Hot Spots in Tcf4 for the Interaction with
-Catenin*
Marina Fasolini
,
Xiaoqiu Wu
¶,
Maria Flocco
,
Jean-Yves Trosset
,
Udo Oppermann
and
Stefan Knapp
||
From the
Pharmacia Corporation Discovery Research Oncology, Department of Chemistry, Viale Pasteur 10, 20014 Nerviano, Italy and
Karolinska Institutet, Medical Biochemistry and Biophysics, S 171 77 Stockholm, Sweden
Received for publication, February 19, 2003
, and in revised form, March 20, 2003.
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ABSTRACT
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The interaction of
-catenin with T-cell factor (Tcf) 4 plays a central role in the Wnt signaling pathway and has been discussed as a possible site of intervention for the development of anti-cancer drugs. In this study, we performed Ala-scanning mutagenesis of all Tcf4 residues in the Tcf-
-catenin interface and studied the binding energetics of these mutants using isothermal titration calorimetry. Binding of Tcf4 was found to be highly cooperative. Single site mutations of most Tcf4 residues resulted in a significant reduction in binding enthalpies but in similar binding constants as compared with wild type Tcf4. Interestingly, this was also true for residues that are disordered in the reported crystal structures. The mutation D16A caused the largest reduction in binding constant (50-fold) accompanied by a large unfavorable enthalpy change (
Hobs) of +8 kcal/mol at 25 °C. In contrast, the mutation of the Tcf residues Glu24 and Glu28, which have been proposed as an interaction hot spot due to their location in a field of strong positive electrostatic potential on the
-catenin surface (charge button), resulted only in a significant reduction of binding enthalpies, which were largely compensated for by unfavorable entropic contributions to the binding. Other mutations that significantly reduced Tcf binding constants were D11A and alanine mutations of the hydrophobic residues Leu41, Val44, and Leu48. The measured thermodynamic data are discussed with the available structural information of Tcf-
-catenin crystal structures and allow us to propose possible sites for development of Tcf antagonists.
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INTRODUCTION
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-Catenin is a crucial component of the cell adhesion machinery as well as an intracellular mediator of the Wnt signaling pathway. These diverse functions are realized through interaction with various binding partners. In its signaling role, cytosolic
-catenin interacts with proteins of the Tcf 1/Lef family of DNA-binding proteins. In the absence of Wnt signaling, target genes are maintained in a quiescent state by Tcf proteins (1, 2). When Wnt signaling is activated, cytosolic
-catenin is stabilized and translocated into the nucleus, where it functions with Tcf proteins as a transcriptional activator for a large number of target genes (3, 4, 5, 6, 7, 8). Transcriptional activation of
-catenin-Tcf target genes is a hallmark of colorectal cancer cells, and the constitutive activation of some of these genes is essential for creating and maintaining the malignant phenotype (9, 10). Consequently, the
-catenin-Tcf complex has emerged as an attractive drug development target.
The first 53 amino acids in Tcf4 constitute the cateninbinding domain (CBD) of this protein. The N-terminal sequence stretch of Tcf4 is highly acidic and is not folded in solution (11). The CBD is necessary and sufficient for high affinity interaction with
-catenin (4, 5, 8, 11). Several crystal structures have been determined to date; one structure is available for the Xenopus xTcf3-
-catenin complex, and two structures have been determined for human Tcf4-
-catenin complexes (12, 13, 14).
Structural alignments of the two human Tcf4-
-catenin complexes and the homologous structure of xTcf3-
-catenin revealed a number of surprising conformational differences, in particular in the N-terminal and the extended regions of Tcf, indicating a high degree of molecular plasticity. Because the Tcf4 ligand is not implicated in crystal packing in any of the published structures, the observed differences might be a result of changes in solvent conditions (12, 13, 14).
