de Groot, B.

Seeliger, D.

Sheldrick, G.

Stegmann, C.

Wahl, M.

English

MPG.PuRe

Structural BiologyDOI: 10.1002/anie.200900481The Thermodynamic Influence of Trapped Water Molecules on aProtein–Ligand Interaction**Christian M. Stegmann, Daniel Seeliger, George M. Sheldrick, Bert L. de Groot, andMarkus C. Wahl*The rational design of protein-binding substances requires anin-depth understanding of the energetics of protein–ligandinteractions, including the free energy (DG), enthalpy (DH),entropy (DS), and the change of heat capacity at constantpressure (DCp) upon binding.[1] Previously, the DCp of bindinghas been correlated with changes in the accessible polar andapolar surface areas of the interacting species,[2,3] suggestingthat hydrophobic interactions are the primary source of heatcapacity effects (for a review, see reference [4]). However,these considerations leave discrepancies between calculatedand observed DCp values in a number of cases.[5] In particular,the effects of solvent (typically water) and solutes (forexample ions) buried upon complex formation are neglected;yet it is well known that these molecules can form discretenoncovalent bonds to both the protein and the ligand, therebyinfluencing recognition processes. For example, interfacewater molecules can act as shape modifiers, hydrogen bondbridges, or as electrostatic screens.[6] Furthermore, solvent andsolute molecules trapped upon a protein–ligand interactionexperience a reduction in amplitude of their vibrationalmodes, thus giving rise to a change in the heat capacity.[7–9] Todate, there is no experimental estimate for the size of thiseffect in a protein–ligand interaction.Herein, we investigated the thermodynamic consequencesof trapping water molecules upon interaction of humancyclophilin G (CypG; an enzyme with peptidyl prolyl cis/transisomerase (PPIase) activity) with the immunosuppressivecyclic peptide cyclosporin A (CsA). Comparison of the CsAbinding of the wild type (wt) CypG to a point mutant allowedus to single out the influence of trapped solvent molecules onthe heat capacity change while excluding a significant differ-ential contribution from buried surface areas.CypG contains an N-terminal cyclophilin domain(CypGPPIase ; residues 1–177) that binds CsA and a C-terminalSer-Arg dipeptide-rich (RS) domain (residues 178–754).[10,11]We determined ab initio crystal structures at 0.75  resolu-tion of CypGPPIase alone and in complex with CsA. CypGPPIasebinds CsA in a similar manner as described for CypA[12,13](Figure 1a,b), without undergoing significant conformationalchanges (root-mean-square deviation of 0.20  for 174 CaFigure 1. a) Crystal structure of CypGPPIase (ribbon plot) in complexwith CsA (sticks, carbon: orange). b) The Arg115 side chain (cyan)adopts a closed conformation when complexed with CsA. c) In theCsA complex, the guanidinium group of Arg115 is well-ordered andhydrogen bonds to the carbonyl oxygen atoms of Arg85 and Gly86 andto the carbonyl oxygen of Abu2 of CsA, thereby burying two watermolecules (red spheres; positions 1 and 3) and a chloride ion (greensphere; position 2). d) In the apo CypGPPIase structure, Arg115 is in anopen conformation (d, major conformation shown). Mesh: sA-weighted electron densities contoured at the 1.5s level.[*] C. M. Stegmann, Dr. D. Seeliger, Dr. B. L. de Groot,Prof. Dr. M. C. WahlResearch Group X-ray Crystallography, Computational BiomolecularDynamics Group, Max Planck Institute for Biophysical ChemistryAm Fassberg 11, 37077 Gttingen (Germany)E-mail: mwahl@gwdg.deHomepage: http://www.mpibpc.gwdg.de/groups/wahl/Prof. Dr. M. C. WahlFreie Universitt Berlin, Institut fr Chemie und Biochemie, AGStrukturbiochemieTakustrasse 6, 14195 Berlin (Germany)Prof. Dr. G. M. SheldrickUniversity of Gttingen, Department of Structural ChemistryTammannstrasse 4, 37077 Gttingen (Germany)[**] We are grateful to Gleb P. Bourenkov, Ehmke Pohl and Ina Dix forhelp with data collection, Ulrich Reidt for initial purification andcrystallization trials of CypGPPIase, Pawel Burkhardt for advice