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STD and TRNOESY NMR Studies on the conformation of the oncogenic protein β-Catenin containing the phosphorylated motif DpSGXXpS bound

to the β-TrCP protein.

Simon Megy ‡, Gildas Bertho ‡, Josyane Gharbi-Benarous ‡, Nathalie Evrard-Todeschi ‡, Gael Coadou ‡, Emmanuel Ségéral #, Catherine Iehle ƒ, Eric Quéméneur ƒ, Richard Benarous #, and

Jean-Pierre Girault *‡

Running title: Binding of a β-Catenin phosphorylated model peptide to the β-TrCP protein.

‡ Université René Descartes-Paris V, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques (UMR 8601 CNRS), 45 rue des Saints-Pères, 75270 Paris Cedex 06, France

# U567-INSERM, UMR 8104 CNRS, Institut Cochin - Département des Maladies Infectieuses, Hôpital Cochin-Paris, Bat. G. Roussy, 27 rue du Faubourg St-Jacques, 75014 Paris, France

ƒ CEA Valrho, DSV/DIEP/SBTN, BP17171, 30207 Bagnols-sur-Cèze, France † This work was supported by grants from ARC (Association pour la Recherche sur le Cancer)

and FRM (Fondation Recherche Médicale).

* To whom correspondence should be addressed. Tel: +33-1 42 86 21 80. FAX: +33-1 42 86 83 87. E-mail: jean-pierre.girault@univ-paris5.fr

β-TrCP is the F-box protein component of an Skp1/Cul1/F-box (SCF)-type ubiquitin ligase complex. Biochemical studies have suggested that β-TrCP targets the oncogenic protein β-Catenin for ubiquitination and followed by proteasome degradation. To further elucidate the basis of this interaction, a complex between a 32-residue peptide from β-Catenin containing the phosphorylated motif DPSGXXPS (P-β-Cat17-48) and β-TrCP was studied using Saturation Transfer Difference (STD) Nuclear Magnetic Resonance (NMR) experiments. These experiments make it possible to identify the binding epitope of a ligand at atomic resolution. An analysis of STD spectra provided clear evidence that only a few of the 32 residues receive the largest saturation transfer. In particular, the amide protons of the residues in the phosphorylated motif appear to be in close contact to the amino acids of the β-TrCP binding pocket. The amide and aromatic protons of the His24 and Trp25 residues also receive a significant saturation transfer. These findings are in keeping with a recently published X-ray structure of a shorter β-

Catenin fragment with the β-TrCP1-Skp1 complex, and with the earlier findings from mutagenesis and activity assays. To better characterize the ligand-protein interaction, the bound conformation of the phosphorylated β-Catenin peptide was obtained using TRansfer Nuclear Overhauser Effect SpectroscopY (TRNOESY) experiments. Finally, we obtained the bound structure of the phosphorylated peptide showing the protons identified by STD NMR as exposed in close proximity to the molecule surface.

The ubiquitin-proteasome pathway of

protein degradation is essential for various important biological processes including cell-cycle

progression, gene transcription, and signal transduction (1,2). This work is based on the study of the oncogenic protein β-Catenin, which plays an essential role in the Wingless/Wnt signaling

pathway and is an important component of cadherin cell-adhesion complexes (Fig. 1A). The abundance of β-Catenin in the cytoplasm is regulated by ubiquitin-dependent proteolysis (3), and Wnt signaling is regulated by the presence or

JBC Papers in Press. Published on May 31, 2005 as Manuscript M501628200

Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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absence of the intracellular protein β-Catenin. When Wnt signal is absent, the signal transduction pathway is OFF because β-Catenin is rapidly destroyed. A large multiprotein machine normally facilitates the addition of phosphate groups to β-Catenin by glycogen synthase kinase-3β (GSK3β). Phosphorylated β-Catenin binds to a protein called β-TrCP, and is then modified by the covalent addition of a small protein called ubiquitin. Proteins tagged with ubiquitin are degraded by the 26S proteasome, the cell's protein-recycling center. When cells are exposed to the Wnt signal, it binds to cell surface receptors. Receptor activation blocks β-Catenin phosphorylation and its subsequent ubiquitination by an unknown mechanism that requires the intervention of the Disheveled protein (Dsh). β-Catenin is thus diverted from the proteasome, it accumulates and enters the nucleus, where it finds a partner, a DNA binding protein of the TCF/LEF family. Together, they activate new gene expression programs. In human colon cancer cells, inappropriate activation of the Wnt pathway initiates cell proliferation by activating genes encoding oncoproteins and cell-cycle regulators (4,5). Thus, the abnormal accumulation of β-Catenin as a result of mutations, either in the adenomatous polyposis coli tumor suppressor protein, or in β-Catenin itself is thought to initiate colorectal neoplasia and has been implicated in various human cancers. The formation of ubiquitin–protein conjugates requires

three enzymes that participate in a cascade of ubiquitin transfer reactions: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). β-Catenin contains a DSGXXS sequence that includes the phosphorylation site required for ubiquitination (3,6). Then proteins polyubiquitinated by these enzymes undergo degradation by the 26S proteasome (7,8). Skp1/Cul1/F-box (SCF) complexes constitute a class of E3 enzymes that are required for the degradation of many key cellular proteins. β-TrCP is the F-box protein which functions as a receptor for target molecules such as phosphorylated β-Catenin, phosphorylated IκBα (the inhibitor of the master transcription factor NFκB) and phosphorylated HIV-1 protein Vpu. The substrate specificity of SCF complexes is thus thought to be determined by the F-box protein (Fig. 1B).

It has been shown that β-TrCP is a member of the WD40 repeat-containing family and specifically only recognizes β-Catenin, IκBα, and Vpu as substrates when they are phosphorylated at both serine residues in the conserved DSGXXS motif (6,9-11). Human β-TrCP is composed of two main domains: an F-box near the N-terminus involved in interaction with Skp1, which is a connecting factor to the other subunits of the SCF E3 ligase complex for ubiquitin-mediated proteolysis; and at the C-terminus, the seven WD repeats necessary and sufficient for binding phosphorylated protein substrates of β-TrCP. Hence, all substrates of β-TrCP, either viral like Vpu, or cellular like IκBα, share this consensus motif which is also present in many cellular signaling proteins. The structure of the SCF β-TrCP ubiquitine-ligase complex and its function are depicted in Fig. 1B. The phosphorylation of the motif on the two serine residues is required for interaction with β-TrCP, and the subsequent targeting of these substrates leads to their degradation by the 26S proteasome. Thus, the elucidation of the mechanism involved in substrate recognition by β-TrCP upon phosphorylation of this DSGXXS motif is essential.

In a previous study, we examined the structural influence of phosphorylation at the two sites Ser33 and Ser37 of the free 32-residues phosphorylated peptide (12) (hereafter referenced as P-β-Cat17-48, Fig. 1A). We now report the β-TrCP-bound conformation of P-β-Cat17-48 and its binding studied by NMR methods. An array of 1D and 2D NMR spectroscopic techniques was investigated for the structural characterization and spectral assignment of the ligand.

