Solution Structure of ASPP2 N-terminal Domain (N ... - Semantic Scholar
J. Mol. Biol. (2007) 371, 948–958
doi:10.1016/j.jmb.2007.05.024
Solution Structure of ASPP2 N-terminal Domain (N-ASPP2) Reveals a Ubiquitin-like Fold Henning Tidow, Antonina Andreeva, Trevor J. Rutherford and Alan R. Fersht⁎ MRC Centre for Protein Engineering, Hills Road, Cambridge CB2 0QH, UK
Proteins of the ASPP family bind to p53 and regulate p53-mediated apoptosis. Two family members, ASPP1 and ASPP2, have pro-apoptotic functions while iASPP shows anti-apoptotic responses. However, both the mechanism of enhancement/repression of apoptosis and the molecular basis for their different responses remain unknown. To address the role of the N-termini of pro-apoptotic ASPP proteins, we solved the solution structure of N-ASPP2 (1–83) by NMR spectroscopy. The structure of this domain reveals a β-Grasp ubiquitin-like fold. Our findings suggest a possible role for the N-termini of ASPP proteins in binding to other proteins in the apoptotic response network and thus mediating their selective pro-apoptotic function. © 2007 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: ASPP2; solution structure; NMR; ubiquitin-like; β-Grasp
Introduction The tumour suppressor p53 is a tetrameric multidomain transcription factor that has several complex functions in the cell. In response to oncogenic stresses, p53-mediated transcriptional activation induces cell cycle arrest or apoptosis.1–4 It is still poorly understood how cells decide between cell cycle arrest and apoptosis during p53 response. Important factors seem to be cell type, degree of DNA damage, and the presence of survival factors, but the exact mechanism remains unknown.5,6 Recently, the ASPP protein family was identified that specifically regulates p53-mediated apoptosis but not cell cycle arrest.7–9 The family comprises two members, ASPP1 and ASPP2, which are pro-apoptotic9–13 and one member, iASPP, which Abbreviations used: ASPP, ankyrin-repeat-, SH3-domain- and proline-rich-region-containing protein; N-ASPP2, N-terminal domain (residues 1–83) of ASPP2; CD, circular dicroism; DSC, differential scanning calorimetry; HSQC, heteronuclear single quantum correlation; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; RDC, residual dipolar coupling; rmsd, root mean square deviation; RBD, Ras-binding domain; TOCSY, total correlated spectroscopy; UDP, ubiquitin-domain protein; UBL, ubiquitin-like modifiers. E-mail address of the corresponding author: [email protected]
is anti-apoptotic.14–16 However, the molecular basis for their different responses upon p53 activation is currently unknown. The domain organization of ASPP proteins consists of an N-terminal domain, which is unique to ASPP1 and ASPP2 and was described as α-helical,9 a predicted coiled-coil domain,17 and a proline-rich region followed by ankyrin repeats and an SH3 domain.18 The latter C-terminal domain is highly conserved among all ASPP family members.9,15 Several interaction partners including p53, BCL2, protein phosphatase 1, and RELA/p65 bind to this region in vivo and in vitro.11,18–22 The N-terminus is only conserved in the proapoptotic members, ASPP1 and ASPP2, being absent from anti-apoptotic iASPP. In the case of ASPP2 the Nterminus is subject to alternative splicing. The shorter (and less frequent occurring) splice variant is lacking the N-terminal 123 residues and is less able to enhance the transcriptional and apoptotic functions of p53.12 Interestingly, the N-terminus also seems to play an important role for its cellular localization. While fulllength ASPP1 and ASPP2 are predominantly cytoplasmic, removal of the N-terminal half of ASPP2 causes its C-terminal part, which contains a nuclear localization signal,23 to localize in the nucleus.12 In addition to its role in cellular localization, the N-terminus of ASPP proteins seems to be important for the transactivation function of p53 with regard to pro-apoptotic response elements like Bax or PIG3.12 While no ternary complex consisting of p53, DNA and the C-terminal domain of ASPP2 (53BP2) exists
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Solution Structure of ASPP2 N-terminal Domain
that could explain the selective up-regulation of proapoptotic response elements,22 it was shown that full-length ASPP proteins are required for maximal transactivation activity.12 This emphasizes the importance of the N-terminus and suggests that its binding to other cellular proteins might be an important factor in the apoptotic response network. To gain insight into the role of ASPP N-termini, we solved the solution structure of the N-terminal domain of ASPP2 by NMR and characterized its properties using biophysical techniques.
Results and Discussion The N-terminus of ASPP2 (N-ASPP2) constitutes a folded domain with high stability Bioinformatics analysis suggested the existence of a folded domain within the N-terminus of ASPP2. This region is conserved amongst ASPP2 orthologues present in vertebrate genomes and shows a very high similarity to the N-terminus of its paralogue ASPP1 (Figure 1). As the functional domain boundaries could not be predicted with certainty, we expressed two constructs of the conserved region, comprising residues 1–83 and 1– 135, respectively. 1D-NMR spectra did not show additional signals in the β-sheet region (N 8.5 ppm) for the longer construct (data not shown). In addition, a comparison of far-UV circular dichroism
949 (CD) spectra of both constructs revealed no additional α-helical signal within the longer construct but a significantly higher proportion of unfolded protein as indicated by the shift of the minimum towards smaller wavelength and a negative signal at 200 nm (Figure 2(a)). Thus, the shorter construct (residues 1–83) encompasses the folded domain and was subsequently used for structure determination. Relaxation analysis using residue-specific R2/R1 ratios24 revealed a correlation time of 6.0 ns being consistent with a molecular mass of 12 kDa, indicating that N-ASPP2 behaves as a monomer in solution. This was confirmed by analytical ultracentrifugation (Figure 2(b)). Thermodynamic stability measurements using thermal denaturation monitored by fluorescence spectroscopy and differential scanning calorimetry (DSC) revealed a midpoint of unfolding at 64 °C (Figure 2(c) and (d)). Solution structure of N-ASPP2 Backbone and side-chain assignments were obtained using standard triple resonance experiments25 with 15N and 13C isotopically labelled samples. A set of 1106 restraints derived from distance, dihedral angle, and hydrogen bond information was used to calculate an ensemble of representative structures using simulated annealing. Structural statistics and analysis are given in Table 1. PROCHECK analysis26 shows a favourable backbone conformation for almost all residues with 77.3% of
Figure 1. Multiple alignment of N-ASPP2 with its paralogue ASPP1 (a) and orthologues (b). Alignments were generated with ClustalW.74 Identical residues are shown in red columns. Conserved and weakly conserved residues are shown in green and yellow columns, respectively. The numbering is based on the sequence of N-ASPP2.
