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J. Biol. Chem., Vol. 281, Issue 2, 1027-1038, January 13, 2006

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Thermodynamics of -Catenin-Ligand Interactions

THE ROLES OF THE N- AND C-TERMINAL TAILS IN MODULATING BINDING AFFINITY^*

From the Departments of Structural Biology and of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5126

Received for publication, October 18, 2005 , and in revised form, November 16, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
-Catenin is a structural component of adherens junctions, where it binds to the cytoplasmic domain of cadherin cell adhesion molecules. -Catenin is also a transcriptional coactivator in the Wnt signaling pathway, where it binds to Tcf/Lef family transcription factors. In the absence of a Wnt signal, nonjunctional -catenin is present in a multiprotein complex containing the proteins axin and adenomatous polyposis coli (APC), both of which bind directly to -catenin. The thermodynamics of -catenin binding to E-cadherin, Lef-1, APC, axin, and the transcriptional inhibitor ICAT have been determined by isothermal titration calorimetry. Most of the interactions showed large, unfavorable entropy changes, consistent with these ligands being natively unstructured in the absence of -catenin. Phosphorylation of serine residues present in a sequence motif common to cadherins and APC increased the affinity for -catenin 300-700-fold, and surface plasmon resonance measurements revealed that phosphorylation of E-cadherin both enhanced its on rate and decreased its off rate. The effects of the N- and C-terminal "tails" that flank the -catenin armadillo repeat domain on ligand binding have also been investigated using constructs lacking one or both tails. Contrary to earlier studies that employed less direct binding assays, the tails did not affect the affinity of -catenin for tight ligands such as E-cadherin, Lef-1, and phosphorylated APC. However, the -catenin C-terminal tail was found to decrease the affinity for the weaker ligands APC and axin, suggesting that this region may have a regulatory role in -catenin degradation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
The protein -catenin serves several roles in the development and maintenance of multicellular organisms. It is a structural component of cell-cell contacts and is also a transcriptional coactivator in the Wnt signaling pathway that controls cell fate determination. The multiple functions of this protein depend upon its interactions with several distinct protein ligands. In adherens junctions, -catenin interacts with the cytoplasmic domain of classical cadherins and with -catenin, which functionally connects the cadherin-catenin complex to the actin cytoskeleton. The levels of nonjunctional -catenin are determined by the presence or absence of Wnt growth factor stimulation. In the absence of a Wnt, cytosolic -catenin is targeted for degradation by a multiprotein complex containing axin and the adenomatous polyposis coli protein (APC).³ The axin-APC complex binds to -catenin and also recruits casein kinase 1 (CK1) and glycogen synthase kinase-3 (GSK-3). These kinases phosphorylate -catenin, flagging it for destruction by the ubiquitin-proteosome pathway. In the presence of a Wnt signal, phosphorylation of -catenin is inhibited. The resulting stabilized pool of -catenin translocates to the nucleus, where it binds to the Tcf/Lef family transcription factors. The -catenin-Tcf complex recruits general transcription factors, resulting in a complex that activates Wnt target genes.

The primary structure of -catenin consists of an N-terminal region of 150 amino acids, followed by a central armadillo (arm) repeat domain of 520 residues and a C-terminal "tail" of about 100 amino acids. Many -catenin ligands, including cadherins, Tcfs, APC, axin, and the transcriptional inhibitor ICAT, bind to the arm repeat domain. This region consists of 12 arm repeats, each of which is composed of three -helices, which pack together to form a highly elongated structure with a positively charged groove (1). Crystal structures of the -catenin arm repeat domain bound to the E-cadherin cytoplasmic domain (2), several Tcfs (3-5), APC (6-8), and ICAT (9, 10) have revealed that an extended peptide present in these ligands binds to the portion of the groove formed by repeats 5-9 (Fig. 1). In addition to this common binding feature, each ligand forms unique interactions with other portions of the -catenin arm domain. For example, sequences in E-cadherin N- and C-terminal to the extended groove-binding peptide interact with all of the other arm repeats, and ICAT has an N-terminal helical domain that interacts with arm repeats 11 and 12. Tcfs contain an amphipathic helix C-terminal to the groove-binding peptide that interacts with repeats 3-5. This region is also exploited when APC 20-mers or cadherin are phosphorylated; phosphorylation of serine residues in the sequence SLSSL shared by these proteins results in ordering of this region and formation of interactions similar to those made by the Tcf helix. In the case of APC, binding of the phosphorylated sequence results in additional contacts made by the 18 amino acids C-terminal to the SLSSL sequence (7, 8). Axin is unique among the known arm-domain binding ligands because it does not interact with the repeat 5-9 groove; instead, it binds as a 17-residue -helix to the groove formed by repeats 3 and 4 (11).

Several studies of the interaction between -catenin and Tcf-4, E-cadherin, and APC have identified key residues or "hot spots" in the ligands responsible for the majority of the binding energy. The extended peptide common to most -catenin ligands can be described by the consensus DXXX_2-7E, where is an aliphatic hydrophobic amino acid, and is an aromatic side chain (9). Mutation and binding studies with Tcf-4 peptides indicate that the consensus Asp and the aromatic side chain confer much of the binding energy in the groove (12, 13). Tcf-4 residues Leu⁴¹ and Leu⁴⁸, which are present on one side of an amphipathic helix that binds to -catenin arm repeats 3-5, are also important determinants of binding energy in this case (Fig. 1) (12, 13). The sequence SLSSL, which is present in both cadherins and the -catenin-binding sequences in APC consisting of homologous 20 amino acid sequences, binds when these sequences are phosphorylated; the two Leu residues make the same packing interactions as Leu⁴¹ and Leu⁴⁸ of the Tcf helix (2, 7, 8).

