Volume 584, Issue 8, Pages 1443-1448 (16 April 2010)

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ABSTRACT

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Structure of the p53 C-terminus bound to 14-3-3: Implications for stabilization of the p53 tetramer

Edited by Hans Eklund

Received 25 November 2009; received in revised form 23 February 2010; accepted 24 February 2010. published online 03 March 2010.

Abstract

The adaptor protein 14-3-3 binds to and stabilizes the tumor suppressor p53 and enhances its anti-tumour activity. In the regulatory C-terminal domain of p53 several 14-3-3 binding motifs have been identified. Here, we report the crystal structure of the extreme C-terminus (residues 385–393, p53pT387) of p53 in complex with 14-3-3σ at a resolution of 1.28Å. p53pT387 is accommodated by 14-3-3 in a yet unrecognized fashion implying a rationale for 14-3-3 binding to the active p53 tetramer. The structure exhibits a potential binding site for small molecules that could stabilize the p53/14-3-3 protein complex suggesting the possibility for therapeutic intervention.

Structured summary

MINT-7711943: 14-3-3 sigma (uniprotkb:P31947) and p53 (uniprotkb:P04637) bind (MI:0407) by X-ray crystallography (MI:0114)

MINT-7711931: 14-3-3 sigma (uniprotkb:P31947) and p53 (uniprotkb:P04637) bind (MI:0407) by isothermal titration calorimetry (MI:0065)

Keywords: 14-3-3 protein, Protein–protein interaction, Stabilizing molecule, p53 regulation, X-ray crystallography

Article Outline

• Abstract

• 1. Introduction

• 2. Materials and methods

• 2.1. Protein purification

• 2.2. Crystallization

• 2.3. Data collection, structure determination and refinement

• 2.4. Isothermal titration calorimetry (ITC)

• 3. Results and discussion

• 3.1. Binding of the p53pT387 peptide to 14-3-3

• 3.2. Structure of the 14-3-3/p53pT387 complex

• 3.3. Comparison of p53pT387 with known 14-3-3 motifs

• 3.4. Role of 14-3-3 for the p53 tetramer

• 3.5. Potential stabilization of the p53/14-3-3 complex by small molecules

• Acknowledgment

• Appendix A. Supplementary data

• References

• Copyright

1. Introduction

The tumour suppressor protein p53 plays a decisive role in cell cycle arrest and apoptosis and is frequently mutated in human cancers [1]. p53 is a homotetrameric transcription factor with each monomer of 393 residues displaying six distinct domains (Fig. 1a) [2]. A multitude of post-translational modifications has been reported that activate p53, among them phosphorylations of the N- and the C-terminus [3]. The major negative regulator of p53 is MDM2, an E3 ubiquitin ligase targeting p53 for proteasomal degradation [4].

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Fig. 1. Binding of the p53pT387 peptide to 14-3-3σ. (a) Domain Structure of p53 with post-translational modification of the C-terminal domain (CTD). TAD=transcriptional activation domain, PD=proline domain, L=linker region, TetD=tetramerization domain, Ac=acetylation, P=phosphorylation, Ub=ubiquitination, S=sumoylation. (b) Isothermal titration calorimetry of 14-3-3σ titrated with the p53pT387 peptide. Top panels, raw heating power over time; bottom panels, fit of the integrated energy values normalized for injected peptides. (c) Ribbon plot of the 14-3-3σ dimer (blue) complexed with the p53pT387 peptide (yellow sticks, rainbow ribbon).

The 14-3-3 proteins are a class of adapter proteins with seven isoforms (β, γ, ε, η, σ, τ, and ζ) in humans. They have been found to bind to the regulatory C-terminal domain of p53, stabilizing the tumour suppressor and enhancing its biological activity [5], [6], [25]. Several crystal structures of 14-3-3 proteins in complex with short (4–10 residues) phosphopeptides [7], [8], [9], a slightly longer (14 residues), unphosphorylated binding motif [10], a 52-residue, dimeric interaction domain [11] and a nearly full-length partner with 200 residues [12] have been reported. As demonstrated by these structures, binding to 14-3-3 employs different peptide motifs (Fig. 2c and d) and association of p53 and 14-3-3 displays some formerly not described features of 14-3-3/phosphopeptide interactions.

