-Pro in theprotease-free assay [29]. The PPIase activity of 0.5 nM CypA was measured in 10 mMHEPES buffer (pH 7.5), 100 mM NaCl at 10 �C using Succinyl-Ala-Ala-Pro-Phe-4-nitroanilide as substrate.

M. Schumann et al. / European Journal of Medicinal Chemistry 46 (2011) 5556e5561 5559

To confirm that the chymotrypsin content in the protease-coupled PPIase assay did not deteriorate the inhibitory nature ofTrp-Gly-Pro, a protease-free PPIase assay was utilized [30]. How-ever, the pattern of the doseeactivity curve was found to be similarto that of the protease-coupled assay, confirming the inert nature ofTrp-Gly-Pro in the CypA activity assays (Fig. 4).

3. Conclusions

We have presented a detailed study of the active site directedinteraction of CypA with linear peptides and cyclosporins with theaim of elucidating the effects of negative charges and cis isomercontent on CypA inhibition. Our results were shown to be incomplete disagreement with the recent predictions of moleculardocking simulations and subsequent experimental findings thata charged linear tripeptide realizes a CsA-like inhibitory efficiencytoward CypA. In contrast, we have shown that short proline-containing peptides behave inert in CypA-catalyzed prolyl isom-erizations up to high micromolar concentrations. Furthermore, wehave examined the question of the charge dependence of CypAinhibition using a 1-position substituted CsA derivative containinga clustered net charge of �7. A small improvement of the inhibitorypotency could be achieved indicating that the active site of CypAtolerates anionic residues in the transition state-like inhibitors.Finally, our findings suggest that endogenous CypAwill maintain itsfull enzymatic activity in the presence of most short oligopeptidesresulting from protein degradation in the cell. Together, thesefindings indicate that CypA-oligopeptide interaction is opposed byelectrostatic repulsion between a negative charge close to thereactive site of a substrate-like inhibitor and CypA and that a similarcharge, if located more distant to the active site, appears toaugment inhibition in the context of a transition state-like peptideinhibitor conformation. Obviously, this conformation cannot berealized by Trp-Gly-Pro. These results also demand reassessment ofprevious findings that the protective effect of Trp-Gly-Pro againstHIV-1IIIB replication is due to cyclophilin inhibition.

4. Experimental protocols

4.1. Peptide synthesis

Peptides were synthesized by solid-phase peptide synthesiswith the robot SYRO II (MultiSynTech, Witten, Germany) using0.15 mmol of preloaded Fmoc-phenylalanine- or Fmoc-proline-2-chlorotrityl resins (Novabiochem, Merck-Chemicals, Darmstadt,Germany). The syntheses were performed by Fmoc strategy andstandard protocol (PyBOP/N-methyl-morpholine in DMF). Piperi-dine (20%) in DMFwas the standard cleavage cocktail used for Fmocdetachment.

Succinylation was done with succinic anhydride, N-ethyl-diisopropylamine and 4-(dimethylamino)-pyridine (Merck-Chem-icals, Darmstadt, Germany) in DMF.

After synthesis, peptides were cleaved from the resin with TFA/TIS/water (95:3:2, v/v/v) for 2 h and purified with a preparativeHPLC (Abimed, Langenfeld, Germany) on an Interchrom Modulo-Cart Strategy 5, C18-2, 250 � 10 column (Interchim, Montlucon,France) using a water/ACN gradient containing 0.1% TFA in thesolvents. Peptides were detected at 220 nm.

The purified peptides were lyophilized and their purity was veri-fied by analytical HPLC using a LiChroCART� column (LiChrospher�

100, RP8, 5 mm; 125� 4 mm) (Merck, Darmstadt, Germany) with thefollowing conditions: gradient of 5e100% ACN in water (with 0.1%TFA) at a flow rate of 1 ml/min over 30 min; and UV-detection at220 nm. Themolecular masses of the peptides were confirmed by ESImass spectrometry.

Succinyl-Ala-Ala-Pro-Phe-OH, Mmcalc 504.5, Mmfound 505.0,Rt ¼ 9.9 min; Trp-Gly-Pro-OH, Mmcalc 358.4, Mmfound 359.0,Rt ¼ 9.7 min.

4.2. VK-053 (MeBmt-SCH2CH2CO-NH(D-Glu)6-Gly-OH]1-CsA)

VK-053was synthesized from CsA as depicted in SupplementaryScheme S1. CsA was purchased from LC Laboratories with >99%purity. Silica and RP18 columns for flash chromatography wereused from Teledyne Isco. All other reagents and chemicals werereceived from commercial sources in analytical grade and wereused without further purification.