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Solution Structure of ASPP2 N-terminal Domain
Figure 2. Biophysical characterization of N-ASPP2. (a) Comparison of different N-ASPP2 constructs with respect to secondary structure content by CD. The far-UV spectrum of N-ASPP2 (1–83) (filled circles) shows characteristics of a folded protein with a minimum at 208 nm while the spectrum of N-ASPP2 (1–135) (open triangles) suggests a higher proportion of unfolded protein as indicated by the shift of the minimum towards smaller wavelength and a negative signal at 200 nm. The spectra were recorded in 25 mM sodium phosphate (pH 7.2), 1 mM DTE. (b) The analytical ultracentrifugation (AUC) equilibrium sedimentation profile of isotopically labelled [15N, 13C]N-ASPP2 fits to a single exponential model with Mr = 12,800 ± 300, indicating that N-ASPP2 exists as a monomer in solution. The measurement was performed at 10 °C and 40,000 rpm. The inset shows residual deviation. (c) Temperature-induced unfolding of N-ASPP2 monitored by fluorescence spectroscopy. The signals at 350 nm (open triangles) and 360 nm (filled circles) reveal a midpoint of unfolding at 61 °C. The main probe for denaturation is Trp49, which is located at the far end of β3strand. (d) Thermal denaturation curve of N-ASPP2 monitored by DSC. The apparent melting temperature is Tm = 64 °C. Buffer conditions for thermal denaturation were 25 mM sodium phosphate (pH 7.2), 150 mM NaCl, 5 mM DTT/β-mercaptoethanol.
the non-glycine and non-proline residues in the most favoured regions and 18.2% in additional allowed regions of the Ramachandran plot. The quality of the structure was assessed using measured and backcalculated 1DNH residual dipolar coupling (RDC) data yielding a Pearson correlation coefficient of 0.97 and a Cornilescu Q-factor of 0.21,27 indicating a good agreement between structure and RDC data. The best-fit superposition of the structures demonstrates a well-defined tertiary structure of N-ASPP2 with rms deviation (rmsd) of 0.66 Å for the entire structured domain (residues 4–83) and 0.27 Å for all secondary structure elements (Figure 3(a)). Compared to the core of the protein, loops L1, L3, and L6, respectively, are less well defined. This is due to higher intrinsic flexibility of the backbone as verified by measurement of {1H}15N heteronuclear nuclear Overhauser effect (NOE) values, T1 and T2 times (Figure 3(b)). The structure of N-ASPP2 has a β-β-α-β-β-α-β secondary-structure organisation and consists of a mixed twisted β-sheet with a strand order 21534 and
α-helices that pack on one side of the β-sheet. Helix H1 (residues R29–C35) packs against strands β1 and β2 (residues F5–L10, and H16–P21) at an angle of ∼ 15°. The slightly longer helix H2 (residues M62– F69) packs against strands β3–β5 (residues H44– W49, S52–V56, R77–R81) at an angle of ∼ 15° (Figure 4(a)). While the strands form a mixed β-sheet, the helices are connected both to this sheet and to each other via the interacting loops L2 (P24–C28) and L5 (D58–R61). A stable hydrogen-bond network exists within the mixed sheet and helix H1 as detected by long-range HNCO connectivities28 and H/Dexchange experiments resulting in protection factors for these residues in the range of 2 × 105–4 × 106. Long-range NOEs were observed between loops L2 and L5 and between both helices and different strands within the sheet. The hydrophobic core is built by residues L6, V8, V22, V31, L45, V56, V65, F69, V76, and F78, which display extensive longrange NOE connectivities among each other. The overall fold of N-ASPP2 belongs to the widespread β-Grasp “superfold”29 that is found in
Solution Structure of ASPP2 N-terminal Domain
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Table 1. Structure calculation statistics for the NMR structure of N-ASPP2 (60 conformers) Structural constraints Intra-residue Sequential Medium-range (2≤|i–j|≤4) Long-range (|i–j|N4) Dihedral TALOS constraints Hydrogen bonds constraints (each contributes 2 constraints) Total Statistics for accepted structures Statistics parameter (±SD) rms deviation for distance constraints (Å) rms deviation for dihedral constraints (°) Mean CNS energy term (kcal mol− 1±SD) E (overall) E (van der Waals) rms deviations from the ideal geometry (±SD) Bond lengths (Å) Bond angles (°) Improper angles (°) Coordinate precision Pairwise rmsd for backbone atoms (residues 4–83) (Å) Pairwise rmsd for heavy atoms (residues 4–83) (Å) rmsd from the mean (residues 4–83) (Å) rmsd from the mean for regular secondary structureb (Å) Ramachandran plot (% residues) Most favoured regions Additional allowed regions Generously allowed regions Disallowed regions
384 252 135 261 47 27 1106
0.024 ± 0.001 0.89 ± 0.04 302 ± 27 120 ± 13 0.0031 ± 0.0002 0.46 ± 0.02 0.41 ± 0.02 0.73 ± 0.15 1.65 ± 0.19 0.66/1.53a 0.27/1.12a
77.3 18.2 3.9 0.6
60 structures out off 90 were accepted where no distance violations were N0.5 Å and no angle violations were N5°. a Backbone atoms/heavy atoms. b Residues 5–10, 16–20, 29–35, 44–49, 52–56, 62–69, 77–81.
