Thermodynamic Dissection of the Binding Energetics of Proline-rich Peptides to the Abl-SH3 Domain: Implications for Rational Ligand Design

doi:10.1016/j.jmb.2003.12.030

J. Mol. Biol. (2004) 336, 527–537

Thermodynamic Dissection of the Binding Energetics of Proline-rich Peptides to the Abl-SH3 Domain: Implications for Rational Ligand Design Andre´s Palencia†, Eva S. Cobos†, Pedro L. Mateo, Jose C. Martı´nez and Irene Luque* Department of Physical Chemistry and Institute of Biotechnology, Faculty of Sciences, University of Granada, 18071 Granada Spain

The inhibition of the interactions between SH3 domains and their targets is emerging as a promising therapeutic strategy. To date, rational design of potent ligands for these domains has been hindered by the lack of understanding of the origins of the binding energy. We present here a complete thermodynamic analysis of the binding energetics of the p41 proline-rich decapeptide (APSYSPPPPP) to the SH3 domain of the c-Abl oncogene. Isothermal titration calorimetry experiments have revealed a thermodynamic signature for this interaction (very favourable enthalpic contributions opposed by an unfavourable binding entropy) inconsistent with the highly hydrophobic nature of the p41 ligand and the Abl-SH3 binding site. Our structural and thermodynamic analyses have led us to the conclusion, having once ruled out any possible ionization events or conformational changes coupled to the association, that the establishment of a complex hydrogen-bond network mediated by water molecules buried at the binding interface is responsible for the observed thermodynamic behaviour. The origin of the binding energetics for proline-rich ligands to the Abl-SH3 domain is further investigated by a comparative calorimetric analysis of a set of p41-related ligands. The striking effects upon the enthalpic and entropic contributions provoked by conservative substitutions at solvent-exposed positions in the ligand confirm the complexity of the interaction. The implications of these results for rational ligand design are discussed. q 2003 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: SH3 domains; ligand design; binding energetics; calorimetry; water-mediated interactions

Introduction The recognition of proline-rich sequences by SH3 and WW domains is one of the most common mechanisms by which specific, transient protein – protein interactions are established within the cell. Polyproline-recognition domains are found in oncoproteins and proteins over-expressed in † A.P. & E.S.C. contributed equally to this work. Abbreviations used: SH3, Src homology 3 region; AblSH3, SH3 domain of the Abl tyrosine kinase; ITC, isothermal titration calorimetry; PPII, polyproline II helix; IPTG, isopropyl-b-D -thiogalactopyranoside; pdb, Protein Data Bank. E-mail address of the corresponding author: [email protected]

deregulated signalling pathways during cancer development and are also intimately involved in the pathogenesis of other diseases such as Alzheimer’s syndrome and muscular dystrophy.1 Additionally, a number of viruses have evolved mechanisms to exploit SH3-mediated cellular processes for their proliferation.2 – 6 Inhibitors of the interactions between SH3 and WW domains and their partners have proved to be promising therapeutic agents,4 – 9 validating these domains as attractive targets for drug design. The interactions between SH3 domains and their natural ligands are relatively weak, with binding affinities in the range of 1 –200 mM, which reflects the need for transient and dynamic interactions within signal transduction pathways. Consequently the development of high-affinity ligands

0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.

528

for these domains has proved to be a difficult task. The success of such classical approaches as synthetic library screening, phage display or mutational analysis has been limited,4 although some encouraging results have been obtained with libraries of N-substituted peptoids, for which nanomolar affinities have been reported.10 In the post-genomic era, a rationalization of the design of SH3 domain ligands is much needed, especially considering the ubiquitous nature of these domains and their key role in cell-cycle regulation and the development of disease. The rational modification of natural peptidic ligands of SH3 domains by molecular modelling has provided some promising, although as yet modest, results.11 The limited success of rational design approaches is due in part to the fact that the origins of the binding energy in SH3 – ligand interactions are not well understood. As shown recently with the HIV1 protease, a complete thermodynamic characterization of the interaction is key to the full understanding of the balance of forces that drive the interaction, and also to the identification of possible lines of action for the development of ligands with the desired characteristics (binding affinity, specificity, response to target heterogeneity, mutations, etc.).12,13 Thus, we present here a complete thermodynamic analysis of the binding energetics of proline-rich ligands to the SH3 domain of Abl. The cellular form of the Abelson leukaemia virus, c-Abl, is a tightly regulated protein tyrosine kinase which causes chronic myelogenous leukaemia when some of the regulation elements are disrupted.14 c-Abl becomes constitutively activated upon the mutation or deletion of the SH3 domain,15,16 and the displacement of the SH3 domain’s interaction by high-affinity ligands has been shown to enhance the inhibitory effect of the anti-tumor drug Gleevec.17,18 A calorimetric analysis of the association of the Abl-SH3 domain and the designed decapeptide p41 (APSYSPPPPP)11 reveals a thermodynamic signature inconsistent with the hydrophobic nature of the interacting surfaces. Both structural and thermodynamic evidence point towards the important role of additional polar interactions mediated by water molecules buried at the binding interface. A comparative analysis of the binding energetics of a set of closely related peptides in which individual substitutions at solvent-exposed positions are introduced provides striking results that confirm the complexity of the interaction. The relevance of these studies for the rational design of improved ligands is discussed.