In order to drive rationally new therapeutic approaches, an understanding of the underlying molecular mechanism of the interaction of phosphorylated β-Catenin with β-TrCP is essential. NMR-based screening methods have been developed in the last few years to characterize the binding process (13). For binding ligands, a NMR approach (14,15), usually referred to as TRansferred Nuclear Overhauser Enhancement (TRNOE) spectroscopy, is successfully applied to investigate the conformation of a ligand in its bound form. With regards to the structural analysis of the β-TrCP-bound conformations, TRNOESY experiments

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were recorded and were further completed by molecular modeling studies to determine the structural properties of the P-β-Cat17-48 peptide when bound to β-TrCP.

A further technique, the Saturation Transfer Difference (STD) NMR method, allows determination of binding epitopes (13,16). This method is based on magnetization transfer by protein signals and their relayed effect to ligand. During the saturation period, progressive saturation transfers from the protein to the ligand protons when the ligand binds to the target. The ligand protons nearest to the protein are most likely to be saturated to the highest degree and therefore have the strongest signal in the STD spectrum; whereas the ligand protons, located further away from are saturated to a lower degree and their STD intensities are weaker. Therefore, the degree of saturation of individual ligand protons reflect their proximity to the protein surface and can be used as an epitope method to describe the target-ligand interactions (13,17,18). Thus, STD NMR experiments were performed in order to define the binding epitopes of the P-β-Cat17-48 peptide.

A crystal structure of the complex involving a peptide derived from β-Catenin sequence and β-TrCP was recently published (19). It was interesting to compare the two structures. Comparing the X-Ray complex with the experimental NMR data could lead to a selection of a particular model, especially in the absence of crystallographic information on the different models of the β-TrCP complexes.

MATERIALS AND METHODS

Reagents. β-Catenin (β-Cat) fragments (17-48): peptides β-Cat

17-48 with the amino acid sequence

DRKAAVSHWQQQSYLDSGIHSGATTTAPSLSG and P-β-Cat

17-48 containing the phosphorylated

sites 33 and 37, Ser(PO3H2), DRKAAVSHWQQQSYLDpSGIHpSGATTTAPSLSG were purchased from Neosystem Lab., Strasbourg. The purity of the peptides (95%) was tested by analytical HPLC and by mass spectrometry. Purification of the WD repeat region from human protein β-TrCP. The β-TrCP protein was expressed as a fusion protein with the glutathione-

S-transferase (GST). The previously described construction (20) resulted in a fusion protein of approximately 60 kDa corresponding to the 218 residues of the GST, a 18-mer linker and the seven WD repeats (residues 251-569) of β-TrCP hereafter named GST-β-TrCP. The expression, separation and purification of GST-β-TrCP were accomplished following a standard GST fusion protein protocol, previously described (21). This amount of purified recombinant protein was then used to prepare the NMR samples.

NMR Spectroscopy. 1H NMR and STD spectra were recorded with a Bruker AMX-500 spectrometer. Standard Bruker software was used to acquire and process the NMR data. NMR samples contained nonphosphorylated β-Cat

17-48 or

phosphorylated P-β-Cat17-48

with or without the GST-β-TrCP protein. They were prepared in phosphate-buffered saline solution, pH 7.1 (20 mM phosphate, 5% D2O, 0.02% NaN3). 1H NMR spectra were recorded at constant temperature (278 K). The NMR samples were adjusted to a protein concentration of 0.02 mM based on the visible absorption at 595 nm. A 150-fold ligand excess (3mM) over binding sites were used throughout the studies. One sample control was prepared containing the β-Cat

17-48 nonphosphorylated

peptide, with the GST-β-TrCP protein at the same ratio 150:1 as that used for the P-β-Cat

17-48/ GST-

β-TrCP sample. A second negative control was done using P-β-Cat

17-48 with the fusion protein

GST-Nef (22). Resonances of the peptide were assigned, based on 2D TOCSY and NOESY experiments. Water suppression was achieved by WATERGATE (23). Chemical shifts were referred to internal TSPD4, 3-(trimethylsilyl)[2,2,3,3-d4] propionic acid, sodium salt (Tables 1 and 2). 2D TRNOESY spectra were recorded to determine the most favorable ratio for TRNOE effects, which was found to be 150:1. TRNOESY experiments were performed with mixing times of 100, 200 and 400 ms, for molar ratios between 600:1 and 25:1. After optimization of the P-β-Cat

17-48 peptide/ protein

ratio, the final sample was prepared with 0.5 mg of GST-β-TrCP protein (0.02 mM protein) and 5.2 mg of the P-β-Cat

17-48 peptide (3 mM; 150:1

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peptide/binding site ratio) in 500 µL of buffer. TRNOESY experiments were acquired in phase-sensitive mode using the States-TPPI method, with 4 k points and 512 t1 increments, a relaxation delay of 1 s. The mixing time of the transferred NOE experiment was set to 200 ms according to the TRNOE build-up curves of P-β-Cat17-48 peptide in presence of the GST-β-TrCP protein.

For STD experiments, the ligand to protein ratio was set to 150:1 (3 mM P-β-Cat

17-48 peptide,

0.02 mM GST-β-TrCP protein). STD NMR spectra were acquired using a series of 40 equally spaced 50 ms gaussian shaped pulses for selective saturation, with 1 ms delay between the pulses, and a total saturation time of approximately 2 s. With an attenuation of 50 dB, the radiofrequency field strength for the selective saturation pulses in all STD NMR experiments was 190 Hz. The frequency of the protein (on-resonance) saturation was set to the protein 1H NMR signals in the low frequency region -3 ppm. The off-resonance saturation frequency was set at 30.0 ppm. Substraction of FID values with on- and off-resonance protein saturation was achieved by phase cycling. As no baseline distortion was observed, no T1ρ filter was applied to eliminate the background resonances of the GST-β-TrCP protein. A total relaxation delay (Aq + d1) of 3.4 s and 8 dummy scans were employed to reduce substraction artifacts. 1k total scans (or 10k for a better signal to noise ratio) were collected. 1D spectra were multiplied with exponential functions (LB = 2 Hz) and zero-filled two times. Relative STD values were calculated by dividing STD signal intensities by the intensities of the corresponding signals in a 1D 1H NMR reference spectrum of the same sample recorded with 512 scans and similarly processed.

For STD experiments, several control samples were prepared to verify the specificity of the interaction. We performed 1D STD NMR experiments with P-β-Cat

17-48 and other fusion

proteins such as GST-Nef (22), or GST-N-Ter, which corresponds to the GST protein fused with the 260 first residues of the full-length β-TrCP protein, including the F-Box but not the 7 WD domains. These experiments resulted in no observable signal, proving the specifity of the interaction observed between P-β-Cat

17-48 and the

GST-β-TrCP fusion protein

Investigation of the time dependence of the saturation transfer with saturation times from 0.2 to 4.0 s showed that 2 s was leading to efficient transfer of saturation from the protein to the ligand protons.