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Fig. 2. Structure of the p53pT387/14-3-3σ complex. (a) Electrostatic interactions (dotted lines) between residues from 14-3-3σ (blue) and p53pT387 (yellow). (b) 2FO−FC electron density map of p53pT387 (yellow sticks) and selected waters (red spheres) contoured at 1σ is shown in black. (c) Comparison of the primary sequence of different 14-3-3 interaction motifs. Phosphorylation sites and amino acids important for directing the peptide backbone are highlighted. (d) Superimposition of p53pT387 and different 14-3-3 interaction motifs bound to 14-3-3, corresponding PDB codes are shown in parentheses.

The initial report of the interaction of 14-3-3 with p53 involved phosphorylation of S378 in p53 with 14-3-3 association leading to enhanced promoter activity of p53 [5]. Furthermore, it was shown that 14-3-3 binding to p53 protects the tumour suppressor from MDM2-dependent degradation and stabilized the tetrameric oligomerization state [6], characterizing 14-3-3 as a positive regulator of p53 activity. Recently, in addition to S378, phosphorylated T387 has been reported to promote the binding of 14-3-3 [13]. We elucidated the structure of the complex with the motif surrounding pT387 bound to 14-3-3σ. The resulting structure can be combined with the tetrameric model of p53 bound to DNA [14] providing a rationale for the observed stabilizing effect of 14-3-3 binding. Furthermore, the structure reveals a pocket with the potential for accommodating a small, protein–protein stabilizing molecule. Such a molecule could enhance the p53 activating effect that results from binding to 14-3-3 proteins.

2. Materials and methods

2.1. Protein purification

Purification of His6-tagged 14-3-3σ protein was carried out using standard procedures. The protein was dialyzed against 20 mM Hepes/NaOH pH 7.5, 100mM NaCl, 10mM MgCl2 and 0.5mM TCEP and stored at −80°C. The phosphopeptide was synthesized by Biosyntan (Berlin, Germany).

2.2. Crystallization

For crystallization of the 14-3-3σ/p53pT387 peptide complex, protein and peptides were mixed in 1:1.5 molar ratio in 20mM Hepes/NaOH pH 7.5, 2mM MgCl2 and 2mM DTT and set up for crystallization in 0.1M Hepes/NaOH pH 7.5, 0.2M CaCl2, 28% PEG 400, 5% glycerol, 2mM DTT at 4°C. Crystals grew within a week and could directly be flash-cooled in liquid nitrogen.

2.3. Data collection, structure determination and refinement

Data collection was performed at the Swiss Light Source, and was processed with XDS [15]. Molecular replacement was carried out with PHASER [16] with the structure of 14-3-3σ (PDB code 1YWT) used as the search model. The obtained model was subjected to iterative rounds of model building and refinement using the programs COOT [17] and REFMAC [18]. Figures were prepared with PYMOL (www.pymol.org).

2.4. Isothermal titration calorimetry (ITC)

Experiments were carried out in buffer containing 20mM MES pH 6.5 with 2mM MgCl2 at 15°C. In the sample cell, a solution of 50μM 14-3-3 protein was placed and titrated stepwise with 6μl aliquots of a 500μM solution of p53pT387. The association constant Ka (Ka=1/Kd), molar binding stoichiometry (N), and the molar binding enthalpy (ΔH°) were determined by fitting the binding isotherm by a single binding site model. The listed Kd value is the average of three independent measurements.