View larger version (21K): [in this window] [in a new window]   FIGURE 1. Overall structures of the -catenin arm domain and bound ligands. The helices of the arm domain of -catenin are drawn as cylinders and colored gray, except for H3 helices that are shown in blue. Each arm repeat is numbered from 1 to 12 as shown below each repeat. All -catenin-ligand complex structures were superimposed using the -catenin coordinates, and the relative position of each ligand is shown separately. The N and C termini of each protein are labeled as N and C, respectively. Dashed lines indicate missing residues in crystal structures. The helices of the ligands are shown as cylinders. The helical domain of ICAT is colored in yellow. The region that becomes ordered upon phosphorylation of Ecyto and APC-R3, is colored in red, and the other regions are shown in green.

In contrast to the wealth of structural knowledge of the -catenin arm domain and its interactions with ligands, little is known about the N- and C-terminal regions. The N- and C-tails of -catenin appear to have important roles in preventing -catenin from binding to desmosomal cadherins, which share significant similarity with classical cadherins found in adherens junctions (23, 24). Several reports suggest that these tails modulate the E-cadherin--catenin interaction by binding directly to the arm domain, thereby competing with the ligand (25, 26). The C-tail appears to interact directly with the arm domain, an interaction that has been mapped to the last 22 amino acids of the protein (26). The N-tail also appears to interact with the arm domain, but only in the presence of the C-tail (26). Although E-cadherin and Tcfs have overlapping interactions with the arm domain, Tcf binding was found to be independent of the C-tail (25, 27).

Although crystal structures have defined the atomic basis of ligand interactions with -catenin, they do not provide any information about thermodynamics. Understanding the strengths of these interactions is important for dissecting the mechanism of adhesion (28, 29), the APC-axin destruction complex (8), and how -catenin can displace corepressors from Tcfs as part of transcriptional activation (30). Moreover, dysregulation of -catenin destruction and consequent inappropriate transcriptional activation by -catenin underlie a number of cancers (31), so there is intense interest in developing inhibitors that can selectively block its interactions with Tcfs while permitting its interactions with components of the destruction complex and cell adhesion molecules (32). However, the interactions of -catenin with many of its ligands have been assessed by qualitative or only semi-quantitative means.

Here we present a systematic, quantitative analysis of -catenin binding to E-cadherin, Lef-1, APC-R3, axin, and ICAT determined by isothermal titration calorimetry (ITC). We also used this method, as well as surface plasmon resonance spectroscopy, to assess the effect of phosphorylation on the cadherin and APC interactions and the effects of the -catenin N- and C-terminal tails on ligand interactions. The thermodynamics correlate well with the structural data. It is found that the -catenin tails influence the binding of some ligands, but the data rule out a direct intramolecular interaction between the tails and the -catenin arm repeat domain.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Construct Design and Overexpression in Escherichia coli—Four different murine -catenin constructs, encoding the full-length protein (-cat-full:1-781), the N-terminal + arm domain (-cat-arm-Nt:1-671), the arm + C-terminal domain (-cat-arm-Ct:134-781), and the arm domain alone (-cat-arm:134-671) were used in these studies (Fig. 2). All were expressed as glutathione S-transferase (GST) fusion proteins. The -cat-arm construct contains a thrombin cleavage site after the GST (1); the other constructs have an N-terminal GST followed by a tobacco etch virus (TEV) protease recognition site using a modified pGEX-KG vector, pGEX-TEV. DNA fragments encoding -cat-arm-Nt and -cat-arm-Ct were obtained by PCR and inserted into pGEX-TEV. The full cytoplasmic domain of murine E-cadherin (E_cyto:577-728) (14) was used for ITC experiments and pulldown assays. Full-length ICAT and the helical domain of ICAT (residues 1-61; ICAT-h) were used for ITC experiments and were expressed as a GST-fused form and an N-terminal His₆-tagged form, respectively, as described (9). Two different murine Lef-1 constructs, Lef-1-(1-65) and Lef-1-(1-131), encoding residues from 1 to 65 and from 1 to 131, respectively, were expressed as N-terminal His₆-tagged forms as described (30). Phosphorylated and nonphosphorylated 20-amino acid repeat 3 of APC (APC-R3:1484-1528) and the -catenin binding domain of human axin fragment (residues 436-498) were used for ITC experiments and were expressed as GST fusion proteins as described previously (8). Each protein was overexpressed in E. coli BL21 cells. Cells were grown at 37 °C until the A₆₀₀ of cells reached 0.6-0.8 and were then induced with 0.5 mM isopropyl thiogalactoside. Cells were harvested by centrifugation after additional growth of cells for 3-4 h at 30 °C, and the cell paste was stored at -80 °C.

Vector Construction for Expression of Biotinylated E_cyto—A new vector, pAN-N1, that adds a biotin ligase consensus site to the N terminus of a protein was constructed from the vector pAN-1 (Avidity, LLC). A consensus cleavage site for the TEV protease was placed between the biotin ligase consensus and the new multiple cloning site (MCS) so that the affinity tag could be removed. Part of the biotinylation consensus, the TEV site, and the MCS was generated by overlap-extension PCR using standard methods and the following three oligonucleotides: 5'-AAATTTCGAAGCGCAGAAAATTGAATGGCATGAAAACCTGTATTTCCAAG-3'; 5'-CATGAAAACCTGTATTTCCAAGGATCCGGAGCTCGAGGCCTGCAGGGC-3'; and 5'-TATAAAGCTTCTAGAATTCCGCGGTACCCGGGCCCTGCAGGCCTCGAGC-3'. The PCR product and the pAN-1 vector were digested with BstBI and HindIII, and the biotinylation/TEV/MCS insert was ligated into pAN-1, generating pAN-N1. The PCR insert, in conjunction with a portion of the biotin consensus tag from pAN-1, encoded the following N-terminal fusion sequence: MSGLNDIFEAQKIEWHENLYFQGSGARGLQGPGTAEF. The biotin ligase consensus site consists of residues 3-17 (33, 34). Biotin-protein ligase covalently couples biotin to residue Lys¹². Residue Glu¹⁷ functions as the C-terminal residue of the biotin ligase consensus and the N-terminal glutamate of the TEV protease consensus site (ENLYFQG) (35, 36). Cloning into the BamHI site leaves a single serine between the C terminus of the TEV consensus site and the sequence of interest.