on ITCexperiments, Kay Diederichs for providing multiprocessor versionsof SHELXL, and Reinhard Lhrmann for generous access to hisfacilities and continued interest. C.M.S. is a fellow of the GermanNational Academic Foundation (Studienstiftung des DeutschenVolkes). This research was supported by a grant from the VWFoundation (VW I/81 649), by the German Research Foundation(DFG-GR 207914), and by the Max Planck Society. Atomiccoordinates and structure factors have been deposited in the RCSBProtein Data Bank (www.rcsb.org) under PDB IDs 2wfi and 2wfj.Supporting information for this article is available on the WWWunder http://dx.doi.org/10.1002/anie.200900481.AngewandteChemie5207Angew. Chem. Int. Ed. 2009, 48, 5207 –5210  2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheimatoms of apo and CsA-bound CypGPPIase). However, Arg115in CypGPPIase replaces an alanine at the corresponding site inCypA (see Supporting Information, Figure S1). Upon com-plex formation, the Arg115 side chain engages in directhydrogen bonds to the carbonyl oxygen atom of Abu2 in CsAand also to the carbonyl oxygen atoms of Arg85 and Gly86 ofCypGPPIase (Figure 1c). These interactions are not available tothe equivalent alanine in CypA. Arg115 thereby seals the so-called Abu pocket,[12] in which two water molecules (atpositions 1 and 3) and a chloride ion (at position 2) becometrapped.The identity of the chloride ion was confirmed by analysisof anomalous difference Fourier maps (Supporting Informa-tion, Figure S2). The two water molecules are tetrahedrallyengaged in hydrogen bonds: the water at position 1 hydrogenbonds to the backbone amides of Gly121 and Gln123, to thebackbone carbonyl of Ala113, and to the water at position 3.The water at position 3 hydrogen bonds to the guanidiniumgroup of Arg115, the carbonyl of Thr119, and the chloride inposition 2. The chloride in turn is coordinated by the amide ofSer122 and the guanidinium group of Arg115 (Figure 1c). Inthe apo form, the Arg115 side chain is partially disordered(Figure 1d), leaving the Abu pocket open. Here, all threepositions are occupied by water molecules. The position 1water forms hydrogen bonds to the backbone NH group ofGly121, the carbonyl groups of Ala113 and Gln123, and thewater at position 3. The water at position 2 hydrogen bonds tothe amides of Ser122 and Gln123, the carbonyl of Gly86, andthe water at position 3 (Figure 1d). Thus, Arg115 seems to actlike a lid that closes and is locked in place upon CsA binding.In CypA, the alanine corresponding to Arg115 cannot form asimilar lid, presumably permitting free flux of solvent andsolutes into and out of the cavity when CsA is bound.As crystallography cannot distinguish between site occu-pancy and residence time, we performed molecular dynamics(MD) simulations of apo wt CypGPPIase, wt CypGPPIase incomplex with CsA, and a CypGPPIase(Arg115Ala) pointmutant in complex with CsA (ten independent simulationsof 10 ns length for each system). The simulations revealedthat a chloride ion at position 2 in the CypGPPIase/CsAcomplex can be exchanged with water and that chlorideoccupied that site owing to its high concentration (400 mm) inthe crystallization setup. At a near-physiological chlorideconcentration (150 mm), the chloride ion was replaced by awater molecule in five out of ten simulations within thesimulation time of 10 ns, whereas the other two watermolecules at positions 1 and 3 remained close to thecrystallographic configuration. The water in position 2engages in alternative bonding schemes to the position 3water and either to Arg115 (Figure 2a) or to the carbonyl ofGly85 (not shown). Thus, at close to physiological conditions,water and chloride bind with similar occupancy at position 2.In agreement with the crystal structures, the MD simu-lations revealed a significant decrease in the flexibility ofArg115 upon CsA binding. Although Arg115 exhibits similarrates of dihedral transitions at its c1 angle in apo CypGPPIaseand in CypGPPIase/CsA, the rates of transitions at c2, c3, and c4are significantly reduced