Structural data as well as co-immunoprecipitation assays have suggested that the main interaction points for the recognition of
-catenin by Tcf are two regions of highly positive electrostatic potential. These so-called charge buttons are formed by the
-catenin residues Lys435, Arg469, His470, Asn426, and Lys508 as well as Lys312 that interact with the xTcf3 residues Asp16, Glu17, and Glu24, respectively (12). The importance of the first charge button for Lef1 binding has been described previously in a mutagenesis study on
-catenin (15). In addition, several hydrophobic contacts such as those formed by the Tcf residues Phe21 and Val48 have been reported to be important for Tcf4 binding to
-catenin (16). Calorimetric, intrinsic fluorescent quenching and surface plasmon resonance binding data have shown that Tcf4 binds to
-catenin armadillo with a binding constant of 3 ± 2 x 108 mol-1. At 20 °C, the interaction is strongly favored by a large negative binding enthalpy that is strongly temperature-dependent and gives rise to a heat capacity change (
Cp) of -1.5 kcal/(mol K) (11).
To define interaction hot spots on Tcf4 that may be mimicked by low molecular weight antagonists and to understand the interactions with
-catenin, we mutated all residues in Tcf that make significant contacts with
-catenin. The binding of the Tcf mutants was subsequently studied by isothermal titration calorimetry, which allowed not only the determination of binding constants in solution but also gave insight into the binding energetics of the Tcf4 mutants.
Our study defined residue Asp16 as the most important interaction point of Tcf4. The mutation D16A results in a large decrease in binding enthalpy and binding constant. Single site mutations of Tcf4 residues interacting with the second charge button on
-catenin resulted in only marginal changes of the binding constant but significantly reduced binding enthalpies. In general, mutations of polar Tcf residues in the Tcf-
-catenin interface resulted in less favorable binding enthalpies. For most mutants, this effect was largely compensated for by entropic contributions to the binding, indicating a large degree of cooperativity in this interaction. Another residue that was found to decrease binding constants and alter binding energetics when mutated to alanine was Asp11, which has been found to be disordered in the crystal structures of the Tcf4-
-catenin complex. This finding demonstrates that even residues that are disordered in crystal structures might significantly contribute to binding affinity and alter binding energetics upon mutation. In addition, an important cluster of hydrophobic residues for the interactions of Tcf4 with
-catenin was defined by the alanine mutants of the residues Leu41, Val44, and Leu48 of the C-terminal helix of the Tcf-CBD.
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MATERIALS AND METHODS
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Cloning, Expression, and Purification of ProteinsThe armadillo repeat region (amino acid residues 134671) in
-catenin was cloned and purified as described previously (11). Tcf4-CBD (residues 156) was cloned into the NdeI and XhoI restriction sites of pET20b. To quantitate the recombinant Tcf4 protein spectroscopically, a tryptophane residue was introduced between the CBD and the C-terminal His6 tag. Sitedirected mutagenesis of Tcf4 was performed using the QuikChange kit (Stratagene) and oligonucleotides that were complementary to 15 bp upstream and downstream of the mutation site on both strands. Tcf4-CBD mutants were transformed into Escherichia coli (BL21-DE3). Transformed bacteria were grown to mid-log phase at 37 °Cin2x yeast tryptone medium, and protein expression was induced by addition of 1 mM isopropyl-1-thio-
-D-galactopyranoside. Induced cells were then grown for 3 h and harvested by centrifugation. Cell pellets were resuspended and lysed in PBS (Sigma) using a French press. The recombinant proteins were purified using pre-charged Ni-Sepharose beads (Pharmacia) and a standard protocol (Novagen). The proteins were judged to be pure after affinity chromatography as assessed by SDS-PAGE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Tcf4 wild type and mutants were quantitated using an absorbance coefficient at 280 nm of 5.69 x 103 mol/cm.