functionally diverse proteins sharing low or no sequence similarity.30 This fold is characterized by a cradle-like shape of the β-sheet that “grasps” around the α-helix and the mixed β-sheet having the strand order 2143. Strand β4 in N-ASPP2 has no structural equivalent in the β-Grasp superfold. N-ASPP2 shows characteristics of Ras-binding (RB)/Ras-association (RA) domains Several members of the family of Ras binding (RB) or Ras association (RalGDS/AF6 Ras associating) (RA) domain proteins share a weak but statistically significant sequence similarity to N-ASPP2. These proteins are putative Ras effector proteins, i.e. they only bind to the activated GTP-bound form of Ras, which plays an important role in various signal transduction pathways such as apoptosis and growth control.31 It has been shown, however, that not all of them bind to Ras proteins, irrespective of whether they are called RA or RB domains, and that they require a key set of residues at the interface for binding.32 Binding of RBDs to Ras is mediated by
edge-to-edge β-sheet association involving a network of backbone H-bonds. Ras-binding is determined by the presence of positively charged, solvent-exposed residues in β-strands 1 and 2 and in α-helix 1, and mutation of these residues has a drastic effect on binding.33 These crucial positively charged residues present in other RDBs are not conserved in N-ASPP2 (Figure 5(a)) (most obvious for strand β1). Indeed, using NMR and ITC-binding experiments we could not detect binding between N-ASPP2 and human HRas (data not shown), confirming the importance of these residues. Members of the RBD-family being similar to N-ASPP2 include the Ras-binding domains of AF634 (Mueller, T. D. et al., DOI: 10.2210/pdb1rax/pdb), GRB7 (Li, H. et al., DOI: 10.2210/pdb1wgr/pdb), c-Raf1 kinase (RAFRBD)35 and RGL.36 The most similar structure to N-ASPP2 is the N-terminal domain of chromosome 12 open reading frame 2 (C12orf2) (Nagashima, T. et al., DOI: 10.2210/pdb2cs4/pdb), which was released during our structure determination. Both proteins superimpose with an rmsd of 2.38 Å (Figure 6). From 71 structurally equivalent residues 21 are identical. The molecular function of C12orf2 is unknown. Its gene maps close to the KRAS2 gene and encodes for RASSF8 (Ras association (RalGDS/AF-6) domain family 8), which was recently proposed to be a novel tumour suppressor for lung cancer.37 Several other proteins containing RA domains have also been described to act as tumour suppressor genes.38,39 N-ASPP2 belongs to the ubiquitin-like superfamily Ras-binding domains belong to the ubiquitin-like superfamily. This superfamily contains also a family of “ubiquitin-like modifiers” (UBLs) that can be conjugated to other proteins in a similar way to ubiquitin and “ubiquitin-domain proteins” (UDPs) that are related to ubiquitin but are not conjugated to other proteins.40 Structures of several UBL and UDP domains have been solved, e.g. ubiquitin,27,41 Sumo-1,42,43 PLCε,44 and HHR23A,45 etc. Other protein families of the ubiquitin-like superfamily that are structurally similar to N-ASPP2 include UBX domains,46,47 GABARAP-like domains,48,49 and FERM domains.50,51 In general, these proteins show a conserved pattern of hydrophobic residues that form the hydrophobic core and are likely to contribute to the fold stability. Most of them show characteristic patterns for amphipathic helices like N-ASPP2 (most obvious for helix α1 in Figure 5(b) and (e) and for helix α2 in Figure 5(b) and (d)). Differences occur in the length of the second helix, which is smaller in ubiquitin-related and FERM proteins, and in the length of the connecting loops (Figure 5). A superposition of the structures of N-ASPP2 with ubiquitin (Figure 6) reveals the conservation of the overall fold. A total of 60 residues are structurally equivalent and superimpose with an rmsd of 2.64 Å. Within the structurally equivalent regions ten residues are identical. The surface electrostatic
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Solution Structure of ASPP2 N-terminal Domain
Figure 3. Solution structure and backbone dynamics of N-ASPP2. (a) Superposition of the 20 lowest energy conformers representing the NMR structure of the N-terminal domain of ASPP2 (PDB code 2uwq). (b) {1H}15N-heteronuclear NOE values (filled circles), T1 (open triangles) and T2 (filled diamonds) times measured at 500 MHz. Residues with NOE values of 0.7 or more are expected to be well-ordered, while values below 0.7 indicate increased backbone dynamics. All data indicate increased backbone dynamics at the N-terminus and in loops L1 (N12-Q15), L3 (P38-C43) and L6 (S71-E75).
potential of N-ASPP2 shows a distinct arrangement into a cluster of positively charged epitopes (R54, R68, R73, R81) and negatively charged epitopes (E19, E25, D30, D33, E37, E40) (Figure 4(b)). While a division into a positive and a negative “face” also occurs in ubiquitin, SUMO, and NEDD8,42,52–54 the presence of an acidic patch comprising strand β2 and helix α1 is unique to N-ASPP2 (Figure 4(b) and (c)). Functional residues are also not conserved. The C-terminal GG-motif in ubiquitin that acts as a recognition site for a protease during conjugation for example is not present in N-ASPP2. Conclusion The present study reports the previously unknown solution structure of the N-terminal domain of ASPP2 and revises the current idea that the N-termini of ASPP proteins are either α-helical or unstructured.9,55 As this domain is the main differentiator between proapoptotic (ASPP1 and ASPP2) and anti-apoptotic (iASPP) members of the ASPP family, it might be possible that its ubiquitin-like structure described here is responsible for the selective pro-apoptotic function of ASPP2. As the N-terminus of ASPP2 is required for maximal transactivation activity12 and most structurally related protein domains are involved in protein–protein interactions we anticipate
that N-ASPP2 is responsible for binding to other proteins in the apoptotic response network and thus plays an important role in tumour suppression. Further analysis may reveal the role of N-ASPP2 within the complex functions of ASPP proteins in cell-cycle control.