Results and Discussion Energetics of p41 binding to the Abl-SH3 domain The energetics of the interaction between the proline-rich peptide p41 (APSYSPPPPP) and the

Calorimetry of Abl-SH3 and Proline-rich Ligands

SH3 domain of Abl were measured directly by isothermal titration calorimetry. Figure 1A shows as an example a calorimetric titration of p41 and AblSH3 at 25 8C in 20 mM sodium phosphate at pH 7.0. The corresponding binding isotherm is shown in Figure 1B together with the best fit to a model of one set of sites (as described in equation (3)). According to the analysis, p41 binds to a single binding site in Abl-SH3 with a dissociation constant of 2.3 mM, in accordance with values obtained by fluorescence spectroscopy reported elsewhere.11 The binding of p41 to Abl-SH3 is driven by a markedly favourable enthalpy change ðDHapp ¼ 221:9 kcal mol21 Þ, which is partially compensated by unfavourable entropic contributions (2TDSapp ¼ 14:2 kcal mol21). The heat-capacity change ðDCp;app ¼ 2174 calðK molÞ21 Þ was determined from the temperature dependence of the

Figure 1. Calorimetric titration of the Abl-SH3 domain with the proline-rich ligand p41 at 25 8C in 20 mM sodium phosphate (pH 7.0). A, (1) Heat effects associated with the injection of p41 (7 ml injections of a 608 mM solution) into the calorimetric cell containing the Abl-SH3 domain at a concentration of 32 mM. (2) Dilution experiment of p41 into the corresponding buffer under the same conditions and identical injection profile. The dilution curve has been displaced in the y-axis for representation purposes. B, Ligand concentration dependence of the heat released upon binding after normalization and correction for the heats of dilution. Symbols represent experimental data and the continuous line corresponds to the best fitting to a model considering one set of binding sites (equation (3)).

529

Calorimetry of Abl-SH3 and Proline-rich Ligands

binding enthalpy under the same pH and buffer conditions. The most salient feature of the binding thermodynamics of p41 to the Abl-SH3 domain is its strongly exothermic character, which contrasts with the highly hydrophobic nature of the binding interface and of the ligand itself. As illustrated in Figure 2, the only polar interactions observed in the three-dimensional structure of the p41/AblSH3 complex are two hydrogen bonds established between p41 and Abl-SH3: (1) a highly conserved hydrogen bond established between Tyr52 in the Abl-SH3 domain and the carbonyl oxygen of Pro8 in the peptide; and (2) a hydrogen bond established between the side-chain of Tyr4 in the ligand with residues Ser12 and Asp14 in the RT loop of the Abl-SH3 domain. The interaction between Abl-SH3 and p41 results ˚ 2 of non-polar area and in the burial of 702(^ 7) A ˚ 2 of ˚ 2 of polar area, of which 438(^ 1) A 250(^ 11) A ˚ 2 of polar area correnon-polar area and 88(^ 6) A ˚ 2 of non-polar spond to the ligand and 263(^ 7) A 2 ˚ area and 162(^ 6) A of polar area are due to the burial of groups in the SH3 domain. These values represent the numerical average of the four different structures found in the asymmetric unit and their corresponding standard deviations. These results (2.81 non-polar/polar ratio) are characteristic of a highly hydrophobic interaction for which an unfavourable, or at best slightly favourable, binding enthalpy would be expected,19 in opposition to the strongly exothermic character of the measured binding enthalpy. Furthermore, at room temperature the interaction between hydrophobic compounds should be associated with a considerable gain in solvation entropy due to the release of ordered water molecules from the ligand and binding site, which contrasts with the unfavourable