pAN-N1 and a construct described previously containing the mouse E-cadherin cytoplasmic tail (E_cyto) (37) were digested with BamHI and EcoRI. The digest product, encoding residues 580-728 of mature E-cadherin, was ligated into pAN-N1 to yield pAN-E_cyto. E. coli strain BL21 containing an IPTG-inducible biotin-ligase gene (birA) in vector pACYC-184 (Avidity, LLC) was transformed with the IPTG-inducible pAN-E_cyto. This system did not reliably produce sufficient quantities of biotinylated E_cyto, possibly because induced biotin ligase expression competed with E_cyto fusion protein synthesis. In an attempt to improve the yield of biotinylated E_cyto fusion protein, the biotinylation/TEV/MCS region of pAN-N1 was moved to vector pET28a (Novagen), which utilizes a strong bacteriophage T7 expression system. The pAN-N1 biotinylation/TEV/MCS region was PCR-amplified using the following oligonucleotides: A, 5'-ATACCATGGCCGGCCTGAACGACATCTTC-3', and B, 5'-ATAAAGCTTCTAGAATTCCGCGGTA-3'. Oligonucleotide A added a 5'-NcoI restriction site and changed the N terminus of the pAN-N1 fusion peptide from Met-Ser to Met-Ala. Amplified insert and pET28a were digested with NcoI and HindIII and the appropriate products ligated to form pET28ahh. pET28ahh was digested with BamHI and EcoRI and ligated with the same BamHI/EcoRI fragment of E-cadherin originally ligated into pAN-N1 (above) to form pET28ahh-E_cyto.

Purification of the Recombinant Proteins—For ITC experiments, -cat-full, -cat-arm, -cat-arm-Nt, -cat-arm-Ct, E_cyto, ICAT, axin, and APC-R3 were purified from their GST fusion forms, and Lef-1-(1-65), Lef-1-(1-131), and ICAT-h were purified from the N-terminal His₆-tagged forms. Protease inhibitor mixture (Complete^TM; Roche Applied Science) was added into thawed cell paste, and the cells were lysed by French press. After centrifugation of lysed cells at 40,000 rpm for 30 min, cleared lysates were removed and incubated with glutathione-agarose beads for GST fusion proteins or Ni²⁺-NTA-agarose beads for His₆-tagged protein for 1 h at 4°C. For GST fusion proteins, glutathione-agarose beads were washed first with phosphate-buffered saline containing 1 M NaCl, 0.005% Tween 20, and 5 mM dithiothreitol (DTT) and then with cleavage buffer consisting of 25 mM Tris-Cl, pH 8.5, 100 mM NaCl, and 2 mM DTT. Cleavage of the GST tag was performed by the addition of TEV protease (-cat-full, -cat-arm-Nt, -cat-arm-Ct, APC-R3, and axin) or thrombin (-cat-arm, E_cyto, and ICAT), and the cleavage reaction was incubated at 4 °C for overnight (TEV protease) or for 2 h (thrombin). The cleaved protein was collected from flowthrough and loaded onto a Mono Q column (Amersham Biosciences) equilibrated with buffer E (25 mM ethanolamine, pH 9.5, 2 mM DTT, and 0.5 mM EDTA) containing 50 mM NaCl for -cat-arm and buffer A (25 mM Tris-Cl, pH 8.5, 2 mM DTT, and 0.5 mM EDTA) containing 50 mM NaCl for others and was eluted with a linear gradient of NaCl. Mono Q-purified protein was loaded onto a Superdex S200 gel filtration column (Amersham Biosciences) equilibrated with buffer T1 (25 mM Tris-Cl, pH 8.0, 2 mM DTT, and 0.15 M KCl), buffer T2 (25 mM Tris-Cl, pH 8.8, 2 mM DTT, and 0.1 M NaCl), or buffer H (25 mM HEPES-NaOH, pH 7.5, 75 mM NaCl and 0.5 mM EDTA) for ITC experiments.

For purification of Lef-1-(1-65), Lef-1-(1-131), and ICAT-h with the N-terminal His₆ tag, Ni²⁺-NTA-agarose beads were washed with 10 mM imidazole, after 1 h of incubation. Each His₆-tagged protein was eluted using 300 mM imidazole, and its His₆ tag was removed by TEV protease. After incubation of the cleavage reaction mixture at 4 °C overnight, imidazole was dialyzed out to reload the cleavage reaction onto Ni²⁺-NTA-agarose beads. Cleaved protein was collected from flowthrough and was further purified using gel filtration chromatography, equilibrated with buffer T2 or H. Each purified protein was spin-concentrated, and the final concentrations were determined by UV absorption at 280 nm.

Phosphorylated E_cyto was prepared as described using purified E_cyto and commercially available casein kinase II (New England Biolabs) (2), and the protein was repurified by Mono Q and gel filtration chromatography. APC-R3 and phosphoAPC-R3 were obtained as described previously (8).