2D STD inverse correlation (1H-

13C)-

phase-sensitive HSQC experiments were recorded using echo/anti-echo gradient selection with decoupling during acquisition (24). The 2D

1H /

13C- correlation were obtained via double INEPT

transfer using TRIM pulse of 0.2 ms duration. One-millisecond half-sinusoid echo/anti-echo gradients of 40, 10 G/cm with recovery delay of 200 µs were used. To select protons attached to carbon, a delay of 1.78 ms was optimum for C-H one-bond couplings: delay = 1 / (4 1JC-H). This technique is optimized to obtain Cα-Hα correlations with a sensitivity enhancement. The spectra were recorded with 512 experiments of 2048 data points and 48 scans per t1 experiment. Subtraction of the on- and off-resonance spectra was made by phase cycling. The acquisition times for the 2D experiments were typically around 20 h with a relaxation delay (Aq + D1) of 2.2 s. All spectra were processed with Bruker XWINNMR software. 2D spectra were multiplied with 90°-shifted squared sine bells in both dimensions and zero-filled two times in the F1 dimension. 512 and 2k points of linear prediction were added in the F1 dimension for NOESY or TRNOESY and for 2D-STD spectra, respectively. Structure Calculation. NMR spectra were analyzed with the FELIX software (Biosym Technologies). P-β-Cat

17-48 chemical shifts were assigned using

standard technique (25). Distance restraints used in the structure calculations were derived from TRNOESY experiments performed with mixing times of 200 ms, as for the free ligand we obtained a NOESY spectrum at 100 ms with very few peaks, and only intra-residue and sequential correlations at 200 ms. They were based on TRNOE peak volumes, and were classified as strong, medium, weak or very-weak restraints, corresponding to distance restraints of 1.8-2.7, 1.8-3.6, 1.8-5.0 and 1.8-6.0 Å, respectively. The distance between the two Tyr-aromatic protons (2.42 Å) was used as a reference for calibration. The final list of distance restraints containing 455 restraints was incorporated for structure calculation with the standard protocol of ARIA 1.2

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(26-28). This program was used to compute the solution structure starting from an extended structure with random side chain conformation. All NOE cross-peaks from the TRNOESY spectra were used as input to ARIA. By default, ARIA calibrates distance restraints by computing the relaxation matrix from the NOE peak volumes and the chemical shift assignments (29). A rotational correlation time of 8 ns for the peptide bound to the GST-β-TrCP protein was used for computation of the relaxation matrix. The simulated annealing protocol consisted of four stages: a high-temperature torsion angle simulated annealing phase at 10000 K (30 ps), a first torsion angle dynamics cooling phase from 10000 K to 50 K (15 ps), a second Cartesian dynamics cooling phase from 2000 K to 50 K (27 ps), and a final minimization phase at 50 K. ARIA runs were performed using the default parameters with eight iterations. Twenty structures were generated each round, and the 10 lowest-energy structures were carried to the next iteration. The peptide structures were determined and further refined using molecular dynamics with the PARALLHDG 5.3 parameters file. Modifications to the force field for the phosphorylated residues (pSer) were made with CHARMM (30) and reported for MD simulations. The residues, except the N-Ter Asp17, Gly34, Gly38 and Pro44, were constrained using 28 φ dihedral angles constraints between –20° and –180° (usually the only range considered in NMR-derived structures) (31), the region of a Ramachandran plot populated by nearly all non-glycyl residues. While ARIA allows the final structures to undergo a procedure in a simulated water box, it was decided that due to the presence of the bound peptide in the hydrophobic recognition sites this step was not applicable. After the final iteration, with ARIA software (OPLS force field) 20 structures were generated, and the 10 best with lower energies were retained as the final structures. The final list of distant restraints contained 455 restraints divided into 213 intraresidue, 150 sequential, 85 medium-range and 7 long-range NOEs. Analysis of the ARIA results (Table 3) was carried out using the aria.overview script (32). The final PDB structures were analyzed and visualized with Procheck-NMR (33) and MOLMOL (34) programs. The set of P-β-Cat

17-48 structures was selected for structural

analysis according to good geometry and few NOE violations, and by eliminating those structures with φ, ψ values in the disallowed regions of the Ramachandran plot specified with Procheck.

RESULTS

NMR assignments for the P-β-Cat17-48

/ β-TrCP protein complex. Proton chemical shifts and resonance assignments were established using two-dimensional 1H, 1H TOCSY, and NOESY experiments and are reported in Table 1. Sequential assignments of 1H resonances were based on characteristic sequential NOE connectivities observed between the α-proton of residue i and the amide proton of residue i+1, i.e. dαN(i,i+1), in NOESY data set. The Arg18 (residue 2 in the peptide numbering) broad amide signal was not identified for the P-β-Cat

17-48 peptide. In a

similar way, it could not be observed in the study of the free peptide (12). This could be attributed to the fast proton exchange with water. Carbon chemical shifts were assigned using 2D inverse correlation (

1H-

13C)-phase-sensitive HSQC

experiments (Table 2). The ability of the nonphosphorylated β-Cat

17-48 and of the

phosphorylated P-β-Cat17-48

peptides to bind to the GST-β-TrCP protein in solution was then tested by NMR spectroscopy. To identify the structural and conformational features of the binding process, STD experiments and TRNOESY spectra were recorded. TRNOESY NMR experiments. To get more information on the bound conformation of the peptide, TRNOESY experiments were recorded for different ratios near 25:1, 50:1, 100:1, 150:1, 300:1 and 600:1. The number and the intensity of the correlations increased when the peptide/GST-β-TrCP molar ratio reached 150:1 and then decreased at the molar ratio 300:1. Thus, the structural properties of the peptide bound to GST-β-TrCP were determined using 2-D TRNOESY experiments in aqueous solution (H2O/D2O, 95:5, v/v, pH 7.1) with a molar ratio equal to 150:1. The P-β-Cat

17-48 peptide gives negative NOEs in the

free state. The observation of supplementary TRNOE cross-peaks, when GST-β-TrCP was added, attested the peptide-protein interaction. Because of the low protein/peptide molar ratio,

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free and bound peptide molecules were in rapid chemical exchange, and only a single set of broadened ligand resonances was observed. In particular, the addition of GST-β-TrCP caused a soft line broadening especially observable on the amide protons of residues pSer33, His36, pSer37 (belonging to the DpSGXXpS motif) and on the amide and aromatic 4H protons of the Trp25. Chemical shift differences relative to the free peptide were also observed, in particular for two different groups of protons (Fig. 2A): the first one, corresponding to the DpSGXXpS motif included the amide protons of the pSer33, Gly34, His36, pSer37, and Gly38; the second one corresponding to the amide protons of His24 (and also its aromatic protons 2H and 4H), Trp25, and Gln26. These two effects (line broadening and shifts), which occurred on the protons of the same residues, gave us an indication of the existence of binding.

The perturbations of the chemical shifts of amide protons of P-β-Cat

17-48 are shown in Fig. 2A. The interaction caused environmental changes on the peptide protein interfaces and hence, affected the chemical shifts of the nuclei in this area. We followed the NH resonances to their bound position and we extracted the binding constant by fitting the fractional shift against an equation depending on total GST-β-TrCP protein and P-β-Cat

17-48 concentrations (35,36). Even if GST fusion proteins use to be dimeric in solution, the calculations were done assuming that the protein was present as a monomer. Fast exchange was measured for the interaction of the phosphorylated P-β-Cat

17-48 peptide to the GST-β-TrCP protein with a dissociation constant estimated around 500 µM.