Analytical HPLC runs were executed on DIONEX equipment orSYKAM equipment. MALDI-TOF mass spectra were measured atUltraflex II TOF/TOF, BrukereDaltonik GmbH, and electrosprayionization (ESI) mass spectra were measured at ESI-IontrapESQUIRE-LC, BrukereDaltonik GmbH.

4.2.1. [O-Acetyl]1-CsACsA (2 g, 1.664 mmol) and 4-(dimethylamino)pyridine

(331.4 mg, 2.71 mmol) were added to acetic anhydride (20 ml) andstirred at room temperature overnight. The solvent was removed invacuo and the residue was purified by flash chromatography(solvent A dichloromethane, solvent B methanol, silica column24 g). Method: 20 �C, flow 30 ml/min, 5 min isocratic 0% B in 7 minto 4.8% B followed by isocratic 9 min with 4.8% B. Yield, 96%, lightyellow solid. Analytical HPLC, solvent A 0.1% TFA/H2O, solvent B0.1% TFA/acetonitrile, RP C8, 250.00 � 4.600 mm. Method: 65 �C,50e100% B in 30 min, flow 1 ml/min.

[O-Acetyl]1-CsA, Mmcalc 1244.9, Mmfound 1245.0, Rt ¼ 16.5 min.

4.2.2. [O-Acetyl]1-CsA bromideAzobisisobutyronitril (10 mg) was added to a solution of [O-

Acetyl]1-CsA (500 mg, 0.402 mmol) and N-bromosuccinimide(1.25 eq., 90 mg, 0.5024 mmol) in carbon tetrachloride (75 ml) andthe mixture was heated to reflux for 3 h. The solvent was removedunder reduced pressure. Residue material was taken up in ethylacetate (150 ml), washed twice with brine, dried over MgSO4 andfiltered. The solvent was removed under reduced pressure. Thecrude product was used for further reactions.

Analytical HPLC: solvent A 0.1% TFA/H2O, solvent B 0.1% TFA/acetonitrile, column RP C8, 250.00 � 4.600 mm. Method: 65 �C,50e100% B in 30 min, flow 1 ml/min.

[O-Acetyl]1-CsA bromide, Mmcalc 1322.8, Mmfound 1344.8[M þ Na]þ, Rt ¼ 16.9 min.

4.2.3. [MeBmt-SCH2CH2COOH]1-CsA

Methyl 3-mercaptopropionate (1.5 eq., 72.6 ml, 0.67 mmol)and Cs2CO3 (1 eq., 130 mg, 0.402 mmol) were added to a solutionof the crude [O-Acetyl]1-CsA bromide (0.402 mmol) in acetoni-trile (50 ml) were added (1 eq., 130 mg, 0.402 mmol dissolved in20 ml water). The solution was stirred at room temperatureovernight.

The solvent was removed under reduced pressure and replacedby methanol (30 ml). After the addition of 0.2 M aqueous LiOH(30 ml) the mixture was stirred at room temperature for 3 h. Thereaction was acidified with diluted HCl and the solvent was evap-orated. The product was purified by flash chromatography. SolventA 0.1% AcOH/H2O, Solvent B 0.1% AcOH/acetonitrile, RP C18, 130 g.Method: 20 �C, flow 75 ml/min, 5 min isocratic 65% B in5 mine100% B, 7 min isocratic 100% B.

Yield, 34% (relative to [O-Acetyl]1-CsA); white solid. AnalyticalHPLC, solvent A 0.1% TFA/H2O, solvent B 0.1% TFA/acetonitrile, RPC8, 125.00 � 4.600 mm. Method: 20 �C, 50e100% B in 30 min, flow1 ml/min.

M. Schumann et al. / European Journal of Medicinal Chemistry 46 (2011) 5556e55615560

[MeBmt-SCH2CH2COOH]1-CsA, Mmcalc 1306.8, Mmfound 1306.9,Rt ¼ 8.7 min.

4.2.4. [MeBmt-SCH2CH2CO-NH(D-Glu)6-Gly-OH]1-CsA

N-ethyldiisopropylamine (5.9 ml, 0.0345 mmol) and 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoropho-sphate (HATU) (4.8 mg, 0.01265 mmol) were added to a solution of[MeBmt-SCH2CH2COOH]1-CsA (15 mg, 0.0115 mmol) in dry DMF(1 ml) and stirred for 5 min at room temperature. Then, a solutionof H-(D-Glu)6-Gly-OH (14.7 mg, 0.01725 mmol) and N-ethyl-diisopropylamine (5.9 ml, 0.0345 mmol) in dry DMF (1 ml) wasadded and the mixture was stirred at room temperature overnight.The reaction was acidified with diluted HCl and the solvent wasevaporated. Preparative HPLC, solvent A 0.05% TFA/H2O, solvent B0.05% TFA/acetonitrile, RP C8 250 � 21 mm. Method: 20 �C, 17 ml/min, gradient 30e80% B in 60 min. Yield: 73%; white solid.Analytical HPLC, solvent A 0.05% TFA/H2O, solvent B 0.05% TFA/acetonitrile, RP C8,125.00� 4.600mm.Method: 20 �C, 5e100% B in30 min, flow 1 ml/min.