Materials and Methods Molecular cloning The genes encoding for amino acid residues 1–83 and 1–135, respectively, of human ASPP2 were extracted from a human cDNA library and subcloned into a pRSET derived plasmid containing an N-terminal fusion of 6xHis/lipoamyl domain/TEV protease cleavage site. Protein expression and purification N-ASPP2 and N-ASPP2 (1–135) were expressed in Escherichia coli BL21 at 25 °C for 4 h and purified using standard His-tag purification protocols followed by TEV protease digestion, a second Ni-affinity chromatography step to separate the HisLipoTEV tag, and a final gel filtration step. 5% (v/v) glycerol was used throughout the purification. The protein was kept in storage buffer (25 mM sodium phosphate (pH 7.2), 150 mM NaCl, 5 mM DTT),
Solution Structure of ASPP2 N-terminal Domain
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Figure 4. Solution structure and surface electrostatic potential of N-ASPP2. (a) Cartoon representation of N-ASPP2 indicating elements of regular secondary structure. The structure is rainbow-coloured from N-terminus (blue) to Cterminus (red). Surface electrostatic potential of N-ASPP2 (b) and ubiquitin (c). The orientation of the molecule is identical in (a) and (b) and derived from superimposition in (c). The surface colour reflects the magnitude of the electrostatic potential: red, negative; blue, positive; white, neutral. Selected electronegative and electropositive residues are labelled. Two views rotated by 180° around a horizontal axis are presented. The charge topology was calculated and displayed using PyMOL.67 and flash frozen in liquid nitrogen. Isotopically labelled (15N, 13C) samples were expressed in E.coli C41 cells56 using M9 minimal media supplemented with vitamin mix and the appropriate nitrogen (1 g/l of 15NH4Cl) and carbon (4 g/l of [13C]glucose) sources. The mass and identity of samples and the completeness of labelling were confirmed by matrix-assisted laser desorption ionization (MALDI) mass-spectrometry. GMPPNP-loaded human H-Ras G12V (residues 1–166) was purified as described.57,58 Circular dichroism spectroscopy Far-UV CD measurements were made using a Jasco J-815 spectropolarimeter (Easton, MD). Spectra were recorded from 260–190 nm (far-UV) using a 1 mm path length cell (Quartz-Suprasil, Hellma UK Ltd) and 20 μM protein. Four scans were acquired using a scan rate of 20 nm/min. Buffer conditions were 25 mM sodium phosphate (pH 7.2), 1 mM DTE. Ellipticity was calculated from the instrument units as described elsewhere.59 Fluorescence spectroscopy Measurements were performed on a Perkin Elmer LS55 luminescence spectrofluorimeter equipped with a temperature device controlled by laboratory software. Intrinsic tryptophan fluorescence anisotropy was measured with excitation at 270 nm and emission range from 300 nm to 400 nm. Individual spectra and thermal denaturations from 20 to 90 °C were carried out using 2 μM protein in a 25 mM sodium phosphate buffer (pH 7.2), 150 mM NaCl, 5 mM DTT.
Differential scanning calorimetry (DSC) DSC experiments were performed using a MicroCal™ VP-Capillary DSC instrument with auto sampler (MicroCal, Northampton, MA) with an active cell volume of 141 μl. Temperatures from 10 to 110 °C were scanned at a rate of 60 °C/h, 125 °C/h, and 250 °C/h, respectively. Protein concentrations ranging from 50 μM to 300 μM were used to exclude aggregation effects. Buffer conditions were 25 mM sodium phosphate (pH 7.2), 150 mM NaCl, 5 mM β-mercaptoethanol. This buffer was also used for baseline scans. Data analysis included subtraction of instrumental and chemical baselines and normalization for concentration and was performed using Origin™ software. Analytical ultracentrifugation (AUC) Equilibrium sedimentation experiments were performed with a Beckman XL-I ultracentrifuge equipped with a Ti-60 rotor and six-sector cells at speeds of 30,000 and 40,000 rpm at 10 °C. Data were collected in interference mode using a 200 μM NMR-sample. Buffer conditions were 25 mM sodium phosphate (pH 7.2), 150 mM NaCl, 5 mM DTT. Sample volume was 110 μl. Samples were considered to be at equilibrium as judged by a comparison of the several scans of each speed. Data were processed and analysed using UltraSpin software† and Kaleidagraph (Abelbeck Software, Reading, PA) for graph plotting. † http://www.mrc-cpe.cam.ac.uk/ultraspin
954 Solution Structure of ASPP2 N-terminal Domain
Figure 5. Structure-based sequence alignment of N-ASPP2 with members of the RBD family (a), ubiquitin family (b), UBX family (c), GABARAP-like family (d), and FERM family (e). The secondary structure elements of N-ASPP2 are indicated at the top. Residues with upper case are structurally equivalent. Residues are colour-coded according to their biophysical properties using ClustalX77 colouring scheme. The numbering is based on the sequence of N-ASPP2. RBD family: C12orf2 (2cs4.A), Grb7 (1wgr.A), Raf-1 (1c1y.B), Ral guanosine-nucleotide dissociation factor (1rax.A), RGL (1ef5.A)/ubiquitin family: ubiquitin (1ubi), NEDD8 (1ndd.B), HHR23A (1p98.A), SMT3C (1tgz.B), hHR23B (1uel.A)/ UBX family: FAF1 (1h8c.A), p47 (1jru.A)/GABARAP-like family: ATG12 (1wz3.A), GATE-16 (1eo6.A), LC3 (1ugm.A), GABARAP (1gnu.A), GABARAP (1kjt.A)/FERM family: ezrin (1ni2.A), radixin (1j19.A), merlin (1h4r.A).