Isothermal Titration CalorimetryCalorimetric measurements were carried out at constant temperatures using a VP-isothermal titration calorimetry titration calorimeter (MicroCal). Samples were extensively dialyzed against PBS, 1 mM DTT. Heats of dilution were measured in blank titrations by injecting the protein into the buffer used in the particular experiment and subtracted from the binding heats. Thermodynamic parameters were determined by non-linear least squares methods using routines included in the Origin software package (MicroCal). Due to the large binding enthalpy of the interaction, a protein concentration in the calorimeter of 2 µM was sufficient to obtain a good signal-to-noise ratio and to reduce "c-values" (c = KB [P] N; [P] = protein concentration; N = stoichiometry) below 500. Measured thermodynamic values are usually a function of solvent conditions. To indicate this, observed binding enthalpy and entropy changes are abbreviated as
Hobs and
Sobs, respectively throughout this publication. The program RIBBON has been used to generate the Figs. 2, 3, 4 (17).
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RESULTS
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In this study we mutated all Tcf4-CBD residues that make significant contacts with
-catenin to alanine and studied the binding of these mutants using isothermal titration calorimetry. The results were analyzed on the basis of the published structures of Tcf-
-catenin complexes. The studied protein domain of Tcf4 contained residues 156, which have been shown to be sufficient for high affinity binding to
-catenin (11, 16). To determine the protein concentration spectroscopically and facilitate purification, we introduced a tryptophane residue and a His6 tag at the C terminus of Tcf-CBD. These additional C-terminal residues did not alter binding affinities for
-catenin (Table I). In this study we noted that the slightly different solvent conditions used (PBS, 1 mM DTT) result in somewhat smaller binding enthalpies than reported in an earlier study in which a buffer of 50 mM sodium phosphate, pH 7.4, 100 mM NaCl, and 2 mM DTT was used. A representative set of experimental data is shown in Fig. 1.
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TABLE I ITC measurements of the binding of Tcf4 mutants to -catenin/armadillo
The errors that are given in the table result from differences between independent experiments, and the data represent the average calculated from all experiments "exp#." All data have been measured in PBS buffer (Sigma), 1 mM DTT at 25 °C.
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Interaction of the Tcf4
-Hairpin and Its Flanking Residues (Residues 215)Most of the
-hairpin formed by the xTcf3 residues 215 has been found to be disordered in human Tcf4 structures. The region that was well defined started with Tcf4 residue Leu12 in the structure determined by Graham et al. (Protein Data Bank accession number 1jdh
[PDB]
, referred to henceforth as Tcf41jdh) (12) and residue Gly13 in the structure determined by Poy et al. (Protein Data Bank accession number 1jpw
[PDB]
, referred to henceforth as Tcf41jpw) (13). This finding is surprising, considering the high sequence similarity between xTcf3 and Tcf4 (Fig. 2). In xTcf3, the main side chain interactions are formed by Asp10, which forms a salt bridge with the
-catenin residue Arg474, and Glu11, which interacts with His578, Arg582, Arg612, and Tyr654 on
-catenin (Fig. 2). In the two Tcf4 structures reported, the location of the N terminus is quite different. Whereas in Tcf41jdh residues 1216 superimpose well with the corresponding residues in xTcf3, the N terminus in Tcf41jpw does not interact with
-catenin and is oriented toward the solvent. The first 12 N-terminal residues in Tcf4 have been shown to be dispensable in solid phase competition assays (enzyme-linked immunosorbent assay) as well as in reporter gene assays (16, 15). Indeed, deletion of the first 9 (ND9) or 12 (ND12) residues of Tcf4 had only a marginal effect on binding constants (Table I), whereas binding enthalpies were found to be reduced by about 5 kcal/mol. This suggests that the Tcf4 N terminus interacts with
-catenin but that it does not contribute to binding free energy change due to enthalpy-entropy compensation mechanisms. A similar effect was observed for the mutation D10A, which bound with a 
Hobs of +6.5 kcal/mol but with a binding constant similar to that of wild type Tcf4. Interestingly, the mutation D11A also reduced the KB of the interaction by about 5-fold. Thus, the mutation D11A has a larger effect on the binding of Tcf4 than deletions of the entire N terminus. It has been observed previously that effects of single mutations may be much stronger than the contribution of the entire site as a whole (18). Cooperative effects are usually invoked to explain such observations. However, the observed data might also be the result of local changes in protein structure, differences in the dynamics of both proteins, or changes in surface hydration. To confirm this result, we also mutated the residues on
-catenin that interact with Glu11 in xTcf3. The mutations R612A and Y654A did not significantly affect binding of wild type Tcf4 (Table II). Surprisingly, we observed an
30-fold decrease in binding constant for wild type Tcf4 for the
-catenin mutations H578A and R582A, suggesting that wild type Tcf4 interacts with this binding site on
-catenin. The Tcf4 residue Asp11 corresponds to a glutamic acid in xTcf3. Possibly, the elongation of the side chain by one CH2 group at this critical position renders this interaction more favorable. Due to the lack of structural information on the Tcf4 N terminus, we can only speculate about the molecular mechanisms that give rise to this binding behavior. However, the measured data demonstrated that under the chosen experimental conditions, the N-terminal residues of Tcf4 do interact in solution with
-catenin and that this interaction involves the
-catenin residues that are contacted by xTcf3.