Significant differences were observed between the NOESY spectra of the P-β-Cat

17-48

peptide with and without GST-β-TrCP, indicating that the cross-peaks observed in presence of GST-β-TrCP are in fact real TRNOEs. These differences between the two experiments were the most visible with short mixing-time such as 200 ms. Thus, the TRNOESY spectra confirmed the results of the STD NMR spectra: P-β-Cat

17-48 does

have binding activity to GST-β-TrCP. By comparing build-up rates of the P-β-Cat

17-48

peptide NOEs with and without GST-β-TrCP, the

mixing time was fixed at 200 ms for the structural calculations, because there was no-observable spin-diffusion at this mixing-time. Furthermore, we could observe much stronger intensities for the TRNOEs peaks (up to 7 times) than for the NOEs peaks of the free peptide. A 2D NOESY NMR experiment of the nonphosphorylated β-Cat

17-48

peptide in the presence of the GST-β-TrCP protein was also performed as a control experiment at the same ratio as that used for the P-β-Cat

17-48/ GST-β-

TrCP sample, and resulted in the absence of any supplementary peak relative to the free nonphosphorylated peptide. A second negative control was done in order to ensure that the observed TRNOEs represent specific interaction, using P-β-Cat

17-48 with the fusion protein GST-Nef

(22), and led to similar results. STD NMR experiments. The 1D saturation transfer difference (STD) NMR experiments were performed on the P-β-Cat

17-48 peptide in the

presence of GST-β-TrCP (ca. from 50 to 600-fold excess of the ligands). The only signals present were those resulting from the transfer of saturation from bound to free ligand, thus permitting their immediate identification. This technique has the great advantage that it can be combined with any NMR pulse sequence and in the 1D mode, the method is fast and reliable (37).

Specific affinity was investigated by STD NMR spectroscopy experiments (38) in order to study the influence of the serine phosphorylation on binding and to describe the region responsible for the binding interaction of the P-β-Cat

17-48

peptide with GST-β-TrCP. Without any GST-β-TrCP protein present, STD spectra did not contain ligand signals, because saturation transfer does not occur without the protein.

The sample containing GST-β-TrCP protein was the only one to show saturation transfer from the protein to the ligand in the STD spectra. We showed that a 2 s saturation time (40 gaussian pulses, each during 50 ms) was sufficient for efficient transfer of saturation from the protein to the ligand protons. The analysis of the relative STD intensities of P-β-Cat

17-48 was accomplished

by measuring the intensity of the dispersed proton resonances and referencing them to the most intensive proton signals. The 1D NMR spectrum shown in Fig. 3A revealed that a direct analysis of

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the intensity of individual proton resonances in the aliphatic region is impeded due to severe signal overlap of the corresponding methyl groups. Nevertheless, the spectral region corresponding to the amino protons is well resolved and can be used to classify the amino acid residues relevant for interaction with β-TrCP. The signals observed in the 1D spectra for the amide protons of the whole peptide are summarized in Fig. 2B. The strongest signals correspond to protons which can be in intimate contact with the protein. This figure highlights the specificity of the interaction between the peptide and the GST-β-TrCP protein.

The STD spectrum (Fig. 3A) clearly demonstrated the involvement of the HN of residues such as Leu31, Asp32, pSer33, Ile35, His 36 and pSer37 which have similar larger STD intensities, ranging from 70% to 100% (Fig. 2B). The saturation transfer also presents maximum intensity for the aromatic protons of residues such as His24, Trp25 and Tyr30 (Fig. 3A). This indicated the proximity of these protons to the protein surface. For the side chains of Gln26, Gln27 and Gln28, the signal of the NεH protons have similar large STD intensities. 2D STD TOCSY NMR spectra were also acquired but resulted in poor additional information; we mainly observed signals of the aromatic protons of Trp25, which were best amplified in the 1D STD spectrum.

The same sample was also used for a 2D STD HSQC experiment. Fig. 3B shows a region of the HSQC spectrum and Fig. 3C shows the very same region on the STD HSQC spectrum. Again, we could observe how signals corresponding to residues in a close proximity to the receptor are saturated to a higher degree, resulting in more intense cross-peaks. The analysis of the relative 2D HSQC-STD volumes of P-β-Cat

17-48 was also

accomplished by integrating the proton resonances and referencing them to the most intensive proton signals, as we did for the 1D STD analysis. The peaks corresponding to Leu31, Ile35 and Ala39 are present in the spectrum Fig. 3C, which means that the aforesaid residues interact with the β-TrCP protein. Conversely, it is interesting to notice, that signals from Ala21, Val22, and Leu46 present in the HSQC spectrum (Fig. 3B) have nearly all disappeared in the STD-HSQC spectrum (Fig. 3C) because of their low degree of saturation.

Surprisingly, signals of the Hγ of Gln27 and Gln28 are weak in the STD-HSQC spectrum, whereas their HN were quite strong in the 1D STD spectrum. But to a large extent, the results are in agreement with those of the 1D STD experiment.

To summarize, two groups of residues seem to be important for binding and seem to be closer to the protein surface than the other protons. The first one is the group of residues of the DpSGXXpS motif (without Gly34, but including Leu31) and those of the 24-28 region. This is consistent with the fact that these two regions have shown the most significant chemical shifts (Fig. 2A).

A 1D STD NMR experiment of the nonphosphorylated β-Cat

17-48 peptide in the

presence of the GST-β-TrCP protein was also performed as a reference experiment, at the same ratio as that used for the P-β-Cat

17-48/ GST-β-TrCP

sample. The addition of the β-Cat17-48

peptide to the GST-β-TrCP protein resulted in the absence of any broadening of the peaks in the control spectrum. Recording STD NMR experiments of the nonphosphorylated β-Cat

17-48 peptide in the

presence of the GST-β-TrCP protein provided a negative control. This control experiment was one experimental way to distinguish here between specific effects of binding peptide to its target and non-specific interactions between ligand and macromolecular complex. In this case, STD spectra does not contain ligand signals. No effects can be observed as enhancements of the signals of protons, because of the absence of contact between β-Cat

17-48 and the β-TrCP protein. Thus, saturation

transfer does not occur without the DpS33

GXXpS37

motif. This showed that the large number of the enhancements observed for the P-β-Cat

17-48 peptide

in the presence of the GST-β-TrCP protein was due to the areas of the ligand actually in contact with the β-TrCP binding site. Other negative controls were made to verify the specificity of the interaction. We performed 1D STD NMR experiments with P-β-Cat

17-48 and other fusion

proteins such as GST-Nef (22), or GST-N-Ter (Gst protein fused with the 260 first residues of the β-TrCP protein). STD spectra did not contain ligand signals, hereby showing that the saturation transfer does not occur without the presence of the 7 WD domains.