Solution Structure of ASPP2 N-terminal Domain
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Figure 6. Backbone superposition of N-ASPP2 (green) with ubiquitin (cyan) (PDB code: 1ubi) and C12orf2 (magenta) (PDB code: 2cs4). Figures were generated with PyMOL67 and are based on the structure-based sequence alignment as shown in Figure 5. NMR spectroscopy All NMR experiments were performed at 298 K in a 25 mM sodium phosphate buffer (pH 7.2), 150 mM NaCl, 5 mM DTT on Bruker DRX500 and Avance800 spectrometers equipped with a cryogenic probe and single-axis gradients. Protein concentrations were 600 μM for triple resonance experiments, 700 μM for nuclear Overhauser effect spectroscopy (NOESY) experiments, 200 μM for RDC measurements, and 400 μM for hydrogen/deuterium-exchange (HDX) experiments. Backbone assignments were obtained using standard HNCA, HNCO, HN(CA) CO, CBCA(CO)NH, and HN(CO)CA triple-resonance experiments.25,60 Two-dimensional [1H, 1H]-DQF-COSY and [1H, 1H]-TOCSY spectra, three-dimensional [15N, 1 H, 1H]-TOCSY spectra, (H)CC(CO)NH, HBHA(CO)NH, CBCA(CO)NH and HNHB spectra enabled almost complete side-chain assignment with some of the Hβ resonances being stereospecifically assigned. Structural NOEs were obtained from two-dimensional [1H, 1H]NOESY spectra. The mixing times chosen were 54 ms (in 8.7 kHz B1) for TOCSY and 100 ms for NOESY spectra at 800 MHz. H/D-exchange fHSQC61 and long-range HNCO28 experiments were used to assign H-bonds. One-bond 1H-15N RDCs were measured using 8% (w/v) C12E6/hexanol bicelles62 as alignment medium to validate the structure of the backbone with 3 s 1H saturation following a 7 s recycle delay. Peak splittings were measured with {1 H, 15 N} IPAP-HSQC, and 1DNH were obtained by subtracting 1JNH values measured for a nonaligned sample. Backbone dynamics were examined by measuring {1H}15N-heteronuclear NOEs.63 Amide proton-
exchange experiments were performed as described.64 Protection factors for backbone amides were extracted according the formula P = kint/kexobs where kint was obtained from elsewhere.65 Time domain NMR data were processed in NMRPipe66 and Figures were generated using PyMOL.67 Structure calculation Distance restraints were obtained from two-dimensional [1H, 1H]-NOESY spectra with interproton distance restraints being classified into four categories corresponding to 1.8–2.8, 1.8–3.6, 1.8–4.7, and 1.8–6.0 Å, respectively, based on their NOESY peak volumes. Backbone ϕ and ψ angular constraints were determined from 13C chemical shifts using the program TALOS.68 The final ensemble of structures was calculated using the program CNS.69 A total of 60 structures out of 90 were accepted with no distance violation N0.5 Å and no angle violation N 5°. Structure calculation statistics are summarized in Table 1. The final structure was validated against measured RDCs using the program PALES.70 Geometries of all structures, as well as elements of secondary structure, were analyzed using MOLMOL71 and PROCHECK.26 PyMOL67 was used for visualization of the structures. Bioinformatics Sequence database search for identification of ASPP2 homologues was performed using PSI-BLAST).72,73 Multiple sequence alignments were generated with
Solution Structure of ASPP2 N-terminal Domain
956 ClustalW.74 Structure-based sequence alignments were produced as described elsewhere.75 Pairwise structure superimpositions were generated using ProSup.76 16. Protein Data Bank accession codes The data are deposited in the RCSB Protein Data Bank and are available under accession code 2uwq.
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Acknowledgements We thank Drs S. Freund, C. Johnson, D. Veprintsev, A. Joerger, M. Bycroft and T. Religa for valuable discussions and help on the experimental setup, and C. Blair for TEV protease. We also thank Drs R. Williams and O. Perisic for the kind gift of H-Ras protein. H.T. was supported by a fellowship from the Boehringer Ingelheim Fonds.