entropic contributions observed for the binding of p41 to Abl-SH3. A similar thermodynamic signature has been observed for other complexes between proline-rich peptides and SH3 domains.20 – 23 Although the binding affinity is of the same order of magnitude as other SH3 – peptide interactions, the mutually compensating enthalpic and entropic contributions that characterize the interaction between p41 and Abl-SH3 are notably bigger (enthalpies ranging from 2 5 kcal mol21 to 2 12 kcal mol21 for other peptides versus 2 21.9 kcal mol21 for p41). This is especially surprising considering the absence of polar or ionisable groups in the p41 peptide, which are present in many of these ligands. From this analysis it becomes clear that there must be additional factors contributing to the binding energetics of p41 to the Abl-SH3 domain, which confer upon an apparently highly hydrophobic interaction the thermodynamic signature typically observed for a polar interaction. There are several factors that might be responsible for the observed thermodynamic behaviour: (a) a coupling of the association process to the ionization of groups in the protein and/or the ligand;24,25 (b) a coupling of the binding of the ligand to a conformational change either in the protein or in the ligand itself;26,27 and (c) incomplete desolvation of the interacting surfaces upon binding and the presence of long-lived water molecules buried at the binding interface.19,28 Effect of protonation/deprotonation on the binding enthalpy Frequently, the binding of a ligand is coupled to a protonation/deprotonation process due to a change in pKa value, which ionisable groups in the ligand and/or the protein undergo upon binding. As a consequence, a pH dependence of the binding thermodynamic parameters (binding affinity, enthalpy and entropy) will be observed19,24,25 and the measured enthalpy change ðDHapp Þ will depend on the ionization enthalpy of the buffer, so that: DHapp ¼ DHbind þ nH DHion

Figure 2. Ribbon representation of the Abl-SH3 domain in complex with p41 (chains A and B in pdb file 1bbz). Protein residues critical for binding are shown in stick representation. Residues in the RT loop involved in the hydrogen bond with Tyr4 in the peptide are shown in purple. Peptide residues are colour-coded: oxygen, red; nitrogen, blue; carbon, grey. Hydrogen bonds are represented as dotted green lines.

ð1Þ

where DHapp is the measured binding enthalpy, both buffer and pH dependent, nH is the number of protons exchanged with the buffer, and DHion is the enthalpy of ionization of the buffer. DHbind , (buffer independent but pH dependent) is the magnitude of interest for ligand design purposes and can be estimated by measuring the binding enthalpies in several buffers that differ in their protonation enthalpies.19,24,25 The results of the calorimetric titrations carried out in three buffers characterized by different ionization enthalpies are summarized in Table 1. At all temperatures the net number of protons exchanged with the buffer upon binding is negligible, although small differences in the heat capacity changes upon binding,

530

Calorimetry of Abl-SH3 and Proline-rich Ligands

Table 1. Thermodynamic analysis of the interaction between p41 (APSYSPPPPP) and Abl-SH3 pH

Temperature (8C)

7.0

15

25 35

n

DH (kcal mol21)

Ka ( £ 1025 M21)

Phosphate Hepes Imidazol Phosphate Hepes Imidazol Phosphate Hepes Imidazol

0.99 1.04 1.04 1.05 1.01 1.02 1.05 0.98 1.03

220.0 218.6 218.8 221.9 221.0 221.0 223.5 223.2 223.8

12.2 9.2 9.9 4.8 3.9 4.3 1.5 1.3 1.1

Buffer

5.0

25

Acetate

0.98

222.0

2.9

3.0

25

Glycine

1.03

222.3

2.1

DCp cal (K mol)21

2174 2228 2247

naH

DH;int a (kcal mol21)

0.1

219.9

0.1

221.9

20.0

223.4

DCp;int a cal (K mol21)

2173

The variability in the experimental values were estimated to be about 1% in the number of binding sites, 5% in the binding enthalpy and 10% in the binding affinity. a These values were obtained considering the ionization values for the buffers reported45,46 for sodium phosphate (DHion ¼ 1224:8 cal mol21 , DCp;ion ¼ 244:7 calðK molÞ21 Þ, Hepes (DHion ¼ 5026:3 cal mol21 , DCp;ion ¼ 11:7 calðK molÞ21 Þ, and imidazol (DHion ¼ 8753:6 cal mol21 , DCp;ion ¼ 23:9 calðK molÞ21 Þ:

DCp;app , obtained with each buffer can be seen.24 An analysis of the temperature dependence of the buffer-independent binding enthalpy gives a value of 2 173 cal(K mol)21, which agrees closely with the heat capacity measured in sodium phosphate. Additionally, no significant changes in the thermodynamic parameters of binding at different pH values was observed (Table 1). These results confirm that the strongly exothermic nature of the binding of p41 to the Abl-SH3 domain reflects the intrinsic properties of the interaction and cannot be rationalized in terms of coupled ionization events. Conformational changes coupled to binding The buffer-independent binding enthalpy of small ligands, DHbind , frequently contains contributions arising from conformational changes in the protein and/or the ligand coupled to the binding event, so that: DHapp ¼ DHbind þ nH DHion ¼ DHint þ DHconf þ nH DHion