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TABLE II ITC measurements of -catenin mutants (Tcf Asp11 binding site)
The errors that are given in the table result from differences between independent experiments, and the data represent the average calculated from all experiments "exp#." All data have been measured in PBS buffer (Sigma), 1 mM DTT at 25°C.
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Interaction of the Tcf4 Extended RegionThis region is structurally very conserved among the three reported Tcf structures and superimposes very well with residues 671686 of the
-catenin ligand E-cadherin as well as the Tcf antagonist ICAT (19, 20, 21). The Tcf extended region binds along a shallow groove with highly positively charged surface patches (charge buttons). Consequently, the electrostatic interactions observed in the
-catenin-Tcf complexes have been discussed as the main interaction sites that drive the binding of Tcf (12). Mutagenesis data on
-catenin showed that mutation of the residues Lys435, Asn426, His470, Arg469, and Lys508 to alanine strongly reduces binding of Lef1 and Tcf4 in co-immunoprecipitation experiments (15). These residues form polar contacts with Tcf4 residues 1619. Asp16 has been identified previously as a critical residue for Tcf binding (11, 16). The Tcf4 residue Asp16 forms hydrogen bonds with the
-catenin residues Lys435 and His470 as well as several water-mediated hydrogen bond networks (Fig. 2). We find in this study that the residue Asp16 defines the most important interaction hot spot on Tcf4. The mutation D16A resulted in a 
G of +2.4 kcal/mol. The neighboring residue Glu17 forms a salt bridge with Lys508. However, upon mutation of Glu17 to alanine, the binding constant decreased only
3-fold (11).
The interaction with the first charge button is followed in Tcf4 by the hydrophobic residues Leu18, Ile19, and Phe21, which interact with hydrophobic groups of the
-catenin residues Thr393, Cys429, Cys466, Ala467, Pro463, and Arg386 (Fig. 3). Single alanine mutants of Leu18, Ile19, and Phe21 did not significantly affect Tcf4 binding in isothermal titration calorimetry experiments. For the related transcription factor Lef1, an important contribution of residue Phe24 for
-catenin binding (Phe21 in Tcf4) has been discussed using a yeast two-hybrid system. This residue has also been shown to be important for the translocation of endogenous
-catenin into the nucleus (15). In addition, the Tcf4 double mutant I19A,F21A has been shown to reduce reporter gene activity by
40% in transient transfection experiments (13). In light of our binding data, it is likely that mechanisms other than
-catenin binding are responsible for the observed effect of these mutants in these cell-based experiments.