Purification and Phosphorylation of Biotinylated E_cyto—The biotin-E_cyto used for E_cyto--catenin BIAcore experiments was purified from a culture of E. coli BL21 containing vector pAN-E_cyto (above) and the birA gene in vector pACYC-184 (Avidity, LLC). Cells were grown in Superbroth containing 150 µg/ml ampicillin and 20 µg/ml chloramphenicol, with biotin added to 50 µM at mid-log growth phase. Protein expression from both vectors was induced with 200 µM IPTG during late log phase, and the cells were harvested after an additional 3 h of growth. Cell pellets were resuspended in minimal volumes of resuspension buffer (phosphate-buffered saline with 1 M NaCl, 0.05% (v/v) Tween 20, and 0.1% (v/v) -mercaptoethanol) and frozen. Phenylmethylsulfonyl fluoride, aprotinin, and pepstatin A (Sigma) were added to thawed cell paste to concentrations of 2 mM and 4 and 2 µg/ml, respectively. Approximately 200 Kunitz units of DNase I (Sigma) were added per ml of cell paste, and the cells were lysed with a French press (SLM-Aminco). Protease inhibitors were added again, including EDTA, which had a final concentration of 2 mM. Lysate was clarified by centrifugation, and the supernatant was incubated for 45 min at 4 °C with UltraLink Immobilized Monomeric Avidin matrix (Pierce) prepared as suggested by the manufacturer. A small column was poured with the matrix/lysate slurry and washed with 10 column volumes of wash buffer (50 mM Tris-HCl, pH 8.0, 75 mM NaCl). Biotinylated E_cyto was eluted with elution buffer (50 mM Tris-HCl, pH 8, 75 mM NaCl, 1.0 mM EDTA, and 2 mM D-biotin) and further purified by size exclusion chromatography utilizing a Superdex 200 Prep-grade 26/60 column (Amersham Biosciences) equilibrated with Superdex buffer (25 mM Tris-HCl, pH 8.5, 150 mM NaCl). E_cyto peak fractions from the Superdex column were pooled, diluted 2-fold with Mono Q buffer A (20 mM Tris-HCl, pH 8.5), and bound to an anion exchange column (Mono Q HR 16/10, Amersham Biosciences). Biotinylated E_cyto was eluted with a gradient from 10 to 30% Mono Q buffer B (20 mM Tris-HCl, pH 8.5, 2 M NaCl).

Phosphorylated biotin-E_cyto (biotin-pE_cyto) was generated using biotin-E_cyto purified from a culture of E. coli BL21 containing vector pET28ahh-E_cyto (above) and the birA gene in vector pACYC-184 (Avidity, LLC). Cells were grown in Luria broth containing 25 µg/ml kanamycin and 15 µg/ml chloramphenicol. Biotin and IPTG were added simultaneously at mid-log phase to concentrations of 50 and 600 µM, respectively, and the culture was harvested 2 h after induction. Cell pellets were resuspended in resuspension buffer (above) and lysed with a French press, and the lysate was clarified by centrifugation. Phenylmethylsulfonyl fluoride, EDTA, aprotinin, and pepstatin A (Sigma) were added to the lysate supernatant to concentrations of 2 and 2 mM and 4 and 2 µg/ml, respectively. Ammonium sulfate was added to 15% of saturation at 4 °C, the solution gently agitated for 1 h at 4 °C, and any precipitate pelleted by centrifugation. Precipitated protein was resuspended in buffered urea (50 mM Tris-HCl, pH 8.5, 150 mM NaCl, 6 M urea) and purified without urea by gel filtration on a Superdex 200 Prep-grade 26/60 column equilibrated with 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM DTT. Additional purification utilized a Mono Q anion exchange column equilibrated with Mono Q buffer A (above). Biotin-E_cyto was eluted as a single well defined peak during a 10-30% gradient of Mono Q buffer B (above). Pulldown experiments with avidin beads demonstrated that a significant fraction of the resulting E_cyto was biotinylated (data not shown).

Biotin-E_cyto was phosphorylated in a 2-ml reaction comprising 0.8 mg/ml biotin-E_cyto, 1x casein kinase II reaction buffer (New England Biolabs), 80 mM Tris-HCl, pH 7.5, 9 mM pH-adjusted ATP, pH 7.3, 10 mM MgCl₂, and 25,000 units of casein kinase II (New England Biolabs). After a 6-h incubation at 30 °C, the reaction was applied to an anion exchange column (Mono Q HR 16/10, Amersham Biosciences) equilibrated with buffer A (50 mM imidazole, pH 7.2, 4 M urea) and eluted with a gradient from 10 to 20% buffer B (50 mM imidazole, pH 7.2, 4 M urea, 2 M NaCl). Mono Q fractions containing the largest single peak of highly phosphorylated pE_cyto were pooled and concentrated. This peak had very modest shoulders and consisted of biotin-E_cyto molecules with 3-5 added phosphate groups, as assessed by mass spectroscopy. The final buffer for the biotin-pE_cyto stock was 25 mM Tris-HCl, pH 8.0, 150 mM NaCl.

Isothermal Titration Calorimetry—ITC measurements were performed at 30 °C using a VP-ITC calorimeter (Microcal, Inc.). All purified ligands and -catenin were concentrated to 200-350 and 8-25 µM, respectively, in H, T1, or T2 buffer. Each titration experiment, except for phosphoE_cyto, was initiated by a 3- or 4-µl injection, followed by 25-40 7- or 8-µl injections. Blank titrations, which were carried out by injecting ligand into H, T1, or T2 buffer depending on the particular experiment, were subtracted from each data set. The association constant K_A, enthalpy change H, and the stoichiometry n were obtained from fitting the data (38) using the Origin software package (Microcal, Inc.). The dissociation constant K_D, the free energy change G, and the entropy change S were obtained from the basic thermodynamic relationships K_D = K_A^-1, G = -RTlnK_A, and G = H - TS. For the titration experiment with the very tight ligand phosphoE_cyto, the displacement method was performed using nonphosphorylated E_cyto as a competitor (39). In this method, a less strongly bound ligand is used as a competitive inhibitor, and its binding parameters, determined by a separate conventional ITC experiment, are used for calculation of thermodynamic parameters of more strongly bound ligand. Before injecting phosphoE_cyto, -catenin in the ITC cell was fully saturated with E_cyto. Then 8 µl of 300 µM phosphoE_cyto was injected 20 times after a first 4-µl injection. The raw ITC data showed a relatively small signal because the measured change in heat was because of the contributions of the heat of binding of the strong ligand phosphoE_cyto and the heat of dissociation of E_cyto from the complex with -catenin, which is of opposite sign. Data analysis were performed by nonlinear regression fitting to the displacement model, which was kindly provided by Dr. B. Sigurskjold and installed in Origin version 5.0. The ITC data for the APC-R3 and phosphorylated APC-R3 interactions have been published previously (8) but are included here for comparison with the other ligands.