ð2Þ

where DHint corresponds to the enthalpy that would be observed if the protein and ligand had the same conformation in the free and bound states and DHconf includes the enthalpic effects associated to any conformational change in the protein and/ or the ligand upon binding. A comparative analysis of the three-dimensional structures of the unliganded Abl-SH3 domain and the complexes with p41 and 3BP1 reveals no significant changes in the conformation of the protein upon binding. Superimposition of the structures results in alpha-carbon rms deviation values ran˚ to 0.96 A ˚ . Only subtle deviations ging from 0.67 A are observed in the RT and n-Src loop regions. The structures of the Abl-SH3 domain in the complexes with p41 and 3BP1 are completely superimposable.

The most important conformational change coupled to the binding event could in any case be associated with the ligand, which must adopt a PPII helix in its C-terminal section upon binding. Previous reports11,29 indicate, however, that a significant fraction of PPII conformation is present in the free peptide in solution. Moreover, p41 (with five consecutive proline residues at the C terminus) has been designed to maximize the PPII helix content in solution in order to decrease the entropic penalty upon binding.11 These observations do not exclude additional conformational changes in the ligand upon binding, although such changes would be unlikely to account for the large magnitudes of the binding enthalpy and entropy observed for p41. Presence of water molecules at the binding interface The desolvation of the ligand and protein interfaces upon binding is not always complete. Longlived, buried water molecules are frequently found at the binding interface in many complexes.30 – 33 These buried water molecules may play a critical role in the binding energetics by mediating the interactions between protein and ligand. The water molecules may serve as adapters that fill non-occupied volumes and optimise van der Waals interactions, satisfy the hydrogen bonding potential of the ligand and the binding site and assist in the dissipation of charges. All these terms might be expected to contribute favourably to the binding enthalpy.19,27 Conversely, the incomplete desolvation of the ligand – protein interface would imply an entropic penalty that would oppose the favourable enthalpic effects, resulting in the effect of the buried water molecules on the Gibbs energy of binding often being no more than modest. In the absence of independent experimental

531

Calorimetry of Abl-SH3 and Proline-rich Ligands

evidence concerning the persistence of water molecules in the complex in solution, considering all ˚ of the fully buried water molecules within 5 – 7 A ligand molecule that bridge its interactions with the protein in the crystal structure seems to provide a good approximation to the situation in solution.19,27 A close inspection of the p41 –AblSH3 complex34 reveals the presence of several water molecules buried at the binding interface that mediate the interactions between the ligand and the Abl-SH3 domain binding site and lead to the formation of a complex hydrogen-bond network. In this case, five water molecules are involved in the binding interaction, three of which are fully buried at the binding interface. The identity of these water molecules and their solvent accessibility in each of the four complexes in the asymmetric unit is summarized in Table 2. A schematic representation of the hydrogen-bond network for one of the complexes is shown in Figure 3. A summary of the distances for the hydrogen bonds in all the complexes, identified by the numerical ID assigned to each interaction in Figure 3, is shown in Table 3. These data clearly show that the water molecules and the interactions in which they participate are preserved in all four complexes with p41 found in the pdb file. Additionally, very similar interactions are found in the complexes of the Abl-SH3 domain with its natural ligand 3BP1. It is interesting to note that most of these water molecules are absent in the structure of the uncomplexed Abl-SH3 domain. Thus, the presence of water molecules at the binding interface is clearly contingent upon the ligand’s binding. This analysis indicates that the p41 and 3BP1 ligands interact with the Abl-SH3 binding site via two different mechanisms: the stacking of proline side-chains with the hydrophobic grooves in the binding site35 – 37 and the formation of an intricate network of hydrogen bonds mediated by water molecules buried at the binding interface. The incomplete desolvation of the ligand and binding site effectively increases the polarity of the interaction and will necessarily be reflected in the binding energetics. It is our hypothesis that the