The cluster of hydrophobic Tcf4 residues is followed by negatively charged residues, which form the interaction with the second charge button defined by the
-catenin residue Lys312. The three-dimensional structure of this region differs significantly in the two Tcf4 crystal structures published. As in xTcf3, in Tcf41jpw the interaction with the second charge button is made by Glu24, whereas in Tcf41jdh, a similar interaction is formed by Glu28. In this structure, Glu24 makes no interaction with
-catenin but points to the solvent space. For the mutant E24A, we found a significant difference in binding enthalpy (
Hobs, +7.4 kcal/mol), which is, however, largely offset by favorable entropic contributions. This mutation resulted in a 
Gobs of only 0.9 kcal/mol and an
4-fold decreased binding constant.
The mutant E28A resulted in only moderate reduction of
Hobs and in similar binding constants with respect to the wild-type protein. In addition to Glu28, Glu29 also makes polar contacts to the second charge button in Tcf41jdh. The observed difference in
Hobs of 5.8 kcal/mol for mutant E29A supports such an interaction, even though binding constants are not altered. This highly acidic region compensates for single mutations as indicated by the structural plasticity shown by the two structures of Tcf4-
-catenin complexes. This hypothesis is supported by co-immunoprecipitation experiments, demonstrating that the mutation of all 4 glutamate residues in that region (Glu24, Glu26, Glu28, and Glu29) abolishes binding to
-catenin, whereas single mutations had either no effect or only a small effect on binding (14).
Mutation N34A only moderately changed
Hobs and had a negligible effect on the binding constants. This residue is disordered in Tcf41jpw and forms a hydrogen bond with Arg342 in Tcf41jdh.
Binding of the entire extended region of Tcf4 that includes residues 928 was probed using a synthetic peptide (Tcf928). The peptide bound with an affinity of 2.4 ± 0.3 x 106 mol-1,an enthalpy change of -9.0 ± 0.2 kcal/mol, and an entropy change close to 0 (Table I). These binding data demonstrate that a large portion of the binding free energy change (about 75%) is due to interactions formed by the extended region of Tcf4.
Interaction of the Tcf4 Helical RegionThe amphipathic
-helix described for all three Tcf structures packs against a hydrophobic surface area on
-catenin defined by the residues Phe253, Phe293, and Ile296 and the aliphatic portions of His219, Lys292, and Lys335 (Fig. 4). The Tcf4 residues Leu41, Val44, and Leu48 form the main hydrophobic contacts in this interface. In general, mutation of the hydrophobic residues significantly reduces binding affinities of Tcf4. We observed an
15-fold reduction for the mutations L41A and V44A as well as a 5-fold reduction for the mutant L48A (Fig. 5) (11).

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FIG. 5. Change in the Tcf4 binding constant (KB) upon mutation of the indicated residues to alanine. Previously published data for the mutations E17A and L48A have been included in the figure for reasons of completeness and comparison (11).
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The Tcf4 residues Asp40 and Lys45 form polar and electrostatic interactions along the edge of the
-catenin groove. Asp40 is positioned between Arg376 and Lys335 in
-catenin, and the Tcf4 residue Lys45 forms a hydrogen bond with the
-catenin residues His260 and Asn261. These interactions are also preserved in the xTcf3 structure. The mutation D40A resulted in an
3-fold reduction of the Tcf4 binding constant. The thermodynamic reasons for the reduced binding constant were mainly due to a reduction of favorable binding enthalpies. Mutation of the Tcf4 residue Lys45 to alanine resulted in slightly larger
Hobs and in similar binding constants (Fig. 6).
Heat Capacity Changes upon Binding of Tcf4 The temperature dependence of the binding enthalpy changes (
Hobs) for the Tcf4 CBD resulted in the determination of a heat capacity change upon binding (
Cp) of -1.45 kcal/(mol K), a value very close to the one that has been published using slightly different buffer conditions (Fig. 7) (11). In contrast to that very large heat capacity change, a linear fit of binding enthalpy data measured on Tcf4928 resulted in a
Cp of only -0.18 kcal/(mol K). Thus, part of the large negative heat capacity change observed upon binding of Tcf4-CBD to
-catenin is a consequence of the formation of the C-terminal helix observed in all Tcf-
-catenin crystal structures.