Surface Plasmon Resonance Data Collection—Data were measured on a Biacore 3000 surface plasmon resonance (SPR) detection system (Biacore AB). Biotin-E_cyto (BE_cyto) and phosphorylated biotin-E_cyto (PBE_cyto) molecules were captured on streptavidin-coated sensor chips (Sensor Chip SA, Biacore AB). A single covalent biotin at a defined N-terminal location in the E_cyto proteins produces an oriented capture system, with the E_cyto domains presented in the orientation one would expect to find in vivo. In each chip, the first of the four flow cells (FC1) did not have any bound E_cyto and was used as a control. Flow cells 2-4 (FC2-FC4) had increasing amounts of captured BE_cyto or PBE_cyto. Single BE_cyto and PBE_cyto chips were used for all of the experiments. The results presented here are derived from FC2, because this had the lowest coupling levels and therefore the lowest levels of mass transport, steric hindrance, and aggregation effects. All experiments were carried out at a flow rate of 100 ml/min to minimize mass transport effects. Each SPR sensorgram consisted of a "blank" buffer injection followed by a 140-s injection of full-length -catenin with a 370-s dissociation period. The E_cyto chip surface had to be regenerated between sensorgrams with two 12-s injections of 10 mM HCl because the SPR signal was not returning to base line in a timely manner.

Each SPR binding experiment tested seven -catenin concentrations, with three replicates per concentration. Binding to BE_cyto utilized -catenin concentrations of 400, 200, 100, 50, 25, 12.5, and 6.25 nM. Binding to phosphoBE_cyto utilized a different set of serial dilutions that included -catenin concentrations of 12.5, 6.25, 3.12, 1.6, 0.8, 0.4, and 0.2 nM. The replicates were measured in a partially randomized order with the same "randomized" order used for each experiment. SPR experiments (7 concentrations and 21 replicates) were carried out using a wide range of KCl concentrations for both BE_cyto and phosphoBE_cyto. Each binding buffer consisted of 25 mM Tris-HCl, pH 8.0, 2 mM dithiothreitol, 0.005% P20 surfactant and KCl at 150, 300, 600, or 900 mM. The Biacore 3000 was cleaned between experiments using the manufacturer's desorbance protocol with the E_cyto-streptavidin chip undocked. Each experiment was preceded by a surface performance test that utilized six cycles of -catenin injection and HCl regeneration steps. The surface performance test provided evidence that a stable base line had been reached after the desorbance procedure.

The BIAevaluation 3.0 software package (Biacore AB) was used for data processing and fitting. The FC1 reference sensorgrams were subtracted from the appropriate data sensorgrams. The blank buffer injection portion of each sensorgram was then subtracted from the subsequent -catenin injection (data) portion of the sensorgram. Sensorgrams were then aligned along the x (time) and y (response unit) axes. It became clear that each injection/regeneration cycle produced a slight reduction in the binding capacity of the chip. This reduction was significant given the large number of sensorgrams collected during each experiment. An average loss of signal per cycle was determined for each flow cell within an experiment, and a correction was applied to each sensorgram. The correction was generally on the order of 0.7%/cycle. Sensorgrams with obvious pathologies were discarded, and the fully adjusted replicate curves for each -catenin concentration were then averaged. The seven averaged curves from each experiment were then simultaneously and globally fit.

View larger version (13K): [in this window] [in a new window]   FIGURE 2. Schematic drawings of the constructs used in ITC experiments. The N-terminal and the C-terminal domains and the arm domain of -catenin were represented as Nt, Ct, and arm, respectively. The numbers shown on top of each construct represent the beginning and ending residue numbers.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

View larger version (32K): [in this window] [in a new window]   FIGURE 3. Experimental calorimetric data for ligand binding to full-length-catenin. All isothermal calorimetric experiments were performed at 30 °C. All ligands, except for Ecyto and phosphoEcyto, were injected into -cat-full in H buffer; for Ecyto and phosphoEcyto, T1 buffer was used. In each figure, the top panel shows the base-line-subtracted heat signal obtained by a series of injections of each ligand into the ITC cell containing -cat-full; the blank injection of ligand into the ITC cell containing only buffer is also shown and has been vertically offset for clarity. The bottom panels show the binding curves calculated using the best fit parameters obtained by a nonlinear least squares fit. For experiments performed in replicate, a representative example is shown.

View this table: [in this window] [in a new window]   TABLE 1 ITC measurements of ligand binding to full-length -catenin All experiments were performed at 30 °C. Titration experiments of Ecyto and phosphoEcyto were carried out in T1 buffer, which is the same used in the BIAcore experiments, in order to compare their results. The other ligands were titrated using H buffer.

View this table: [in this window] [in a new window]   TABLE 2 Buried surface area and number of contacts The total number of residues and the number of residues at the interface represent the number of amino acids of each ligand used for crystallization and the number of residues that are involved in the interaction with -catenin, respectively. The buried area and number of contacts were calculated in various -catenin complex structures using CNS (42). The maximum distance between two atoms is 3.4 Å for polar contacts and 4.0 Å for apolar contacts.

Most of the reactions studied, apart from APC-R3 and axin, display unfavorable negative entropy and favorable negative enthalpy changes. The unfavorable entropies of most of the interactions correlate with the ligand being unstructured in the absence of -catenin; presumably there is a large configurational entropy penalty associated with forming the ordered, bound structure. The magnitude of the unfavorable entropy change correlates well with the number of ligand residues that are immobilized in the interface (Table 2; Fig. 4B). Most interestingly, the ICAT helical domain has a small unfavorable binding entropy, despite the fact that it, unlike the other ligands, is structured in isolation (9). The reactions with APC-R3 and axin have favorable positive entropy changes, but their affinities for -catenin are relatively weak, with K_D values of 3.1 and 1.3 µM, respectively. The positive S values for these ligands may reflect the relatively small number of ordered residues in these ligands, such that the loss in configurational entropy upon binding does not dominate the favorable entropy of the hydrophobic effect, displacement of bound water from solvated polar groups, and other factors.