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3D Structure calculations. A summary of sequential d(i, i+1) and short range d(i, i+2), d(i, i+3) and d(i, i+4) 1H-1H NOE connectivities of the peptide in the bound state is shown in Fig. 4A. A great number of NOEs (455) were observed when the phosphorylated peptide was bound to GST-β-TrCP, suggesting a rather rigid conformation in the bound state. Multiple TRNOESY connectivities between side chain protons of the aromatic residues (His24, Trp25, Tyr30, and His36) and the main backbone, as well as the side chain protons of other residues, appeared upon addition of GST-β-TrCP (for instance, between 2H of Trp25 and 2H of His24, or between 2H of His36 and Hα of Gly38) suggesting that the aromatic side chains rings are constrained. The numerous NOE connectivities observed for the side chain protons indicate that binding of the peptide by GST-β-TrCP tends to freeze the rotation of the peptide side chains.

The TRNOE spectrum exhibits a great number of NOEs including intense and medium (i, i+1) connectivities suggesting the presence of secondary structures. The presence of multiple αN(i, i+2), αN(i, i+3), αN(i, i+4) (21-25 and 23-27) and αβ(i, i+3) connectivities attests that the phosphorylated peptide adopts a well-defined and folded structure in the presence of GST-β-TrCP. In particular, in region involving residues 21 to 32, the six αN(i, i+3) (Fig. 4A) connectivities are indicative of a helical conformation located just before the DpS33GIHpS37G motif. In this motif, the three αN(i, i+2) ( 32-34, 33-35, and 37-39) and αN(i, i+3) (32-35) connectivities argue in favor of a bend conformation, while the C-terminal part seems to be less structured. It should be noted that the spatially constrained aromatic rings of H24 and W25 (ie, NOEs between 4H of Trp25 and Hα of Val22, or between 4H of His24 and Hβ* of Ala21) are both incorporated within the helical part of the phosphorylated peptide.

The structures generated using ARIA resulted in TRNOE restraint files consisting of 350 unambiguous and 105 ambiguous distance constraints (Table 3). Among these TRNOEs, those that were possibly ambiguous were assigned with the most logical assignment, as determined by the sequence. Various runs were performed with ARIA to utilize as many unambiguous and ambiguous restraints as possible from the 500

MHz TRNOESY spectra and the lowest-energy structures were retained. Twenty solution structures were generated using the combined ARIA/CNS protocol described above. The structural statistics for the ten lowest energy structures, generated by ARIA in the final iteration, are shown in Table 3. The ensemble of the ten lowest energy structures exhibits a backbone rmsd of 5.6 ± 0.9 Å, but for the 21-36 region, this value decreases to 2.00 ± 0.9 Å (Table 3). This is confirmed by the plot of the rmsd of the backbone dihedral Φ and Ψ angles values for the family of the 10 conformers of the bound structure of P-β-Cat

17-48 (Fig. 4B). This graphic highlights a

better definition of residues 20 to 37 (except for Gly34). This is consistent with the total number of NOE constraints observed per residue as illustrated in Fig. 4C. The reduced number of inter-residue NOEs observed in the C-terminal part of the peptide suggests a less well-structured region. Interestingly, the well-ordered part of the peptide (20-37) corresponds to the residues which were shown to be the most involved in the binding surface as evidenced in the STD-NMR experiments.

DISCUSSION

Structure description. The ensemble of the seven lowest energy structures is shown in Fig. 5A. The structures are superposed from residue 21 to residue 36. The bound peptide showed a helical structure in its N-terminal part for residues 20 to 31 and a large bend from residue Asp32 to residue Gly38 in the DpS

33GIHpS

37G motif. At low

temperatures, the NMR structures of peptides can indicate the important structural features responsible for a ligand's ability to bind a receptor (39). We observe that the overall shape of the bound structure of β-Catenin resembles that of the free peptide at 278 K (Fig. 5B). At low temperatures, the NMR structures of peptides can indicate the important structural features responsible for a ligand's ability to bind with a receptor (39). The N-terminal helical structure is followed by a first turn in the DpS33G motif, the two XX residues, and finally, a second turn due to the pSer37. As previously observed (12), the phosphorylation of β-Catenin strongly modifies the peptide structure, which results in a negative

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charge surface (in red, Fig 5C) which could interact with the positively charged groups of β-TrCP. However, the way the β-Catenin protein interacts with β-TrCP cannot be summed up to a simple electrostatic interaction.

Hydrogen bonds and hydrophobic interaction may play a fundamental role in the stabilization of the complexes with the β-TrCP protein. As the β-Catenin peptide only binds when it is in its phosphorylated state, it seems that hydrophobic interactions can play a secondary role in the stability of the complex. The two amino acids (His24 and Trp25) create a surface upstream of the DpSGXXpS motif which could make additional hydrophobic and/or π-π interactions (in white, Fig. 5C). Epitope mapping. The STD amplification factor of the amide protons (Fig. 2B) were first classified (Fig. 5D). The strongest ones have been labeled in red, on the TRNOE derived P-β-Cat17–48 bound structure. Nearly all of them are found in the DpSGXXpS motif where the phosphate groups of pSer33 and 37 are pointing out of the structure. The Trp25 and His36 aromatic amino acids of P-β-Cat

17-48 make contacts with β-TrCP as evidenced

by STD-NMR experiments. The STD intensities observed for aromatic protons, which classically possess larger T1 relaxation rates, could be overestimated (18). We have shown in our experiments that, even when the relaxation delay was increased up to seven times T1 (the average value of T1 was around 500 ms), signals of Trp25 and essentially those coming from the aromatic chain were always the strongest we observed in STD experiments. Tryptophan is a rarely found aromatic residue (1.2 %) but Trp25 from β-Catenin is very well conserved in other organisms. It is present in β-Catenins from Human, Mouse, Rat, sea urchin and clawed frog. Another interesting fact is that a mutation on this single amino-acid in β-Catenin has been found to be involved in some human intestinal gastric cancers (40). Trp 25 is also conserved in the protein Armadillo from house fly and in the protein Plakoglobin from Human, rat, pig, and zebrafish. Hence the position of Trp25, upstream of the DSG motif, could reflect the particular role that tryptophan can play in the interaction of β-Catenin with β-TrCP. Comparison between the bound and the free P-β-Cat17-48 structures. The phosphorylated free

peptide is characterized by a bend in the motif region and a N-terminal helix (12). In the bound structure of β-Catenin, the DpSGXXpS motif and N-terminal part are well-defined regions characterized by low rmsds for their Φ and Ψ backbone dihedral angles, and these regions are involved in the binding surface contact of β-Catenin with the β-TrCP protein. As evidenced by STD experiments, the C-terminal Ala39-Gly48 region of the P-β-Cat

17-48 peptide has only few

contacts with β-TrCP. These results indicate that the region after the DSGXXS motif is less important for binding. Both in the free and in the bound structures of the P-β-Cat

17-48 peptide, the C-

terminal region (starting from Ala39) appears to have fewer NOEs constraints and a higher rmsd for Φ and Ψ angles. These could be consistent with a higher mobility of pSer37 which could contact any accessible positively charged residues spanning the β-TrCP surface (Fig. 6A).