extremely negative enthalpy associated to the binding of p41 to Abl-SH3 domain is related to the presence of buried water molecules at the interface and to the interactions that they mediate. Additionally, the hydrogen bond network at the binding interface can influence the intramolecular interactions within the SH3 domain and assist in the propagation of the binding interactions throughout the structure, which have been shown to contribute to the measured energetics in other SH3 –ligand complexes.23,38 Energetics of binding of p41-related peptides to the Abl-SH3 domain Several p41-related ligands for the Abl-SH3 domain have been selected to test our hypothesis. This set of ligands includes the peptide corresponding to the binding sequence of the natural ligand of the Abl-SH3 domain 3BP1 (APTMPP PLPP) and four other 3BP1-derived peptides rationally designed to improve binding affinity and specificity for the Abl-SH3 domain.11 The sequences of these peptides (p0 (APTYPPPLPP); p17 (APTYSPPLPP); p7 (APTYPPPPPP); and p40 (APTYSPPPPP)) differ in specific positions (5 and 8) that are exposed to the solvent in the complex and do not participate in the hydrophobic interactions established with the protein. Specifically, Ser5 is replaced by Pro and Pro8 replaced by Leu (as observed in the natural peptide 3BP1) in different backgrounds. The replacement of Ser or Leu by Pro at solvent-exposed positions can only be expected to result in a more favourable binding entropy due to the restrictions imposed by the proline side-chain on the conformational space of the free ligand, whilst no enthalpic effects are to be expected. A recent experimental illustration of these effects is provided by the thermodynamic analysis of the binding of variants of a SOS peptide to the Sem-5 SH3 domain, where the substitution of exposed Pro residues by Ala and Gly resulted in changes in the binding affinity ranging between 0.6 kcal mol21 and 1.2 kcal mol21 entirely due to entropic effects.21 The binding energetics for all ligands to Abl-SH3

Table 2. Water molecules at the Abl-SH3/ligand binding interface and their solvent accessibilities P41 (a/b)a ˚ 2) w1015 (0.03 A ˚ 2) w1064 (0.00 A ˚ 2) w2103 (5.55 A ˚ 2) w1082 (2.37 A ˚ 2) w1105 (0.00 A

P41 (c/d)a

P41(e/f)a

P41(g/h)a

3BP1 (a/c)b

˚ 2) w1036 (1.12 A ˚ 2) w1025 (0.00 A ˚ 2) w2016 (16.73 A ˚ 2) w1067 (4.89 A ˚ 2) w1060 (0.00 A

˚ 2) w1019 (3.76 A

˚ 2) w2036 (2.85 A ˚ 2) w1028 (0.00 A ˚ 2) w1003 (8.93 A ˚ 2) w1001 (6.25 A ˚ 2) w1018 (0.54 A

˚ 2) w8 (8.22 A ˚ 2) w35 (9.78 A ˚ 2) w60 (7.19 A ˚ 2) w42 (6.51 A

˚ 2) w1097 (0.00 A ˚ 2) w1061 (0.00 A ˚ 2) W1089 (0.00 A ˚ 2) w2082 (1.14 A

3BP1 (b/d)b ˚ 2) w26 (10.55 A

FreeAbl/SH3c ˚ 2) w8 (36.86 A

˚ 2) w82 (6.27 A ˚ 2) w88 (4.65 A

Water molecules are labelled according to their number in the original pdb files. Values in parentheses are the solvent accessibilities in the complex. a Four structures for the p41–Abl-SH3 complex in the asymmetric unit (1bbz). b Two structures for the 3BP1–Abl-SH3 complex in the asymmetric unit (1abo). c Free Abl-SH3 domain (1abq).

Figure 3. Schematic representation of the hydrogen-bond network mediated by the water molecules buried at the binding interface. Water molecules are represented as filled spheres, intermolecular hydrogen bonds are depicted as thick broken lines and intramolecular hydrogen bonds are shown as thin dotted lines. Each interaction has been assigned a numeric ID for discussion purposes.

533

Calorimetry of Abl-SH3 and Proline-rich Ligands

Table 3. Distances for direct and water-mediated hydrogen bonds in Abl-SH3 complexes Interaction IDa 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 a b c d

P41(A/B)b

P41(C/D)b

P41 (E/F)b

P41(G/H)b

3BP1(A/C)c

3BP1(B/D)c

Free Abl-SH3d

2.57 3.23 2.67 3.08 3.22 3.91 3.07 2.76 2.86 2.81 3.30 3.31 2.49 2.74 3.65 2.70 2.49 2.64 2.88 3.02 3.12

3.55 2.31 3.14 2.82 3.34 3.95 3.28 2.98 3.15 2.87 3.46 3.94 2.45 2.71 3.38 – – 2.81 3.18 3.06 –

3.07 4.49 2.90 – – – – – – 2.80 3.56 – 2.55 2.65 3.16 3.13 2.51 2.73 3.19 3.07 2.78

3.90 3.00 2.71 2.78 3.18 3.33 2.98 2.82 3.02 2.82 3.58 3.48 2.64 2.68 3.51 2.60 2.51 2.90 3.24 2.90 3.05