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DISCUSSION
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Binding data of a number of Tcf4 and
-catenin mutants have been reported in the literature using several diverse in vivo and in vitro assay techniques, which makes it difficult to compare the reported experimental data. We used titration calorimetry to generate a comparable set of Tcf4 mutant binding data that describes the effect of single alanine mutations for all Tcf4 residues in the
-catenin-Tcf interface. The single mutations show that Tcf4 binds in a bi-dentate mode: the largest effects of single alanine mutants were observed for residue Asp16 and for hydrophobic contacts of the C-terminal helix of the CBD (Fig. 5). The strength of a thermodynamic analysis of a binding event is that calorimetric data give a deeper insight into the nature of the interaction than binding constants alone. It has been observed that the altered interaction made by a mutant often does not result in a change in binding constant, even though large differences in binding energetics have been observed. In this study, most mutations of polar residues in the Tcf4-
-catenin interface resulted in a significant change in binding enthalpy, which was compensated for by more favorable entropic contributions to the binding. As a consequence, only minor changes in the free energy of binding and in the resulting binding constants were observed (Fig. 6). This phenomenon of enthalpy-entropy compensation is a general hallmark of the binding energetics of mutated protein-protein interfaces and indicates a large degree of cooperativity in these interactions (22). On a molecular level, enthalpy-entropy compensation can be explained by the large energetic cost of side chain dehydration and by the unfavorable entropic terms that result from the reduction of side chain mobility upon formation of a polar interaction. It has recently been shown in a detailed study (23) on the contribution of electrostatic interactions of single residues to the binding energy in protein-protein interfaces that electrostatics strongly opposed binding in two of the four cases studied. One case had a net effect close to zero, and another case provided a significant contribution to the binding free energy (23). Structural features that explained favorable electrostatic interactions in that study were more favorable desolvation terms due to already partially buried charged groups in the free monomer or the formation of partially exposed ion pairs in the complex as well as the formation of salt bridge networks. Because Tcf4 is unstructured in solution, a mechanism that invokes already buried charged groups is not likely. However, the fact that a large number of residues interact in an extended conformation with an interface that consists mainly of charged and polar interactions will favor the formation of networks and partially exposed ion pairs. Indeed, residue Asp16 forms multiple polar contacts in a network of direct and water-bridged hydrogen bonds.
Upon mutation of a polar side chain to alanine, one binding partner remains hydrated and is not restrained when the complex forms. The thermodynamic consequences of this are that binding enthalpies are reduced because polar interactions have been broken, but favorable entropic terms arise from the gained flexibility of the remaining binding partners. In general, mutations of most polar or charged residues in an interface of two proteins result in complex destabilization. This is the case even if the polar interaction itself is destabilizing. Consequently, mutagenesis data of charged residues reflect primarily the effect of the removal of a charge but do not indicate whether these polar interactions per se stabilize or destabilize protein-protein interactions (24).
An interesting aspect of the thermodynamic data generated in this study is that residues that are disordered in crystal structures may have a significant influence on binding thermodynamics. In particular, such contributions were seen for mutations of the N-terminal region that corresponds to a
-hairpin loop in Tcf3 and is disordered in both crystal structures of the Tcf4-
-catenin complex and in the region between the Tcf4 residues Glu24 and Glu29 that is disordered from residue 26 on in Tcf41jpw and forms an
-helix in Tcf41jdh. Cooperative contributions have been observed for polar residues that are separated by less than 7 Å. Consequently, at least for the Tcf N terminus, such relatively short-range cooperative effects can be ruled out. This indicates that the N terminus of Tcf4 physically interacts with
-catenin and in particular with the residues Arg612, Tyr654, His578, and Arg582 that were mutated in this study.