Comparison with Previous Studies—Thermodynamic analyses of -catenin binding to Tcf-4 (12, 15), APC 20-mers (40), and a number of peptides from -catenin ligands (13) have been reported. Each of these studies used buffer conditions and/or temperatures somewhat different from those used here and from one another, but in general the agreement of these studies is good. (The effect of buffers, in particular pH, on the strength of the interactions is discussed below.) The conditions of the present study and those used by Knapp and co-workers (12, 15) for Tcf-4 are similar, and their results, as well those of Gail et al. (13), indicate that Tcf-4 binds with 3- or 4-fold higher affinity than Lef-1. There are no structural data for the Lef-1 interaction, and given the similarity of the Lef-1 and Tcf-4 sequences, the basis of their affinity difference is unclear.

Thermodynamics of E-cadherin Binding—The E-cadherin cytoplasmic domain interacts with the entire -catenin arm repeat domain (2) (Fig. 1). The 151-amino acid domain is unstructured in isolation (14), but most of the last 100 residues become structured upon binding to -catenin (2). The interaction involves five distinct regions of the cadherin primary structure. Region 2, residues 639-666, contains a helix that binds to arm repeats 11 and 12. This is followed by region 3 (residues 667-684), which is the conserved peptide that binds to arm repeats 5-9. Region 4, residues 685-694, binds to -catenin only when Ser⁶⁸⁶ and Ser⁶⁹² are phosphorylated. Cadherin region 5 (residues 698-723) consists of two helices that bind to the first two -catenin arm repeats.

View larger version (13K): [in this window] [in a new window]   FIGURE 4. Relationships between thermodynamic and structural parameters. The buried areas and the numbers of residues in the interface were calculated using -catenin-ligand complex crystal structures, as shown in Table 2. Each data point, except for ICAT-h and xTcf-3, is shown as a square and used to calculate the straight regression line. The values for ICAT-h and xTcf-3 are shown as triangles and were not included in the calculation of the regression line. A, G values versus the surface area buried upon the formation of the complex. The calculated regression has R2 = 0.84. B, TS values versus the numbers of residues in the interface. The calculated regression has R2 = 0.80.

Phosphorylation of E_cyto results in larger favorable enthalpy and unfavorable entropy changes, but the enthalpy change dominates to give an affinity increase of almost 3 orders of magnitude (Table 1). This interaction was too strong to measure in a direct titration experiment but could be obtained by the displacement method in which -catenin was saturated with nonphosphorylated E-cadherin, whose binding constant had been determined (see "Experimental Procedures") (39). The dominance of the more favorable enthalpy change (H = -14 kcal mol^-1) over the more modest entropy change ((TS) = -10 kcal mol^-1) can be rationalized by the crystal structures of the nonphosphorylated and phosphorylated cadherins (2). Phosphorylation leads to the specific binding of E-cadherin residues 685-694 that are otherwise disordered, and these residues form a number of polar and nonpolar contacts. Several of these polar interactions are buried, which would be expected to contribute favorably to the binding enthalpy. Although an additional 10 residues become ordered upon phosphorylation, the sequences on either side of the phosphorylated region adopt a single, bound structure regardless of the phosphorylation state of E-cadherin. Thus, the phosphorylated region is already constrained by interactions on either side, so the structuring upon binding of this region probably involves relatively little extra loss of conformational entropy.

The effect of phosphorylation on the interaction of -catenin was further investigated by surface plasmon resonance spectroscopy. The E-cadherin cytoplasmic domain was expressed with an N-terminal recognition sequence for the E. coli BirA gene product, such that the construct could be enzymatically biotinylated at a single site outside of the cadherin sequence. The biotinylated protein was immobilized on a streptavidin surface on a BIAcore chip. Full-length -catenin flowed over the chip, and the change in binding as a function of time was measured. Fitting the data with simple 1:1 binding models (Table 3) gave K_D values of 18 nM and 81 pM for nonphosphorylated and phosphorylated cadherin, respectively, which are very close to the values obtained by ITC (Fig. 5, A and B). The good agreement in the K_D values indicates that although the 1:1 models do not give particularly good fits to the data (probably reflecting more complicated kinetic processes associated with the structuring of cadherin upon binding to -catenin), they are nonetheless reasonable approximations. The fitted rate constants show that phosphorylation increases the on rate 13-fold and decreases the off rate 18-fold (Table 3).

View this table: [in this window] [in a new window]   TABLE 3 Fitting of surface plasmon resonance data for Ecyto and phosphoEcyto binding to -catenin The heterogeneous ligand model assumes two distinct Ecyto species on the chip (designated with subscripts 1 and 2), each of which binds to the ligand with distinct rate constants. For the Langmuir 1:1 binding model, only a single species is fit. NA indicates not applicable.

View larger version (22K): [in this window] [in a new window]   FIGURE 5. BIAcore experiments of unphosphorylated and phosphorylated Ecyto. Sensorgrams of full-length -catenin binding to biotinylated Ecyto immobilized on streptavidin chips. Background subtracted data are shown in black, and the fit curves shown in red. A, biotinylated Ecyto data fit with a simple 1:1 binding model. B, biotinylated phosphoEcyto data fit with a simple 1:1 binding model. C, biotinylated Ecyto data fit with a model in which two distinct species of Ecyto are present on the chip.

Compared with the other -catenin ligands, the entropy penalty for Lef-1 binding is smaller than would be expected from the number of residues observed in the interface of XTcf-3 (3) and human Tcf-4 (4, 5) structures. This may be due to the fact that -catenin Lys³¹² can form alternative interactions with acidic residues at the C-terminal end of the conserved groove-binding sequence of the ligand, as shown in the two crystal structures of human Tcf-4 bound to -catenin (4, 5). This would be expected to contribute favorably to the binding entropy, and thereby partially offset the penalty of structuring the Tcf/Lef sequence.