– – 3.73 2.78 3.04 – 3.56 2.78 2.60 3.02 – 3.24 – 2.68 – – – 2.87 3.30 3.10 2.83

– – 2.83 3.23 3.75 – 2.75 – – 2.80 – – 2.74 2.89 3.06 3.20 2.67 3.10 3.36 3.22 2.96

– – – – – – – – – 2.67 – – – – – – – – – – –

Interactions are numbered according the ID assigned in Figure 3. Four structures for p41– Abl-SH3 in the asymmetric unit (1bbz). Two structures for 3BP1–Abl-SH3 in the asymmetric unit (1abo). Free Abl-SH3 domain (1abq).

were measured by isothermal titration calorimetry at 25 8C in sodium phosphate (pH 7.0). Because the substitutions in this set of peptides included no ionisable groups, the pH dependency and ionization contributions to the binding energetics were expected to parallel those of the related peptide p41. Thus, the binding enthalpies of the different peptides in phosphate buffer could be compared directly. Table 4 summarizes the results of the thermodynamic analyses for the six peptide ligands. It is clear that the binding of all the peptides is characterized by a common thermodynamic signature: i.e. a large and favourable binding enthalpy partially counterbalanced by an unfavourable entropic contribution. Within this trend important differences can be seen in the magnitude of the enthalpic and entropic contributions to the Gibbs energy of binding. For the solvent exposed positions, these differences are maximum when Ser at position 5 and Pro at position 8 are simultaneously introduced into the sequence (DDHapp ðp40=p0Þ ¼ 4:1 kcal mol21 and It is 2TDDSapp ðp40=p0Þ ¼ 23:3 kcal mol21 ). important to emphasize that these are remarkable

and unexpected effects for conservative substitutions at solvent-exposed positions, not directly implicated in the hydrophobic interactions at the binding site. These results indicate that a complex mechanism must exist by which substitutions at these positions influence the binding energetics. For clarity, the effects of the replacements at positions 5 and 8 in the enthalpy and entropy of binding are represented in Figure 4 as a thermodynamic cycle. The replacement of Ser by Pro at position 5 results in the loss of the intramolecular hydrogen bond established between the hydroxyl moiety in the side-chain of Ser5 and the carbonyl oxygen atom of Pro6 (see Figure 3). These substitutions exert no effect upon binding affinity. The favourable entropic effect due to the reduction in the conformational space available for the free peptide when the Pro residue is introduced is completely compensated by unfavourable enthalpic contributions, probably arising from the loss of the hydrogen bond. This type of intramolecular hydrogen bond between short polar side-chains and the carbonyl oxygen atom of the following residue in

Table 4. Thermodynamic analysis of the binding of p41-related peptides to the Abl-SH3 domain Peptide

Sequence

p41 p40 p7 p17 p0 3BP1

APSYSPPPPP APTYSPPPPP APTYPPPPPP APTYSPPLPP APTYPPPLPP APTMPPPLPP

DGapp (kcal mol21)

DHapp (kcal mol21)

2TDSapp (kcal mol21)

27.7 27.7 27.6 26.9 26.9 26.1

221.9 221.1 218.3 217.5 217.0 216.7

14.2 13.4 10.7 10.6 10.1 10.6

All reported values were obtained in 20 mM sodium phosphate (pH 7.0) at 25 8C.

534

Calorimetry of Abl-SH3 and Proline-rich Ligands

Figure 4. Thermodynamic cycle summarizing the effects of substitutions at positions 5 and 8 on the proline-rich ligands on the thermodynamic parameters of binding to the Abl-SH3 domain. The values associated with the vertical arrows correspond to the substitutions at position 5 and those associated with the horizontal arrows to the substitutions at position 8. The diagonal arrow corresponds to the simultaneous replacement of both positions.

the sequence is very common39 and is thought to be responsible for the unusually high PPII propensities of these type of residues.40 A very striking feature made apparent by these experiments is the existence of some kind of cooperativity between positions 5 and 8, which amplifies the enthalpic and entropic effects of substitutions at position 5 when Pro is present at position 8, and the effects of substitutions at position 8 when Ser is present at position 5. In both positions the enthalpic and entropic differences are increased by 2.2 kcal mol21. Although the entropic effect of introducing Pro at position 5 with Leu at position 8 (2 0.5 kcal mol21) is comparable with values reported elsewhere for Ala/Pro substitutions,21 the 2 2.7 kcal mol21 value obtained when Pro is at position 8 cannot be easily rationalized, and reflects the existence of additional interactions, not apparent from the crystal structure. The most interesting effects are those generated by the replacement of Leu8 by Pro. In this case, a significant increase in binding affinity is observed, which is in accord with the results of fluorescence spectroscopy experiments reported elsewhere. These results were initially rationalized in terms of conformational entropy effects.11 Considering the fact that Leu is a good PPII former and that the insertion of a non-proline residue in the middle of a polyproline helix does not significantly disrupt the PPII conformation,40 the substitution of Leu8 by Pro might be expected to have a small, though favourable, entropic effect. Our thermodynamic