Binding of Tcf does not significantly alter the overall conformation of
-catenin compared with its unbound state (12, 13). Main chain root mean square deviation between
-catenin without ligand and the two published Tcf4-
-catenin complexes were only 0.7 Å, confirming the role of the armadillo repeat region as a rigid structural platform. However, binding to
-catenin induces structural changes in the Tcf ligand, which is mainly unstructured in solution (11). Thus, folding events in Tcf4 upon binding to the
-catenin surface additionally modulate the binding energetics of Tcf4
-catenin complex formation. This study showed that one effect of such folding events is the determined large negative heat capacity increment of -1.45 kcal/(mol K). This effect can be largely assigned to folding events because the extended region of Tcf4 alone (residues 928) bound with a heat capacity change of only -0.18 kcal/(mol K).
A large number of thermodynamic studies showed that binding energetics can be calculated on the basis of polar and apolar surface area changes upon complex formation (25, 26). However, surface area changes calculated on the basis of the xTcf3-
-catenin complex predicted a heat capacity increment of -0.067 kcal/(mol K) (11), and the new structural data on Tcf4-
-catenin complexes did not significantly change these calculated values. Large deviations of calculated values and experimental heat capacity changes have been described for many other systems, and possible mechanisms for such discrepancies have been discussed by us and by others (11, 27, 28, 29, 30). The comparison between experimental heat capacity increments of Tcf4-CBD and Tcf 928 suggests that folding events upon binding are largely responsible for the large negative heat capacity increment.
Implications for Drug DevelopmentBecause of the central role of the Tcf/Lef
-catenin interaction in Wnt signaling, the development of a low molecular weight antagonist is an interesting strategy for anti-cancer therapy. In contrast to many active sites in proteins that can be efficiently inhibited by small inhibitors, protein interaction sites usually span much larger surface areas. However, a number of studies have shown that these large interfaces often contain interaction hot spots that may be used as binding sites for much smaller antagonists (15, 31). In this study, we showed that, at least in vitro and on a single amino acid residue basis, interference with the contact site of the Tcf residue Asp16 would be the most promising strategy. A hydrophobic binding pocket on
-catenin in the vicinity of Asp16 that could harbor a small molecule has been described (15). However, such an inhibitor would also compete with the
-catenin ligand cadherin, which would lead to the retention of the cadherin in the endoplasmic reticulum and degradation of the protein (32). A recent structural study on the Tcf antagonist ICAT showed that even though ICAT, cadherin, and Tcf have partially overlapping binding sites, ICAT competed with Tcf binding but not with cadherin binding in vivo (19, 21). Possible explanations for this observation could be that cadherin binds much more strongly to
-catenin than Tcf and ICAT or that the binding is regulated in vivo by local concentration differences and/or by post-translational modifications. Recently, it has been reported that cadherin binding is modulated by phosphorylation, which increases its binding constant to
-catenin by 3 orders of magnitude (19, 21). A large body of structural information on
-catenin complexes has been reported recently (12, 13, 14, 19, 21). This knowledge will certainly aid the structure-based design of Tcf antagonists. However, due to the large binding constants of Tcf4, the observed structural plasticity, the high degree of cooperativity, and the electrostatic nature of the interaction, the development of efficient antagonists will be challenging.
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FOOTNOTES
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* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
¶ Supported by Karolinska Institutet Fonder. 
|| To whom correspondence should be addressed. Tel.: 39-02-4838-5072; Fax: 39-02-4838-3965; E-mail: stefan.knapp{at}pharmacia.com.
1 The abbreviations used are: Tcf, T-cell factor; xTcf, T-cell factor from Xenopus; Lef, lymphocyte enhancer binding factor; CBD, catenin-binding domain; PBS, phosphate-buffered saline; DTT, dithiothreitol; ICAT, inhibitor of
-catenin and Tcf. 
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ACKNOWLEDGMENTS
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We thank Jan Malyszko for the synthesis of oligonucleotides and DNA sequencing, Dr. M. Sigvardsson (Lund University) for the His6-
-catenin expression system, and Dr. A. Schnuchel (Pharmacia Corp.) for providing us with the Tcf4-W-His6 wild type expression system.
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