3. Results and discussion

3.1. Binding of the p53pT387 peptide to 14-3-3

ITC measurement was used to quantify the affinity of the pT387 region (385FKpTEGPDSD393-COOH) of the p53 C-terminus to 14-3-3σ. The phosphorylated peptide p53pT387 binds with a Kd of 16.3±0.7μM (Fig. 1B). This value fits nicely with the value recently reported by others [13], who measured the binding of a similar peptide to 14-3-3γ (Kd=14±3μM) and 14-3-3ε (Kd=11±2μM) using fluorescence anisotropy titration. Our Kd determined by ITC is similar to those of previously reported 14-3-3/phosphopeptide interactions when dissociation constants were obtained with the same method [8], [19]. As expected, the stoichiometry from our ITC experiments indicates that two phosphopeptides bind to one 14-3-3 dimer.

3.2. Structure of the 14-3-3/p53pT387 complex

To elucidate the structural basis of the p53/14-3-3 interaction in atomic detail, we solved the crystal structure of 14-3-3σ in complex with the p53pT387 peptide to 1.28Å by molecular replacement (start model: 1YWT). For the 14-3-3σ protein we found interpretable density for 230 out of 231 residues. For the p53pT387 peptide the final density allowed building of 7 (385FKpTEGPD391) out of 9 residues. Data and refinement statistics are summarized in Supplementary Table 1. The 14-3-3 dimer displays the characteristic W-like shape with each of the monomers harbouring one p53pT387 peptide (Fig. 1c). In accordance with previously solved 14-3-3/phosphopeptide structures the p53pT387 peptide is bound in an extended conformation and is coordinated predominantly by electrostatic interactions (Fig. 2a and b).

3.3. Comparison of p53pT387 with known 14-3-3 motifs

14-3-3 proteins have initially been described as adapter proteins that recognize their partner proteins by either a mode 1 (RSXpSXP) or a mode 2 motif (RXF/YXpSXP) [20], [21]. Since then, several additional interaction motifs have been reported, among them a so-called mode 3 motif that ends C-terminally from the phosphorylated serine/threonine [22], as well as the histone H3 [9] and beta2 integrin [10] specific motifs. Although all these motifs contain a central phosphorylated amino acid, they employ different residues at different positions to exit the central binding channel of the 14-3-3 proteins. The mode 1 and 2 motifs display an invariant proline at position +2 from the phosphorylated amino acid that redirects the peptide backbone (Fig. 2c and d). Deviating strongly from this arrangement, in the 14-3-3 interaction motif from beta2 integrin a threonine is found at the +2 position which preserves the direction of the peptide backbone. Interestingly, the main-chain nitrogen of the +2 threonine (T760) in beta2 integrin overlays nicely (R.M.S.D.=0.61Å) with that of the +2 glycine (G389) in p53 (Fig. 2d). In the histone H3 interaction motif two consecutive glycines at positions +2 and +3 are leading the peptide away from the primary phospho-acceptor site. In p53 the +2 residue is also a glycine (G389) introducing a kink in the polypeptide backbone, but it is followed by a proline (P390) thus creating a rather unique 14-3-3 interaction which is further characterized by the fact, that the polypeptide chain ends at the +5 position (D393, Fig. 1a). Thus, in contrast to most other 14-3-3 protein partners with interaction motifs located rather at the N-terminus or in the middle of the protein like Raf1 (S259, S621 of 648 residues) or Cdc25C (S216 of 473 residues), the polypeptide of p53 is neither expected to exit the 14-3-3 binding groove nor to occupy further space in the 14-3-3 channel. This has important consequences for the possible modulation of the p53/14-3-3 interaction by small molecules addressing the interface of the proteins.

3.4. Role of 14-3-3 for the p53 tetramer

Full-length p53 has eluded structural elucidation by X-ray crystallography so far. Nevertheless, the structures of the p53 DNA-binding domain [23] and the tetramerization domain [24] have been solved by X-ray crystallography or NMR and were fitted into the EM volume of full-length p53 in complex with a 60-bp dsDNA [14]. The resulting model positions the DNA-binding and the tetramerization domain approximately 40Å apart, connected by the unstructured linker region. The four C-termini exit the tetramer as pairs of two in opposite directions. Based on this model, we depict a possible constellation of how two 14-3-3 dimers could bind to the p53 tetramer (Fig. 3 and Supplementary data S1). This hypothetical model fits well to the results that binding of 14-3-3 proteins shift the equilibrium of p53 oligomerization to the active tetrameric form resulting in an enhanced affinity for target promotors [5], [13]. This very mechanism has recently been shown by the group of Fersht [25]. Furthermore 14-3-3 binding in our model masks parts of the regulatory C-terminal domain that otherwise is prone to ubiquitination leading to proteasomal degradation of p53 [6] which is especially true for lysine K386 as shown by our structure.