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Llanos, S., Ali, S. et al. (2004). The N-terminus of a novel isoform of human iASPP is required for its cytoplasmic localization. Oncogene, 23, 9007–9016. Bergamaschi, D., Samuels, Y., O'Neil, N. J., Trigiante, G., Crook, T., Hsieh, J. K. et al. (2003). iASPP oncoprotein is a key inhibitor of p53 conserved from worm to human. Nature Genet. 33, 162–167. Zhu, Z., Ramos, J., Kampa, K., Adimoolam, S., Sirisawad, M., Yu, Z. et al. (2005). Control of ASPP2/ (53BP2L) protein levels by proteasomal degradation modulates p53 apoptotic function. J. Biol. Chem. 280, 34473–34480. Gorina, S. & Pavletich, N. P. (1996). Structure of the p53 tumor suppressor bound to the ankyrin and SH3 domains of 53BP2. Science, 274, 1001–1005. Naumovski, L. & Cleary, M. L. (1996). The p53binding protein 53BP2 also interacts with Bc12 and impedes cell cycle progression at G2/M. Mol. Cell. Biol. 16, 3884–3892. Helps, N. R., Barker, H. M., Elledge, S. J. & Cohen, P. T. (1995). Protein phosphatase 1 interacts with p53BP2, a protein which binds to the tumour suppressor p53. FEBS Letters, 377, 295–300. Yang, J. P., Hori, M., Takahashi, N., Kawabe, T., Kato, H. & Okamoto, T. (1999). NF-kappaB subunit p65 binds to 53BP2 and inhibits cell death induced by 53BP2. Oncogene, 18, 5177–5186. Tidow, H., Veprintsev, D. B., Freund, S. M. & Fersht, A. R. (2006). Effects of oncogenic mutations and DNA response elements on the binding of p53 to p53 inding protein 2 (53BP2). J. Biol. Chem. 281, 32526–32533. Sachdev, S., Hoffmann, A. & Hannink, M. (1998). Nuclear localization of IkappaB alpha is mediated by the second ankyrin repeat: the IkappaB alpha ankyrin repeats define a novel class of cis-acting nuclear import sequences. Mol. Cell. Biol. 18, 2524–2534. Kay, L. E., Torchia, D. A. & Bax, A. (1989). Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease. Biochemistry, 28, 8972–8979. Sattler, M., Schleucher, J. & Griesinger, C. (1999). Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Prog. NMR Spectrosc. 34, 93–158. Laskowski, R. A., Rullmannn, J. A., MacArthur, M. W., Kaptein, R. & Thornton, J. M. (1996). AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR, 8, 477–486. Cornilescu, G., Marquardt, J. L., Ottiger, M. & Bax, A. (1998). Validation of protein structure from anisotropic carbonyl chemical shifts in a dilute liquid crystalline phase. J. Am. Chem. Soc. 120, 6836–6837. Cordier, F. & Grzesiek, S. (1999). Direct observation of hydrogen bonds in proteins by interresidue (3(h)J(NC′) scalar couplings. J. Am. Chem. Soc. 121, 1601–1602. Andreeva, A., Howorth, D., Brenner, S. E., Hubbard, T. J., Chothia, C. & Murzin, A. G. (2004). SCOP database in 2004: refinements integrate structure and sequence family data. Nucl. Acids Res. 32, D226–D229. Orengo, C. A., Jones, D. T. & Thornton, J. M. (1994). Protein superfamilies and domain superfolds. Nature, 372, 631–634. Bourne, H. R., Sanders, D. A. & McCormick, F. (1990). The GTPase superfamily: a conserved switch for diverse cell functions. Nature, 348, 125–132.
Solution Structure of ASPP2 N-terminal Domain 32. Kiel, C., Wohlgemuth, S., Rousseau, F., Schymkowitz, J., Ferkinghoff-Borg, J., Wittinghofer, F. & Serrano, L. (2005). Recognizing and defining true Ras binding domains II: in silico prediction based on homology modelling and energy calculations. J. Mol. Biol. 348, 759–775. 33. Wohlgemuth, S., Kiel, C., Kramer, A., Serrano, L., Wittinghofer, F. & Herrmann, C. (2005). Recognizing and defining true Ras binding domains I: biochemical analysis. J. Mol. Biol. 348, 741–758. 34. Kuriyama, M., Harada, N., Kuroda, S., Yamamoto, T., Nakafuku, M., Iwamatsu, A. et al. (1996). Identification of AF-6 and canoe as putative targets for Ras. J. Biol. Chem. 271, 607–610. 35. Nassar, N., Horn, G., Herrmann, C., Scherer, A., McCormick, F. & Wittinghofer, A. (1995). The 2.2 Å crystal structure of the Ras-binding domain of the serine/threonine kinase c-Raf1 in complex with Rap1A and a GTP analogue. Nature, 375, 554–560. 36. Kigawa, T., Endo, M., Ito, Y., Shirouzu, M., Kikuchi, A. & Yokoyama, S. (1998). Solution structure of the Ras-binding domain of RGL. FEBS Letters, 441, 413–418. 37. Falvella, F. S., Manenti, G., Spinola, M., Pignatiello, C., Conti, B., Pastorino, U. & Dragani, T. A. (2006). Identification of RASSF8 as a candidate lung tumor suppressor gene. Oncogene, 25, 3934–3938. 38. Dammann, R., Li, C., Yoon, J. H., Chin, P. L., Bates, S. & Pfeifer, G. P. (2000). Epigenetic inactivation of a RAS association domain family protein from the lung tumour suppressor locus 3p21.3. Nature Genet. 25, 315–319. 39. Hesson, L., Dallol, A., Minna, J. D., Maher, E. R. & Latif, F. (2003). NORE1A, a homologue of RASSF1A tumour suppressor gene is inactivated in human cancers. Oncogene, 22, 947–954. 40. Jentsch, S. & Pyrowolakis, G. (2000). Ubiquitin and its kin: how close are the family ties? Trends Cell Biol. 10, 335–342. 41. Vijay-Kumar, S., Bugg, C. E. & Cook, W. J. (1987). Structure of ubiquitin refined at 1.8 Å resolution. J. Mol. Biol. 194, 531–544. 42. Bayer, P., Arndt, A., Metzger, S., Mahajan, R., Melchior, F., Jaenicke, R. & Becker, J. (1998). Structure determination of the small ubiquitin-related modifier SUMO-1. J. Mol. Biol. 280, 275–286. 43. Song, J., Zhang, Z., Hu, W. & Chen, Y. (2005). Small ubiquitin-like modifier (SUMO) recognition of a SUMO binding motif: a reversal of the bound orientation. J. Biol. Chem. 280, 40122–40129. 44. Bunney, T. D., Harris, R., Gandarillas, N. L., Josephs, M. B., Roe, S. M., Sorli, S. C. et al. (2006). Structural and mechanistic insights into ras association domains of phospholipase C epsilon. Mol. Cell, 21, 495–507. 45. Mueller, T. D. & Feigon, J. (2003). Structural determinants for the binding of ubiquitin-like domains to the proteasome. EMBO J. 22, 4634–4645. 46. Yuan, X., Shaw, A., Zhang, X., Kondo, H., Lally, J., Freemont, P. S. & Matthews, S. (2001). Solution structure and interaction surface of the C-terminal domain from p47: a major p97-cofactor involved in SNARE disassembly. J. Mol. Biol. 311, 255–263. 47. Buchberger, A., Howard, M. J., Proctor, M. & Bycroft, M. (2001). The UBX domain: a widespread ubiquitinlike module. J. Mol. Biol. 307, 17–24. 48. Knight, D., Harris, R., McAlister, M. S., Phelan, J. P., Geddes, S., Moss, S. J. et al. (2002). The X-ray crystal structure and putative ligand-derived peptide binding properties of gamma-aminobutyric acid receptor