A further indication was obtained by observing the possible role of the Trp25 region. The short helix observed for the residues around Trp25 was not observable in the crystal structure of β-Catenin. But this region, just upstream of the DSGXXS motif, is found both in the free and the bound states of β-Catenin and Vpu peptides. As shown by the intense STD response of some residues in the helix part, this region could be determinant in the stabilization of the complex with the peptide containing the consensus DpSGXXpS motif to the β-TrCP protein.

The main structural changes are observed in the motif region of the P-β-Cat

17-48 peptide,

which is modified upon binding, and becomes more compact in the bound state than in the free one as reflected by the Cα-Cα interatomic distance of the two serines (mean distance around 11 Å for the free structure, and around 8 Å for the bound structure). We also observe different orientations for the side chain of these residues. Hence, in the free P-β-Cat

17-48 peptide structure, the phosphate

groups have a nearly coplanar orientation while in the bound structure they have an opposite coplanar orientations closer to that of the crystal structure (Fig. 6A). Interestingly, the different orientation of the phosphorylated residue pSer33 is the main difference observed between free and bound

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structures of the β-Cat peptide interacting with β-TrCP (Fig. 6A).

The area downstream of the motif is probably less relevant in the binding with low STD response and less TRNOEs constraints. Comparison between the NMR and the X-Ray bound structures. A crystal structure (19) of the complex between β-TrCP and β-Catenin was recently published (30-40 corresponding residues). As the region upstream of the DSG motif is not present in the crystal structure, these contacts were not observable. We superimposed residues 33 to 36 of the lowest energy structure of the bound peptide and of the recent crystal structure (Fig. 5E). A good agreement is found between the two structures, essentially for the DpSGX region of the consensus DpSGXXpS motif. In both cases, the side chain of the first phosphoserine pSer33 points in the same direction, and the phosphate groups are found close together. However, in the consensus DpSGXXpS motif, the XpS region containing the second phosphoserine is found to be different in the two structures. Interestingly, this result highlights the strong implication that the first phosphoserine pSer33 of β-Catenin can have with β-TrCP. In the crystal structure, the phosphate group of pSer37 may also interact with many positively charged aminoacids. This is not the case for residue pSer33, the phosphate group of which can only contact few Arg or Lys residues. Thus, the different positions of the phosphorylated residue pSer37 represent the main difference observed between all the known bound structures of peptides interacting with β-TrCP (Fig. 6B).

We reported our STD results on the surface representation of the top face of the β-TrCP WD40 domain with the bound DpSGXXpS motif from the crystal structure of human β-TrCP-SkP1 complex bound to a β-Catenin substrate peptide (Fig. 5F). We decided to look at the smallest interatomic distances found between the β-Catenin substrate peptide residues and those of the β-TrCP WD40 domain in the crystal structure. We measured the distances between the amide protons of P-β-Cat and the nearest atoms of the β-TrCP protein. The shortest distances are 1.9 Å and 2.4 Å for the distances HN pSer33-OH Tyr271 and HN His36-OD1 Asn394 respectively, which correspond to the residues with the strongest STD responses (Fig. 2B). In the case of the second

phosphorylated serine, the STD response of which is only in the medium range, we measured a large distance of 5.9 Å between the ligand and the receptor (HN pSer37-Cα Gly408). Thus, the STD results are in keeping with the crystal structure. An excellent agreement is found between the amide protons with the strongest STD amplification factors, and the corresponding shortest distances in the crystal structure. Thus, either by using STD NMR experiments or by inspecting the crystal structure, the DSG motif was found to make the closest contacts with β-TrCP.

The bound structure of the P-Vpu peptide, which binds β-TrCP and contains the DSGXXS motif, has been recently published (21). We followed it up by superimposing on the DpSGXXpS motif, supposed to make close contact with the β-TrCP protein, the three known peptide bound structures i.e. the P-β-Cat

17-48 bound

peptide, the β-Catenin fragment of the crystal structure and the P-Vpu bound structure (Fig. 6B). Structural similarities were observed for the three structures. Interestingly, the DpSGXX motif structure is conserved with a bend in the DSG domain which exposes the first phosphoserine to make contacts with the β-TrCP protein, whereas the second phosphoserine was seen to possess different orientations. These results are in excellent agreement with the fact that the atoms of the peptide detected by STD experiments make close contact with the macromolecular protein receptor and are essential in the binding of the ligand studied so far. Given that the β-Catenin peptide only binds in its phosphorylated state, it seems that hydrophobic interactions may play a role in the stability of the complex. His24, Trp25 and Tyr30 involved in this helical segment define a hydrophobic cluster which could fit into a hydrophobic binding pocket localized on the β-TrCP surface and supposed to be located at the interface between the WD1 and WD2 domains.

A recent publication (41) suggests that Vpu is a strong competitive inhibitor of β-TrCP that impairs the degradation of SCF−β-TrCP substrates as long as Vpu has an intact phosphorylation motif and can bind to β-TrCP. These results indicate that the ligands of β-TrCP have common features for interaction and may even have identical epitopes.

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CONCLUSION

In this study, we demonstrated that the doubly phosphorylated β-Cat

17-48 peptide, containing the

phosphorylation site DpSGXXpS, efficiently binds to GST-β-TrCP. When bound to the GST-β-TrCP protein, the phosphorylated peptide adopts a folded structure, close to those observed in the free peptide; it reflects the local rearrangement of the DpSGXXpS phosphorylation site at the interface with β-TrCP. Our results suggest that two regions of the peptide seem to be required for the interaction with β-TrCP: the 32DpSGXXpS37 phosphorylated motif, as previously suggested (19), and also the helical region located just before the phosphorylated motif. The side chains of the three aromatic residues His24, Trp25 and Tyr30 involved in this helical segment define a hydrophobic cluster which could fit into a hydrophobic binding pocket localized on the β-TrCP surface. The residues which are the most relevant to binding were identified: Leu31, Asp32, pSer33, Ile35, and His36 in the bend region including the DpSGXXpS motif, and His24, Trp25, Gln26, Gln27 and Gln28 in the helical region located just before. Interestingly, we observed that the residues located in these two regions had upon addition of β-TrCP the most important shift of their amide proton resonance, and also had the largest STD responses. This indicates that the binding process significantly modifies the chemical environment of these residues. Finally, our results show that the first phosphorylated serine is reoriented upon binding and must be important for the interaction of peptides containing the consensus motif with β-TrCP. The second one is mobile and could accommodate different positively charged residues on the β-TrCP binding surface. The study of shorter peptides will elucidate the role of the different region in the interaction. It is clear that the epitope mapping drawn from STD experiments and the bound structure obtained from TRNOESY experiment are in keeping with the crystallographic data.