ICAT Binding—ICAT was identified as an inhibitor of the interaction of -catenin with Tcf (41), and it binds competitively with Tcfs, cadherins, and the general transcription factor CBP/p300 (9, 10). ICAT binds to -catenin with a K_D of 3 nM, stronger than Lef-1 or E-cadherin and comparable with Tcf4. The helical domain of ICAT also binds to -catenin with high affinity (K_D = 12 nM), which is consistent with the notion that this region serves as a high affinity anchor that allows the extended C-terminal tail to compete effectively with other ligands bound in the groove of -catenin (9, 10). The ICAT helical domain is unique among -catenin ligands in that it is structured in the absence of -catenin, which implies that it has no loss of conformational entropy upon binding. This is reflected in the small, albeit slightly unfavorable, S value (Table 1) and likely explains its high affinity even though it buries a relatively small amount of surface area relative to other -catenin ligands (Table 2). The interaction with the ICAT helical domain occurs largely through formation of helix-helix packing interactions, in particular the hydrophobic interactions of ICAT residues Tyr¹⁵, Val²², Leu²⁶, and Met²⁹ with hydrophobic residues of -catenin repeat 12. The extended region of ICAT alone is not sufficient for binding to -catenin (9, 10), even though it buries 1700 Ų of surface upon binding and forms similar contacts with the arm repeat 5-9 groove seen as those present in the E_cyto, Tcf, and APC complexes. This appears to be a general feature of these -catenin ligands; and even though they share a highly conserved, critical set of interactions in the -catenin arm repeat 5-9 groove, high affinity depends upon flanking regions that form interactions unique to each ligand.

Axin and APC Binding—Only 17 of the 70 amino acids present in the -catenin binding domain of axin used in the crystallographic analysis were visible in -cat-arm/axin structure (11), and 14 of 47 residues are ordered in -cat-arm-APC-R3 complex structure (8). The small number of ordered residues associated with these interactions is consistent with a relatively small loss of translational, rotational, and conformational entropy associated with complex formation. This loss of entropy is overcome by entropically favorable hydrophobic interaction between -catenin and APC-R3 or axin, resulting in the overall positive entropy changes. In the -cat-arm-axin structure, 5 hydrophobic residues of axin, Ile⁴⁷², Leu⁴⁷³, Val⁴⁷⁷, Val⁴⁸⁰, and Met⁴⁸¹, form hydrophobic contact -catenin arm repeats 3 and 4. In the case of APC-R3, Leu¹⁴⁸⁸, Leu¹⁴⁸⁹, Phe¹⁴⁹¹, Ala¹⁴⁹², and Pro¹⁴⁹⁷ interact with -catenin arm repeats 5-8. In addition to hydrophobic interactions, there are hydrophilic and hydrogen bond interactions that contribute to the favorable enthalpy change. Asp⁴⁷⁴ from axin and Asp¹⁴⁸⁶ and Glu¹⁴⁹⁴ from APC-R3 form salt bridges with -catenin Lys²⁹², Lys⁴³⁵, and Lys³¹², respectively. However, the surface areas buried in the interface with -catenin are much smaller for axin and APC than those of the other ligands, which correlates with their relatively small binding enthalpies (Tables 1 and 2).

Phosphorylation of six serine residues of APC-R3 dramatically increases its affinity to a K_D of 10 nM, which corresponds to 300 times tighter binding than the nonphosphorylated form (7, 8). Phosphorylation results in the ordering of an additional 25 amino acids relative to the nonphosphorylated R3 structure, which likely accounts for the change to an unfavorable S. The unfavorable entropy change is compensated by a much larger, favorable enthalpy change because of formation of many new contacts with -catenin (Tables 1 and 2) (8).

Effects of the -Catenin N-terminal and C-terminal Tails—The C-terminal tail of -catenin has been reported to diminish the affinity of -catenin for E-cadherin by binding directly to the arm repeats and directly competing with E-cadherin (25, 26). These experiments were performed by incubating GST fusion proteins of -catenin constructs lacking one or both tails with E-cadherin or the purified tail, affinity-purifying the resulting complexes on glutathione-agarose beads, and detecting the bound proteins by Western blotting with an antibody to cadherin or the tail. By using similar methods, it was reported that the C-tail inhibits binding of cadherin but not Tcf-4 to -catenin (27). Direct binding to the tail by the arm repeats was also detected by blot overlay assay, in which -catenin is run on SDS-PAGE, transferred to nitrocellulose, incubated with the tail domain, and Western-blotted with antibody to the tail. The arm repeats were reported to bind to a peptide comprising the last 22 amino acids of -catenin covalently coupled to Sepharose beads.

Because pulldown and blot overlay assays do not give absolute affinities, ITC was used to compare the affinities of four different constructs of -catenin, with or without one or both tails (-cat-full, -cat-arm, -cat-arm-Nt, and -cat-arm-Ct), for E_cyto, Lef-1-(1-65), the ICAT helical domain (ICAT-h), APC-R3, and axin (Tables 4, 5, 6, 7, 8). The poor solubility and tendency of the -catenin arm domain to aggregate at pH 7.5 or 8.0 required that these comparative experiments be carried out at pH 8.8. Comparison of the affinities of full-length -catenin at these pH values with those at more neutral pH shows little or no effect on binding. The affinities of some of the ligands are independent of pH in this range, whereas others, in particular E_cyto and APC-R3, show slightly weaker affinities corresponding to very small energy differences at the higher pH. Therefore, comparison of different -catenin constructs binding to the same ligand at pH 8.8 provides a meaningful comparison set for assessing the effect of the -catenin tails.

View this table: [in this window] [in a new window]   TABLE 4 ITC measurements of Ecyto binding to -catenin ITC experiments were performed at pH 8.0 and pH 8.8. In cases in which two or more measurements were performed, the data shown represent the averaged values of all experiments. All experiments were performed at 30 °C.

View this table: [in this window] [in a new window]   TABLE 5 ITC measurement of Lef-1 (1–65) binding to -catenin All experiments were performed in T2 buffer at 30 °C. Except for the -cat-arm data, the numbers in this table represent the averaged value of two independent experiments.