analysis, however, has uncovered a completely different scenario: the observed increase in binding affinity is enthalpic in origin and the introduction of Pro at position 8 is entropically unfavourable. These results, particularly the large and favourable enthalpic changes, cannot be rationalized in the context of a purely hydrophobic interaction and provide evidence that additional effects come into play. In light of the structural information available, the origin of this behaviour is likely to be linked to the presence of the previously described water molecules at the binding interface and the hydrogen-bond network they mediate. Proline residues in PPII helix conformation are known to be ideal hydrogen bonding sites due to the fact that their backbone carbonyl groups are electron-rich and poorly hydrated as a consequence of the conformational restrictions imposed by their cyclic sidechain.1,40 The observed increase in the favourable enthalpic contributions might reflect the tightening of the hydrogen-bond network when Pro is introduced, as well as the intensification of the ligand effects upon the intramolecular hydrogen bonds within the SH3 domain itself. Changes in the properties of the hydrogen bonds and in the backbone dynamics of the Src-SH3 domain when the ligand binds have been reported elsewhere.23,38 Changes in dynamics of the SH3 domain together with the trapping of water molecules at the binding interface are also consistent with the unfavourable entropic effects. Further studies to test the persistence of the buried water molecules in solution as

535

Calorimetry of Abl-SH3 and Proline-rich Ligands

well as a comparative analysis of the influence of the peptide sequence on the dynamics of the complex will contribute to a fuller understanding of these effects. Implications for ligand design The thermodynamic data presented above indicate that the binding affinity of proline-rich peptides to the Abl-SH3 domain is the result of a delicate balance of mutually compensating contributions. An apparently simple interaction based on the stacking of hydrophobic residues has a complex thermodynamic behaviour indicative of the existence of other factors such as water molecules buried at the binding interface or the propagation of binding interactions throughout the SH3 domain, which are important for the binding energetics. A complete thermodynamic analysis has proved to be essential for understanding the forces that drive the interaction, making it patent that apparently irrelevant substitutions in terms of binding affinity produce considerable alterations in the interactions within the complex. Uncovering these effects is the key to the efficient rational design of new and improved ligands. Within the set of 3BP1-derived peptides analysed in this work, the replacement of Met4 by Tyr, which leads to the formation of hydrogen bonds between the peptide and residues at the RT loop in the protein, constitutes an example of the ideal situation in ligand design. This substitution results in modest but favourable effects in both the enthalpy and entropy of binding that are translated into maximum effects on binding affinity. The opposite is found for substitutions at positions 5 and 8, which induce surprisingly large changes in the enthalpic and entropic contributions that cancel each other out and result in a modest or null improvement to binding affinity. The thermodynamic analysis has uncovered important features of the interaction that could be exploited for the design of improved ligands. To this end, for example, the introduction of new moieties in the ligand to replace some of the water molecules buried at the binding interface whilst maintaining the hydrogen bond interactions might provide a way of obtaining entropic benefits due to the release of water molecules, whilst maintaining the favourable enthalpic effects. Additionally, the existence of cooperative effects between different positions in the peptide provides alternative strategies for design: i.e. substituting Ser5 by another short polar residue such as Gln with stronger hydrogen bonding capability.

Materials and Methods Expression and purification of the Abl-SH3 domain The plasmid pET3d containing the Abl-SH3 domain gene was a generous gift from L. Serrano (EMBL, Heidel-