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FOOTNOTES

This work was supported by grants from ARC and FRM. Emmanuel Ségéral is supported by ARC and Simon MEGY by FRM. We thank Béatrice Berna (Centre for Technical Languages, Université René Descartes-Paris V) for her critical reading of this manuscript. The abbreviations used are: β-TrCP, Transducin Repeat Containing Protein; Cul1, Cullin 1; GST, glutathione-S-transferase; HSQC, heteronuclear single quantum correlation; IκBα, Inhibitor of nuclear factor kappa B alpha; INEPT, insensitive nuclei enhanced by polarization transfer; MD, molecular dynamics; NFκb, Nuclear Factor Kappa B; NOESY, nuclear Overhauser effect spectroscopy; P-β-Cat, Phosphorylated β-Catenin peptide; PBS, phosphate-buffered saline; rmsd, root mean square deviation; SCF, Skp1-Cullin-FBox; Skp1, Suppressor of kinetochore protein; STD, saturation transfer difference; TRNOESY, transferred nuclear Overhauser effect spectroscopy; TOCSY, total correlation spectroscopy; TPPI, time proportional phase incrementation; WATERGATE, water suppression by gradient-tailored excitation. Keywords: β-Catenin; oncogenic protein; phosphorylated peptide P-β-Cat17-48; P-β-Cat17-48/ β-TrCP complex; STD-NMR; TRNOESY; restrained molecular dynamics; bound structure; binding fragment; epitope.

FIGURE LEGENDS Fig. 1. (A) Schematic representation of β-Catenin. (a) The three-dimensional structure of a protease-resistant fragment of β-Catenin containing the armadillo repeat region. The core region of β-Catenin is composed of 12 copies of a 42 amino acid sequence motif known as an armadillo repeat. The 12 repeats form a super helix of helices that features a long positively charged groove of the proteolysis resistant fragment (42). The structure of the N and C terminal domains remain unresolved. (b) Primary structure sequence of the full β-Catenin protein. The 12 armadillo repeats are shown in green. The phosphorylation site containing the consensus motif DpSGXXpS is shown in yellow. (c) The sequence (32 residues) of the

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phosphorylated β-Catenin fragment, which was investigated in the present work. (B) Structure of the SCF β-TrCP ubiquitine-ligase complex. A model of regulation of β-Catenin degradation is depicted: β-TrCP recruits the Skp1/Cul1/E2 ubiquitination apparatus (shown respectively in grey, yellow, and blue) to the complex, leading to multi-ubiquitination of phosphorylated β-Catenin. The multi-ubiquitinated β-Catenin protein is then recognized by 26S proteasome and degraded (8). Fig. 2. (A) Difference for NH resonances of the 32 residues of P-β-Cat

17-48 between the chemical shifts in

presence of the GST-β-TrCP protein and the chemical shifts of a given resonance free in buffer solution and plotted against residue number at 278 K and pH 7.1,. The strongest difference shifts (>0.01 ppm) are shown in black. One can observe two different groups of residues concerned by this effect: 24-25-26 and 33-34-36-37 of the DpSGXXpS motif and also Gly38. (B) Relative STD intensities for P-β-Cat

17-48. The

integral value of the largest signal of the P-β-Cat17-48

peptide, the Ile35 HN proton, was set to 100%. The relative degree of saturation for the individual protons normalized to that of the Ile35, can be used to compare the STD effect (13). Relative STD intensities for the amide protons of Asp17 and Arg18 could not be determined due to the fast exchange of the protons in the N-terminal region of the peptide. The relative intensities have been classified arbitrary in three categories (dashed lines). Fig. 3. (A) 1D 1H spectrum (top) and 1D 1H STD-NMR spectrum (middle) of the P-β-Cat

17-48 peptide in

association with the GST-β-TrCP protein. Protons making close contacts with the protein interaction site show significant enhancements of their resonances. In particular, the two groups of residues 24-25-26 and 33-34-35-36-37-38 show strong enhancement signals. In the other hand, protons like Arg18 or Lys19 show very low intensity peaks. (bottom) 1D 1H spectrum of the GST-β-TrCP protein (0.02 mM). As no baseline distortion was observed, no T1ρ filter was then applied to eliminate the background resonances of the GST-β-TrCP protein. The symbol I identifies an impurity signal (acetate), which becomes biphasic in the 1D 1H STD-NMR spectrum. (B) 2D HSQC region of the complex P-β-Cat

17-48/ GST-β-TrCP protein. (C) Corresponding 2D STD HSQC region

spectrum. Ala21 and Val22 show low intensity signals in the STD-HSQC experiment. Other strong signals are identified with the one letter amino acid code. Threonine residues 40, 41 and 42 are identified with an asterisk, because resonance overlaps. Fig. 4. (A) Some sequential and medium range TRNOE connectivities in the P-β-Cat

17-48 peptide

(sequence at the top) in the presence of the GST-β-TrCP protein at 278K and pH 7.1. The thickness of the lines reflects the relative intensities of the NOEs within the individual plots. dNN(i, i+1) and dαN(i, i+1) represent sequential NOE connectivities; dαN(i, i+2), dαN(i, i+3), dαN(i, i+4), and dαβ(i, i+3) are medium-range NOE connectivities between α- and amide protons or α- and β- protons. (B) Backbone dihedral Φ and Ψ angles rmsd values for the family of the 10 best structures of P-β-Cat17–48 bound to the GST-β-TrCP protein. (C) Number of NOE restraints per residue. The restraints are displayed as intra-residue, sequential, medium and long-range constraints. Fig. 5. TRNOE derived structures of P-β-Cat17–48. (A) Twenty structures of P-β-Cat17–48 were generated after eight iterations with ARIA software. The best 7 are displayed. The molecules are superposed from residue 21 to residue 36. (B) Comparison between the lowest energy conformers of the free peptide (down, in yellow) and the bound peptide (top, in gray). (C) Surface representation of the best conformer of bound P-β-Cat17–48. The surface is colored according to the electrostatic potential, calculated with the MOLMOL software. (D) The amide protons with the strongest STD amplification factor are displayed as colored spheres, in red for the 5 strongest of them, and in blue for the 8 others. The phosphate groups of pSer33 and 37 are shown, and point out of the structure. (E) Superimposition of the lowest energy structure of the bound peptide (in grey) and the 30-40 corresponding residues obtained in a recent crystal structure (in green) (19). The two peptides are superposed from residue 33 to 36. (F) Surface

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representation of the top face of the β-TrCP WD40 domain with the bound DpSGXXpS motif from the crystal structure of the human β-TrCP-SkP1 complex bound to a β-Catenin substrate peptide (19). The protein surface is colored (red, negative region and blue, positive surface) according to the electrostatic potential calculated with the MOLMOL software. STD results have been added to the crystal structure: the amide protons with the strongest STD amplification factors are displayed as colored spheres (green for the 5 strongest of them, and light green for the rest). Fig. 6. (A) Superimposition of the 32DpSGXXpS37 fragment for the P-β-Cat

17-48 bound peptide (in gray),

the free P-β-Cat17-48

peptide (in yellow) (12) and the crystal structure (in green) (19). The 3 structures are superposed from residue 33 to 36. (B) Superimposition of the 32DpSGXXpS37 fragment of the P-β-Cat

17-48

bound peptide (in gray), the crystal structure (in green) (19) and the Vpu bound structure (in purple) (21). The 3 structures are superposed from residue 33 to 36 in β-Catenin numbering and from residue 52 to 55 in Vpu numbering.