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Solution Structure of ASPP2 N-terminal Domain
958 65. Bai, Y., Milne, J. S., Mayne, L. & Englander, S. W. (1993). Primary structure effects on peptide group hydrogen exchange. Proteins: Struct. Funct. Genet. 17, 75–86. 66. Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J. & Bax, A. (1995). NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR, 6, 277–293. 67. DeLano, W. L. (2002). The PyMOL Molecular Graphics System. DeLano Scientific, San Carlos, CA, USA. 68. Cornilescu, G., Delaglio, F. & Bax, A. (1999). Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J. Biomol. NMR, 13, 289–302. 69. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W. et al. (1998). Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Crystallog. sect. D, 54, 905–921. 70. Zweckstetter, M. & Bax, A. (2000). Prediction of sterically induced alignment in a dilute liquid crystalline phase: aid to protein structure determination by NMR. J. Am. Chem. Soc. 122, 3791–3792. 71. Koradi, R., Billeter, M., & Wuthrich, K. (1996). MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 14, 51–55, 29–32.
72. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl. Acids Res. 25, 3389–3402. 73. Schaffer, A. A., Aravind, L., Madden, T. L., Shavirin, S., Spouge, J. L., Wolf, Y. I. et al. (2001). Improving the accuracy of PSI-BLAST protein database searches with composition-based statistics and other refinements. Nucl. Acids Res. 29, 2994–3005. 74. Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl. Acids Res. 22, 4673–4680. 75. Andreeva, A., Prlic, A., Hubbard, T. J. & Murzin, A. G. (2007). SISYPHUS-structural alignments for proteins with non-trivial relationships. Nucl. Acids Res. 35, D253–D259. 76. Feng, Z. K. & Sippl, M. J. (1996). Optimum superimposition of protein structures: ambiguities and implications. Fold. Des. 1, 123–132. 77. Jeanmougin, F., Thompson, J. D., Gouy, M., Higgins, D. G. & Gibson, T. J. (1998). Multiple sequence alignment with Clustal X. Trends Biochem. Sci. 23, 403–405.
Edited by M. F. Summers (Received 24 March 2007; received in revised form 7 May 2007; accepted 7 May 2007) Available online 13 May 2007
doi:10.1016/j.jmb.2007.05.024
Solution Structure of ASPP2 N-terminal Domain (N-ASPP2) Reveals a Ubiquitin-like Fold Henning Tidow, Antonina Andreeva, Trevor J. Rutherford and Alan R. Fersht⁎ MRC Centre for Protein Engineering, Hills Road, Cambridge CB2 0QH, UK
Proteins of the ASPP family bind to p53 and regulate p53-mediated apoptosis. Two family members, ASPP1 and ASPP2, have pro-apoptotic functions while iASPP shows anti-apoptotic responses. However, both the mechanism of enhancement/repression of apoptosis and the molecular basis for their different responses remain unknown. To address the role of the N-termini of pro-apoptotic ASPP proteins, we solved the solution structure of N-ASPP2 (1–83) by NMR spectroscopy. The structure of this domain reveals a β-Grasp ubiquitin-like fold. Our findings suggest a possible role for the N-termini of ASPP proteins in binding to other proteins in the apoptotic response network and thus mediating their selective pro-apoptotic function. © 2007 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: ASPP2; solution structure; NMR; ubiquitin-like; β-Grasp
Introduction The tumour suppressor p53 is a tetrameric multidomain transcription factor that has several complex functions in the cell. In response to oncogenic stresses, p53-mediated transcriptional activation induces cell cycle arrest or apoptosis.1–4 It is still poorly understood how cells decide between cell cycle arrest and apoptosis during p53 response. Important factors seem to be cell type, degree of DNA damage, and the presence of survival factors, but the exact mechanism remains unknown.5,6 Recently, the ASPP protein family was identified that specifically regulates p53-mediated apoptosis but not cell cycle arrest.7–9 The family comprises two members, ASPP1 and ASPP2, which are pro-apoptotic9–13 and one member, iASPP, which Abbreviations used: ASPP, ankyrin-repeat-, SH3-domain- and proline-rich-region-containing protein; N-ASPP2, N-terminal domain (residues 1–83) of ASPP2; CD, circular dicroism; DSC, differential scanning calorimetry; HSQC, heteronuclear single quantum correlation; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; RDC, residual dipolar coupling; rmsd, root mean square deviation; RBD, Ras-binding domain; TOCSY, total correlated spectroscopy; UDP, ubiquitin-domain protein; UBL, ubiquitin-like modifiers. E-mail address of the corresponding author: [email protected]
is anti-apoptotic.14–16 However, the molecular basis for their different responses upon p53 activation is currently unknown. The domain organization of ASPP proteins consists of an N-terminal domain, which is unique to ASPP1 and ASPP2 and was described as α-helical,9 a predicted coiled-coil domain,17 and a proline-rich region followed by ankyrin repeats and an SH3 domain.18 The latter C-terminal domain is highly conserved among all ASPP family members.9,15 Several interaction partners including p53, BCL2, protein phosphatase 1, and RELA/p65 bind to this region in vivo and in vitro.11,18–22 The N-terminus is only conserved in the proapoptotic members, ASPP1 and ASPP2, being absent from anti-apoptotic iASPP. In the case of ASPP2 the Nterminus is subject to alternative splicing. The shorter (and less frequent occurring) splice variant is lacking the N-terminal 123 residues and is less able to enhance the transcriptional and apoptotic functions of p53.12 Interestingly, the N-terminus also seems to play an important role for its cellular localization. While fulllength ASPP1 and ASPP2 are predominantly cytoplasmic, removal of the N-terminal half of ASPP2 causes its C-terminal part, which contains a nuclear localization signal,23 to localize in the nucleus.12 In addition to its role in cellular localization, the N-terminus of ASPP proteins seems to be important for the transactivation function of p53 with regard to pro-apoptotic response elements like Bax or PIG3.12 While no ternary complex consisting of p53, DNA and the C-terminal domain of ASPP2 (53BP2) exists
0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.