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Fig. 3. Model of the DNA-bound p53 tetramer with two C-terminally bound 14-3-3 dimers. The DNA-binding domain is shown as green ribbon bound to DNA (magenta), the tetramerization domain as rainbow-colored ribbons and 14-3-3 as blue ribbons with the extreme C-terminus of p53 displayed as yellow sticks. The maximal length of the 28 residues connecting the 14-3-3 dimers to the C-termini of the individual p53 chains of the tetramerization domain is represented as transparent grey spheres with a radius of 70Å.

3.5. Potential stabilization of the p53/14-3-3 complex by small molecules

We previously solved the 14-3-3 complex structures of Fusicoccin and Cotylenin stabilizing a 14-3-3 protein-protein interaction [11], [19], [26]. A prerequisite for binding of these molecules is the accessibility of a conserved pocket in the interface of the 14-3-3/partner protein complex. In the majority of 14-3-3 complexes this pocket is partially occupied by residues of the 14-3-3 target protein. However, in our 14-3-3/p53pT387 structure a pocket for a stabilizing molecule is established (Fig. 4). It can be speculated that this binding site also exists in the 14-3-3 complex with full-length p53. Fusicoccin itself cannot bind to the 14-3-3/p53pT387 complex due to sterical and electrostatic incompatibility of p53 E388 and Fusicoccin (Supplementary Fig. S1). However, other molecules binding to this site and contacting both protein partners simultaneously might be interesting compounds to modulate p53 activity in a yet unexplored way with the potential of a therapeutic benefit in cancers with deregulated p53 activity (Fig. 4). To gain information on drug-binding ability of this complex we analysed it using the method SCREEN for prediction of potential drug-binding cavities [27]. Fig. 5a shows the prediction of a cavity at the site of interest. The geometric and physicochemical parameters are in the same range of known drug-binding pockets (see Supplementary Table S2). Using this information we performed a virtual screening of 10000 small molecules onto the predicted binding pocket (Fig. 5a and b, Supplementary Fig. S3). Both, the pocket analysis and the predicted binding modes of numerous compounds encourage further exploration of this strategy.

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Fig. 4. The 14-3-3σ/p53pT387 interface displays a potential drug-binding pocket. (a) A potential drug-binding pocket identified with SCREEN is established at the interface of 14-3-3σ (marine ribbon) and p53pT387 (yellow sticks). The surface of this pocket is formed by both the 14-3-3 protein (red surface) and the phosphopeptide derived from the regulatory C-terminus of p53 (green surface). (b) Example of two molecules from a virtual screening, ZINC19931902 and ZINC04879460 (c), binding to the identified pocket.

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Fig. 5. Possible effect of a small molecule stabilizing the p53/14-3-3 protein-protein interaction. (a) p53 is subject to rapid degradation that counteracts its tumour suppressor activity. (b) Binding of 14-3-3 proteins leads to stabilization of the p53 tetramer, protection against MDM2-mediated degradation and increases its tumour suppressor activity. These effects might be enhanced with small molecules that stabilize the binding of 14-3-3 to the C-terminus of p53.

Acknowledgements

We thank the staff at the Swiss Light Source, beamline X10SA, for support during crystallographic data collection.

Appendix A. Supplementary data

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Supplementary data. Supplementary information

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a Chemical Genomics Centre of the Max-Planck-Society, Otto-Hahn-Strasse 15, 44227 Dortmund, Germany

Corresponding author. Fax: +49 (0) 231 9742 6479.

1 These authors contributed equally to this work.

PII: S0014-5793(10)00169-9

doi:10.1016/j.febslet.2010.02.065

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