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Table 1: Proton chemical shift assignments (278 K, pH 7.1, in 20 mM sodium phosphate buffer) of the P-β-Cat17-48

peptide

with β-TrCP in ppm from TSPD4. a

Residue NH CαH CβH Other

Asp (D17) NH3+ 4.23 2.75/2.84

Arg (R18) - 4.35 1.84/1.78 CγH 1.63 ; CδH 3.18 ; NεH 7.38 ; NηH 6.53

Lys (K19) 8.61 4.27 1.80 CγH 1.45 ; CδH 1.68 ; CεH 2.99

Ala (A20) 8.43 4.27 1.39

Ala (A21) 8.46 4.31 1.38

Val (V22) 8.28 4.11 2.05 CγH 0.92/0.88

Ser (S23) 8.50 4.42 3.73/3.79

His (H24) 8.58 4.61 3.07 4H 6.99 ; 2H 8.07

Trp (W25) 8.14 4.56 3.24 NεH 10.26

2H 7.24 ; 4H 7.53 ; 5H 7.15 ; 6H 7.25 ; 7H 7.48

Gln (Q26) 8.15 4.10 1.77/1.92 CγH 2.09 ; NεH 6.93/7.51

Gln (Q27) 8.26 4.13 1.99/2.04 CγH 2.34 ; NεH 7.00/7.66

Gln (Q28) 8.53 4.28 1.96/2.00 CγH 2.30; NεH 6.96/7.61

Ser (S29) 8.43 4.42 3.79

Tyr (Y30) 8.30 4.55 2.95/3.03 CδH 7.07; CεH 6.79

Leu (L31) 8.21 4.32 1.53 CγH 1.45; CδH 0.82/0.87

Asp (D32) 8.22 4.61 2.75

pSer (S33) 9.26 4.45 4.12/4.18

Gly (G34) 8.63 3.92

Ile (I35) 8.08 4.03 1.79 Cγ1H 1.14/1.44 ; Cγ2H 0.81 ; CδH 0.81

His (H36) 8.81 4.81 3.15/3.31 4H 7.23 ; 2H 8.45

pSer (S37) 9.40 4.47 4.11

Gly (G38) 8.78 4.01

Ala (A39) 8.30 4.41 1.44

Thr (T40) 8.46 4.44 4.25 CγH 1.23

Thr (T41) 8.40 4.46 4.25 CγH 1.22

Thr (T42) 8.38 4.33 4.16 CγH 1.22

Ala (A43) 8.59 4.59 1.38

Pro (P44) 4.45 2.33/1.91 CγH 2.03/2.05 ; CδH 3.67/3.83

Ser (S45) 8.62 4.45 3.88

Leu (L46) 8.64 4.45 1.66 CγH 1.72 ; CδH 0.89/0.95

Ser (S47) 8.42 4.48 3.87/3.90

Gly (G48) 8.14 3.80

a Ratio P-β-Cat17-48

/ β-TrCP = 150. Resonances of protons marked by - were not visible.

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Table 2: 13C chemical shift assignments (278 K. pH 7.2. in 20

mM sodium phosphate buffer) of the P-β-Cat17-48 peptide with

β-TrCP in ppm. a Residue 13Ca 13Cß Other

Asp (D17) 53.5 40.3

Arg (R18) 56.5 30.9 Cγ 27.1 ; Cδ 43.4

Lys (K19) 56.4 33.0 Cγ 25.0; Cδ 29.3; Cε 42.2

Ala (A20) 52.4 19.3

Ala (A21) 52.4 18.0

Val (V22) 62.3 33.2 Cγ1 21.1; Cγ2 20.7

Ser (S23) 58.4 64.0

His (H24) 56.4 30.2 Cε1 137.5 ; Cδ2 119.8

Trp (W25) 58.0 29.5 Cδ1 127.5 ; Cε3 120.6 ; Cζ3 122.3 ;

Cη2 124.8 ; Cζ2 114.7

Gln (Q26) 56.0 29.3 Cγ 33.6

Gln (Q27) 56.2 29.4 Cγ 33.8

Gln (Q28) 56.3 29.4 Cγ 32.1

Ser (S29) 58.4 64.0

Tyr (Y30) 58.0 38.5 Cδ 133.3 ; Cε 118.2

Leu (L31) 54.5 42.8 Cγ 27.2 ; Cδ 23.6/23.8

Asp (D32) 53.9 41.9

pSer (pS33) 58.4 65.9

Gly (G34) 45.1

Ile (I35) 61.1 38.5 Cγ 17.4 ; Cd 12.6

His (H36) 54.9 30.0 Cδ2 120.5 ; Cε1 137.3

pSer (pS37) 58.4 65.7

Gly (G38) 45.4

Ala (A39) 54.9 19.5

Thr (T40) 61.7 70.2 Cγ 21.6

Thr (T41) 61.8 70.1 Cγ 21.6

Thr (T42) 61.9 70.3 Cγ 21.6

Ala (A43) 50.7 19.3

Pro (P44) 63.4 29.3 Cγ 29.5 ; Cδ 50.4

Ser (S45) 58.3 64.0

Leu (L46) 55.5 42.4 Cγ 27.2 ; Cδ 25.2

Ser (S47) 58.5 64.0

Gly (G48) 46.2

a Ratio P-β-Cat17-48

/ β-TrCP = 150. Resonances of carbons marked by - were not visible.

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Table 3: Structural statistics of the final 10 NMR structures of

P-β-Cat17-48 bound to the β-TrCP protein

ARIAoutput

no. of exptl distance restraints

unambiguous NOE

ambiguous NOE

total NOEs

divided into

intraresidue NOE

sequential NOE

medium-range NOE

long-range NOE

no. of exptl broad dihedral restraints

NOE violations > 0.3 Å per structure

rmsd a (Å) to a mean molecule

Backbone (all residues)

heavy atoms (all residues)

Backbone (residues 21-36)

heavy atoms (residues 21-36)

Ramachandran Plot of Residues b (%)

In most favoured regions

In additional allowed regions

In generously allowed regions

In disallowed regions

350

105

455

213

150

85

7

28

1.6

5.6 ± 1.9

6.0 ± 1.7

2.0 ± 0.9

2.4 ± 0.8

35.2

57.0

7.4

0.4 a Calculated by MOLMOL. b Calculated by PROCHECK

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Figure 1

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Figure 2

A

B

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Figure 3

(A)

(B) HSQC (C) STD-HSQC

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Figure 4

A

B

C

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Figure 5

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Figure 6

(A)

(B)

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Jean-Pierre GiraultCoadou, Emmanuel Ségéral, Catherine Iehle, Eric Quéméneur, Richard Benarous and

Simon Megy, Gildas Bertho, Josyane Gharbi-Benarous, Nathalie Evrard-Todeschi, Gaelprotein

-TrCPβ-catenin containing the phosphorylated motif DpSGXXpS bound to the βSTD and TRNOESY NMR studies on the conformation of the oncogenic protein

published online May 31, 2005J. Biol. Chem. 

  10.1074/jbc.M501628200Access the most updated version of this article at doi:

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