in the complex (Figure 2b). WhenArg115 adopts an elongated conformation and forms hydro-gen bonds with the carbonyl oxygen atoms of Arg85 or Gly86,a cavity is formed in which water molecules become trapped(Figure 2a; Supporting Information, Figure S3). Owing to theFigure 2. a) Representative locations and hydrogen bonding of trappedwater molecules at positions 1, 2, and 3 during a simulation of theCypGPPIase/CsA complex. Dashed lines indicate hydrogen bonds. b) His-togram of dihedral angle transitions of Arg115 in apo wt CypGPPIase(black bars) and in the CypGPPIase/CsA complex (gray bars). Theflexibility of the side chain is significantly reduced upon binding ofCsA. c) Residence times of water molecules at the three binding sitesin the Abu pocket. Whereas the water at position 1 does not exchangein any of the systems, the water molecules at positions 2 and 3 showincreased exchange rates upon mutation of Arg115 to Ala (i.e. removalof the “lid” of the Abu pocket) and in the absence of CsA (i.e. upon“unlocking” the lid).Communications5208 www.angewandte.org  2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2009, 48, 5207 –5210reduced flexibility of Arg115 in the CypGPPIase/CsA complex,the lifetime of this conformation is increased and, as aconsequence, the residence times of two of the three watermolecules in the cavity are significantly prolonged (Table 1;Supporting Information, Figure S3).The exchange rate of water molecules at position 1 is verylow in all systems and in all simulation runs (0.2 exchanges per10 ns in the wild-type complex; 0.5 exchanges per 10 ns in apowt CypGPPIase and the mutant complex; Figure 2c). Althoughthe residence times of the position 1 water molecules in wtCypGPPIase/CsA simulations appear to be longer than in theother simulations (Figure 2c), the absolute numbers ofexchanges are too low to accurately calculate differences inthe exchange rates. Longer simulations of the three systemsrevealed that a water molecule can remain at position 1 for upto 180 ns.In contrast, the exchange rate at position 2 is stronglydependent on both CsA binding and the Arg115Ala muta-tion. Whereas the exchange rate at position 2 is also very lowin the wt CypGPPIase/CsA complex (0.5 exchanges per 10 ns), itincreases eightfold in the apo form (to 4.1 exchanges per10 ns; Figure 2c). A comparable increase in the exchange rate(to 4.3 exchanges per 10 ns) is observed in the simulations ofthe mutant CypGPPIase(Arg115Ala)/CsA complex (Figure 2c).Similarly, in the wt CypGPPIase/CsA complex, a watermolecule at position 3 has a very low exchange rate (0.1exchanges per 10 ns). In the apo CypGPPIase simulations, incontrast, we did not observe a prolonged residence time ofwater molecules at this position (Figure 2c). Furthermore, nowater was trapped at this position in the simulations of themutant CypGPPIase(Arg115Ala)/CsA complex. Therefore, oursimulations show that the reduced flexibility of Arg115 in thewt CypGPPIase/CsA complex has a major effect on theresidence times of two of the three trapped water moleculesin CypGPPIase/CsA, whereas the water molecule in position 1appears to be structurally relevant and is neither affected byCsA binding nor by mutation of Arg115.The above analyses suggest that an arginine instead of analanine at position 115 contributes negatively to the DCp ofthe complex formation with CsA by mediating the trapping ofwater molecules. To test this hypothesis, we performedcalorimetric titrations at a range of temperatures with wtCypGPPIase and the CypGPPIase(Arg115Ala) point mutant. At25 8C, CsA binding to wt and mutant CypGPPIase has a similarbinding free energy owing to enthalpy/entropy compensationeffects (Table 2). However, a plot of the interaction enthal-pies versus temperature reveals that the DCp of binding ofCsA to CypGPPIase decreases by 114 calmol1K1 compared tothe DCp of binding of CsA to the mutant (Figure 3).The protein surface areas buried upon CsA binding are426 2 in wt CypGPPIase and 444 2 in CypGPPIase(Arg115Ala)(model based on the wt crystal