View this table: [in this window] [in a new window]   TABLE 6 ITC measurement of the helical domain of ICAT binding to -catenin All measurements were performed in T2 buffer at 30 °C.

View this table: [in this window] [in a new window]   TABLE 7 ITC measurement of APC-R3 binding to -catenin All experiments with APC-R3 were performed in T2 buffer at 30 °C. Titration experiments of phosphoAPC-R3 into -cat-arm and -cat-full were carried out in T2 buffer and H buffer, respectively. In the case that duplicate experiments were performed, data were averaged.

View this table: [in this window] [in a new window]   TABLE 8 ITC measurement of axin binding to -catenin All measurements were performed in T2 buffer at 30 °C. The data of -cat-full are the averaged values of two independent experiments.

We also tested whether the C-tail can bind to the -catenin arm domain in trans, as reported earlier. Using Coomassie-stained gels in GST pulldown assays, we could not detect binding of the tail to the arm domain even when a 170-fold molar excess of the C-tail (1.4 mM) was added to 8 µM GST--cat-arm that had been immobilized on glutathione-agarose (data not shown). To more closely replicate earlier experiments, these pulldown experiments were also analyzed by Western blotting using a monoclonal antibody to the C-tail of -catenin. Although C-tail was detected, the intensity of each bound band was similar to that of the background binding control of the C-tail to glutathione-agarose beads (data not shown). Castaño et al. (26) suggested that the C-terminal 22 amino acids of -catenin specifically bind to the arm domain with similar interactions with those observed in E_cyto--cat-arm structure, based on the sequence alignments of C-tail (760-781) and E_cyto (655-677). This proposal aligns key hydrophobic and acidic residues present on the face of a helix in E-cadherin (region 2; see Ref. 2), and the arm repeats bound to this peptide immobilized on agarose. However, this alignment places the C-tail sequence Pro-Pro-Gly sequence in the middle of the -helix in the E_cyto structure, which is extremely unlikely. We speculate that the interaction observed is because of the basic nature of the arm repeats, which might bind to the acidic peptide; the negative control with unmodified agarose beads might not show this interaction.

E_cyto, Lef-1, and APC-R3 form virtually identical interactions with the arm repeat 5-9 groove, yet only APC-R3 appears to be affected by the presence of the C-tail. These results indicate that the proposed mechanism in which the C-tail binds back to the arm domain and acts as a competitive inhibitor cannot be correct. If there is an equilibrium between an "open" form of -catenin in which the arm domain is accessible to a ligand and a "closed" form in which the tail binds in the repeat 5-9 groove, then the fraction of accessible open form will be the same for all ligands under a given experimental condition. In this case, the equilibrium constants for all of the ligands that interact with this site would have to be diminished by the same factor. However, E_cyto and Lef-1, both of which bind to the groove at arm repeats 5-9, show no dependence on the tails, whereas APC-R3, which also binds to this region, is affected. Indeed APC-R3 and axin bind to nonoverlapping regions of the arm domain and show effects of the C-tail, but none of the other ligands that shares one or both of these sites (E_cyto, Lef-1) shows this dependence. Thus, the tails must affect binding either through interaction with the ligand, or they exert some other allosteric effect on the arm domain. The data also rule out the suggestion that the tail binds to repeats 11 and 12 (27), as the ICAT helical domain also shows no dependence on the tails (Table 5). It is true, however, that E_cyto, Lef-1, and APC-R3 have slightly different interactions with -cat-arm even though their binding sites mostly overlap. Thus, we cannot absolutely rule out the possibility that the C-tail affects interaction sites specific to APC-R3 and axin that do not affect the overall binding affinities of E_cyto and Lef-1 to the arm domain. Nonetheless, the fact that we cannot detect binding of the C-tail to -catenin or the effects of the tail on the other ligands makes it less likely that this region binds to the -catenin arm domain. An alternative explanation is that the C-tail might interact with APC or axin in a manner that diminishes their affinity toward the -catenin arm domain.

Overall, the thermodynamics of ligand binding to -catenin clearly show that the -catenin tails can affect the affinity of the interaction of the destruction complex ligands axin and APC with the arm domain, which may imply a role in regulating -catenin turnover. However, the results rule out models in which the tails bind to the arm domain and thereby act as competitive inhibitors. Further studies will be required to understand the molecular basis of this effect.

	FOOTNOTES

 
^* This work was supported by Grant R01 GM56169 from the National Institutes of Health. 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.

¹ Present address: FFA, LLC, 3550 General Atomics Ct., Bldg. 2, Ste. 129, San Diego, CA 92121.

² To whom correspondence should be addressed: Dept. of Structural Biology, Stanford University School of Medicine, 299 Campus Dr. West, Stanford, CA 94305-5126. Tel.: 650-725-4623; Fax: 650-723-8464; E-mail: bill.weis{at}stanford.edu.

³ The abbreviations used are: APC, adenomatous polyposis coli protein; arm, armadillo; -cat full, full-length -catenin; -cat-arm, -catenin armadillo repeat domain; -cat-arm-Nt, -catenin N-tail + arm domain; -cat-arm-Ct, -catenin arm domain + C-tail; BE_cyto, biotinylated E-cadherin cytoplasmic domain; CK1, casein kinase 1; E_cyto, E-cadherin cytoplasmic domain; GSK-3, glycogen synthase kinase-3; ICAT, inhibitor of catenin and TCF; ICAT-h, ICAT helical domain; ITC, isothermal titration calorimetry; SPR, surface plasmon resonance spectroscopy; DTT, dithiothreitol; TEV, tobacco etch virus; MCS, multiple cloning site; GST, glutathione S-transferase; IPTG, isopropyl 1-thio--D-galactopyranoside; NTA, nitrilotriacetic acid.

	ACKNOWLEDGMENTS

 
We thank Sofiya Fridman for technical assistance and Dan Herschlag and Sabine Pokutta for discussions and comments on the manuscript.

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