[MeBmt-SCH2CH2CO-NH(D-Glu)6-Gly-OH]1-CsA, Mmcalc 2138.1,Mmfound 2138.2, Rt ¼ 16.4 min.

4.3. PPIase assays

4.3.1. Protease-coupled assayHuman recombinant CypAwas prepared as described elsewhere

[25]. This assay was done according to a published procedure [15]at 10.0 �C in 10 mM HEPES buffer pH 7.5 (AppliChem A1069,Darmstadt, Germany), 100 mM NaCl, 2 nM bovine serum albumin(SigmaeAldrich A1900, Steinheim, Germany).

4.3.2. Protease-free assaySuccinyl-Ala-Ala-Pro-Phe-4-nitroanilide (18.7 mg, Bachem L-

1400, Bubendorf, Switzerland) was dissolved in 1 ml anhydrous0.55 M LiCl/TFE solvent mixture and stored at 4 �C. Solvent jumpswere initiated by addition of an aliquot of stock solution of peptidein 0.55 M LiCl/TFE into 10 mM HEPES buffer pH 7.5 (AppliChemA1069, Darmstadt, Germany), 0.5 nM human recombinant CypA,2.0 nM bovine serum albumin (SigmaeAldrich A1900, Steinheim,Germany) resulting in a final peptide concentration of 60 mM at10 �C. Absorbance at 390 nm was measured on a Hewlett PackardHP 8452A diode array spectrometer. A water-jacket cell holderconnected to a cryostat was used for temperature control using anexternal thermostat. In order to calculate first-order rate constants,a monoexponential function was fitted to the reaction progresscurves. Assays were done in the presence and absence of inhibitor.Enzyme activities were obtained from the time course of the cis/trans isomerization using a calculated first-order rate constant kappwith kapp ¼ k0 þ [E] kcat/KM, where k0 is the rate constant of theuncatalyzed reaction, [E] is the PPIase concentration and kcat andKM are the turnover number and the Michaelis constant, respec-tively. Great care must be taken in keeping assay constituents freeof protease contamination.

4.4. NMR

NMR spectra were acquired using a Bruker DMX 500 spec-trometer operating at 500.13 MHz proton resonance frequency andequippedwith a 5mm triple-resonance 1H{13C/15N} probe. All NMRmeasurements were performed with an approx. 10 mM peptidesample in 50 mM phosphate buffer pH 7.5 at 25 �C. For chemicalshift assignments, standard 2D 1H/1H-TOCSY, 1H/1H-ROESY, 1H/13C-HSQC and 1H/13C-HMBC spectra were collected with the carrierplaced in the center of the spectrum on the water resonance, whichwas suppressed by applying either presaturation or a WATERGATE

sequence. 1H and 13C chemical shifts were referenced to external2,2-dimethyl-2-silapentane-5-sulfonate.

4.5. Isothermal titration calorimetry

Titration experiments were performed in 10 mM HEPES bufferpH 7.5 (AppliChem A1069, Darmstadt, Germany), 100 mM NaClusing a MicroCal VP-ITC at 20 �C. Protein samples were dialyzedagainst the batch of buffer used for titration. A peptide stocksolution was prepared, and prior to the experiment, it was dilutedinto the same batch of buffer used for dialysis. All solutions usedwere degassed before filling the sample cell and syringe. The ITCstirring speed was set to 310 rpm. For titration of CsA with CypA300 ml of 100 mMCypAwere added to 1.4 ml of a 10 mMCsA solutionin 15 ml steps. For titration of Trp-Gly-Pro with CypA 300 ml ofa 100 mM peptide solution were added to 1.4 ml of 10 mM CypA in15 ml steps. Since the initial injection generally delivers inaccuratedata, only 2 ml were injected in this step. For ITC of CsA with CypA,the recorded data of this first step were discarded. Referencetitration of buffer versus buffer, CypA versus buffer and peptideversus buffer were separately obtained and subtracted from thethermogram of the sample titrations. Binding isotherms were fittedaccording to a one-site model of binding. Errors correspond to thestandard deviations of the non-linear least-squares fit of the datapoints of the titration curve.

Acknowledgments

This study was supported by the DFG SFB 610 and the BMBFProject ProNet3. We are grateful to Dr. Angelika Schierhorn andSuzanne Roß for experimental support.

Appendix. Supplementary material

Supplementary data related to this article can be found online atdoi:10.1016/j.ejmech.2011.09.019.

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