structure). On the basis ofchanges in the accessible polar and apolar surface areasalone,[3] this increase in buried surface area would result in anincrease in DCp of binding. Instead, we observe a decrease inDCp of binding, which we attribute to the differential trappingof two water molecules in the wt complex. Although theexchange rates of the water molecules at positions 2 and 3increase to different extents upon removal of the Arg115 sidechain (Figure 2c), the current statistics do not allow us toquantify individual contributions to DCp of binding. Ourresults, therefore, provide an experimental value of (5721) calmol1K1 for the average heat capacity change owingto the sequestration of one water molecule upon formation ofa protein–ligand complex. This experimental estimate agreesvery well with theoretical figures and studies of modelcompounds. Holdgate and co-workers[14] suggested a valueof (48 31) calmol1K1. Cooper[5] calculated a change inDCp of at least 18 calmol1K1 per sequestered watermolecule based on simple lattice models. Habermann andMurphy[15] determined a value of (60 8) calmol1K1 persequestered water molecule based on the dissolution of c(AS)diketopiperazine crystals (a dipeptide model compoundcontaining one water molecule in its crystalline form).Taken together, our findings demonstrate that one aminoacid substitution in the active site of a well-conserved enzymecan cause the burial of solvent molecules and thereby elicit aTable 1: Exchanges per 10 ns at positions in the Abu pocket.Simulation Pos 1 Pos 2 Pos 3wt CypGPPIase/CsA 0.2 0.5 0.1apo wt CypGPPIase 0.5 4.1 >10CypGPPIase(Arg115Ala)/CsA 0.5 4.3 >10Table 2: Thermodynamic data from isothermal titration calorimetry.Titrant CypGPPIase CypGPPIase(Arg115Ala)T [8C] 25 25KD [nm] 70 64DHobs [calmol1] 1279073 1336081DS [calmol1K1] 10.2 11.9TDS [calmol1] 3039 3546DG [calmol1] 9750 9813DCp [calmol1K1] 58031 46611Figure 3. Integrated enthalpies of calorimetric titration experiments(DHobs) plotted against the temperature (T) for the wt CypGPPIase/CsAand the CypGPPIase(Arg115Ala)/CsA interaction. The differential changein heat capacity (DCp) upon CsA binding is given by the slopes of thecurves.AngewandteChemie5209Angew. Chem. Int. Ed. 2009, 48, 5207 –5210  2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.orgchange in the thermodynamic signature of the interactionwith ligands.Received: January 25, 2009Revised: March 18, 2009Published online: June 4, 2009.Keywords: biophysics · drug design · isomerases ·molecular dynamics · structural biology[1] R. Perozzo, G. Folkers, L. Scapozza, J. Recept. Signal Trans-duction Res. 2004, 24, 1.[2] K. P. Murphy, D. Xie, K. C. Garcia, L. M. Amzel, E. Freire,Proteins Struct. Funct. Genet. 1993, 15, 113.[3] R. S. Spolar, J. R. Livingstone, M. T. Record, Jr., Biochemistry1992, 31, 3947.[4] N. V. Prabhu, K. A. Sharp,Annu. Rev. Phys. Chem. 2005, 56, 521.[5] A. Cooper, Biophys. Chem. 2005, 115, 89.[6] C. S. Poornima, P. M. Dean, J. Comput.-Aided Mol. Des. 1995, 9,500.[7] J. M. Sturtevant, Proc. Natl. Acad. Sci. USA 1977, 74, 2236.[8] J. E. Ladbury, J. G. Wright, J. M. Sturtevant, P. B. Sigler, J. Mol.Biol. 1994, 238, 669.[9] S. Bergqvist, M. A. Williams, R. OBrien, J. E. Ladbury, J. Mol.Biol. 2004, 336, 829.[10] L. Cavarec, T. Kamphausen, B. Dubourg, I. Callebaut, F.Lemeunier, D. Metivier, J. Feunteun, G. Fischer, N. Modjtahedi,J. Biol. Chem. 2002, 277, 41171.[11] F. P. Nestel, K. Colwill, S. Harper, T. Pawson, S. K. Anderson,Gene 1996, 180, 151.[12] V. Mikol, J. Kallen, G. Pflugl, M. D. Walkinshaw, J. Mol. Biol.1993, 234, 1119.[13] G. Pflgl, J. Kallen, T. Schirmer, J. N. Jansonius, M. G. Zurini,M. D. Walkinshaw, Nature 1993, 361, 91.[14] G. A. Holdgate, A. Tunnicliffe, W. H. Ward, S. A. Weston, G.Rosenbrock, P. T. Barth, I. W. Taylor, R. A. Pauptit, D. Timms,Biochemistry 1997, 36, 9663.[15] S. M. Habermann, K. P. 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The thermodynamic influence of trapped water molecules on a protein-ligand interaction.

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