berg). Expression levels of the Abl-SH3 domain were optimized by re-cloning the Abl-SH3 domain gene, coding for the sequence MENDPNLFVA LYDFVASGDN TLSITKGEKL RVLGYNHNGE WCEAQTKNGQ GWVP SNYITP VNS, into the pBAT4 plasmid41 by polymerase chain reaction and digestion with the Nco I and HindIII restriction enzymes. The Abl-SH3 domain was expressed and purified as described elsewhere.29,42 Briefly, plasmidencoded Abl-SH3 domain was expressed in Escherichia coli BL21(DE3) strain (Novagen) using IPTG as inducing agent. Cells were resuspended in 100 mM Tris (pH 9.0) buffer and broken with two passes through a French pressure cell. The Abl-SH3 domain was precipitated from the supernatant with ammonium sulphate (75% saturation) and resuspended in 10 mM phosphate buffer, 500 mM sodium chloride (pH 6.5). The protein was further purified on a Superdex-75 column equilibrated and eluted with the same buffer. Abl-SH3-bearing fractions were pooled, concentrated and stored at 220 8C at 11.7 mg ml21 in the same buffer. Under these conditions, the Abl-SH3 domain is stable for several months. Protein purity was checked by SDS-PAGE and mass spectrometry and estimated to be higher than 99%. Peptide ligands Peptide ligands were provided by the EMBL peptide service with the exception of peptides p40 and p41, which were bought from DiverDrugs (Barcelona, Spain). All the peptides were acetylated and amydated at their N and C termini, respectively. They were synthesized in the solid phase in an MPS column and their molecular mass was confirmed by mass spectrometry. Peptide purity (. 95%) was assessed by analytical HPLC. Determination of protein and peptide concentrations Abl-SH3 domain concentration was determined by absorbance at 280 nm using an extinction coefficient of 16,894 M21 cm21. Peptide concentration was determined by absorbance at 278 nm using an extinction coefficient of 1450 M21 cm21. Extinction coefficients were calculated using Gill and von Hippel’s method.43 Isothermal titration calorimetry Isothermal titration calorimetry was performed using a high-precision MCS titration calorimetric system (Microcal Inc., Northampton, MA). Frozen aliquots of Abl-SH3 domain were thawed on ice. The protein was incubated with 100 mM dithiothreitol to reduce any possible intermolecular disulphide bonds formed during storage and was extensively dialyzed against the titration buffer. All solutions were filtered, properly degassed to avoid bubble formation, and equilibrated to the required temperature prior to each experiment. The Abl-SH3 solution (at about 100 mM) in the calorimetric cell was titrated with the appropriate ligand (between 0.608 mM and 1.0 mM) dissolved in the dialysis buffer. Titrations were made by injecting 7 – 8 ml of all peptide– ligand solutions with the exception of 3BP1, where, due to its relatively low binding affinity, a profile of injection volumes from 4 ml to 20 ml was used to define the titration curve more clearly. The heat evolved after each peptide injection was obtained from the integral of the calorimetric signal. The heat produced by the binding reaction between the Abl-SH3 domain and the peptide ligand was obtained as the difference between the heat

536

Calorimetry of Abl-SH3 and Proline-rich Ligands

of reaction and the corresponding heat of dilution, as obtained from independent titrations of the peptide ligand into the buffer. The resulting binding isotherms were analysed by non-linear least-squares fittings of the experimental data to a model corresponding to a single set of identical sites, according to the equation:

Q¼


n½Mt DHV0 ½Xt  1 1þ þ n½Mt  nKa ½Mt  2

sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi  2  Xt 1 4½Xt  2 1þ þ 2 nKa ½Mt  n½Mt  n½Mt 

ð3Þ

where Q is the net heat of binding, n is the number of binding sites, Ka is the association constant, V0 is the active cell volume, and ½Mt  and ½Lt  are the total concentrations of macromolecule and ligand, respectively. In all the least-squares analyses, the number of binding sites, association constant, and binding enthalpy were considered as floating parameters. The data were performed using Microcal Origin (OriginLab Corporation, Northampton, MA) together with software developed in this laboratory. Experiments were performed at least twice. Typically the variability in the experimental values were estimated to be about 1% in the number of binding sites, 5% in the binding enthalpy and 10% in the binding affinity. Accessible surface area calculations Changes in accessible surface area were calculated according to the Lee and Richards’ algorithm.44 In all cal˚ and a slice width of culations a solvent radius of 1.4 A ˚ were used. In order to better define differences in 0.25 A solvent accessibility, 64 different orientations of the ligands and proteins with respect to the slicing plane, generated by rotating the molecule around the x, y, and z axes, were considered in the calculations. The solvent accessibility for each atom was obtained as the numerical average of the values calculated for all molecular orientations.

Acknowledgements This research was funded by grants BIO20001459 and BIO2003-04274 from the Spanish Ministry of Science and Technology and grant HPRN-CT2002-00241 from the European Union. A.P. was supported by a research contract funded by the Andalusian Government. E.S.C. was supported by a pre-doctoral grant from the Spanish Ministry of Education and Culture. I.L. was supported by a research contract from the University of Granada and is the recipient of a Ramo´n y Cajal research contract from the Spanish Ministry of Science and Technology. We thank our collegue Dr John Trout for revising the English text. We also thank Dr M. T. Pisabarro and Dr F. Conejero-Lara for helpful discussions.

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Edited by F. Schmid (Received 22 September 2003; received in revised form 1 December 2003; accepted 1 December 2003)