Solution Structure of ASPP2 N-terminal Domain
that could explain the selective up-regulation of proapoptotic response elements,22 it was shown that full-length ASPP proteins are required for maximal transactivation activity.12 This emphasizes the importance of the N-terminus and suggests that its binding to other cellular proteins might be an important factor in the apoptotic response network. To gain insight into the role of ASPP N-termini, we solved the solution structure of the N-terminal domain of ASPP2 by NMR and characterized its properties using biophysical techniques.
Results and Discussion The N-terminus of ASPP2 (N-ASPP2) constitutes a folded domain with high stability Bioinformatics analysis suggested the existence of a folded domain within the N-terminus of ASPP2. This region is conserved amongst ASPP2 orthologues present in vertebrate genomes and shows a very high similarity to the N-terminus of its paralogue ASPP1 (Figure 1). As the functional domain boundaries could not be predicted with certainty, we expressed two constructs of the conserved region, comprising residues 1–83 and 1– 135, respectively. 1D-NMR spectra did not show additional signals in the β-sheet region (N 8.5 ppm) for the longer construct (data not shown). In addition, a comparison of far-UV circular dichroism
949 (CD) spectra of both constructs revealed no additional α-helical signal within the longer construct but a significantly higher proportion of unfolded protein as indicated by the shift of the minimum towards smaller wavelength and a negative signal at 200 nm (Figure 2(a)). Thus, the shorter construct (residues 1–83) encompasses the folded domain and was subsequently used for structure determination. Relaxation analysis using residue-specific R2/R1 ratios24 revealed a correlation time of 6.0 ns being consistent with a molecular mass of 12 kDa, indicating that N-ASPP2 behaves as a monomer in solution. This was confirmed by analytical ultracentrifugation (Figure 2(b)). Thermodynamic stability measurements using thermal denaturation monitored by fluorescence spectroscopy and differential scanning calorimetry (DSC) revealed a midpoint of unfolding at 64 °C (Figure 2(c) and (d)). Solution structure of N-ASPP2 Backbone and side-chain assignments were obtained using standard triple resonance experiments25 with 15N and 13C isotopically labelled samples. A set of 1106 restraints derived from distance, dihedral angle, and hydrogen bond information was used to calculate an ensemble of representative structures using simulated annealing. Structural statistics and analysis are given in Table 1. PROCHECK analysis26 shows a favourable backbone conformation for almost all residues with 77.3% of
Figure 1. Multiple alignment of N-ASPP2 with its paralogue ASPP1 (a) and orthologues (b). Alignments were generated with ClustalW.74 Identical residues are shown in red columns. Conserved and weakly conserved residues are shown in green and yellow columns, respectively. The numbering is based on the sequence of N-ASPP2.
950
Solution Structure of ASPP2 N-terminal Domain
Figure 2. Biophysical characterization of N-ASPP2. (a) Comparison of different N-ASPP2 constructs with respect to secondary structure content by CD. The far-UV spectrum of N-ASPP2 (1–83) (filled circles) shows characteristics of a folded protein with a minimum at 208 nm while the spectrum of N-ASPP2 (1–135) (open triangles) suggests a higher proportion of unfolded protein as indicated by the shift of the minimum towards smaller wavelength and a negative signal at 200 nm. The spectra were recorded in 25 mM sodium phosphate (pH 7.2), 1 mM DTE. (b) The analytical ultracentrifugation (AUC) equilibrium sedimentation profile of isotopically labelled [15N, 13C]N-ASPP2 fits to a single exponential model with Mr = 12,800 ± 300, indicating that N-ASPP2 exists as a monomer in solution. The measurement was performed at 10 °C and 40,000 rpm. The inset shows residual deviation. (c) Temperature-induced unfolding of N-ASPP2 monitored by fluorescence spectroscopy. The signals at 350 nm (open triangles) and 360 nm (filled circles) reveal a midpoint of unfolding at 61 °C. The main probe for denaturation is Trp49, which is located at the far end of β3strand. (d) Thermal denaturation curve of N-ASPP2 monitored by DSC. The apparent melting temperature is Tm = 64 °C. Buffer conditions for thermal denaturation were 25 mM sodium phosphate (pH 7.2), 150 mM NaCl, 5 mM DTT/β-mercaptoethanol.
the non-glycine and non-proline residues in the most favoured regions and 18.2% in additional allowed regions of the Ramachandran plot. The quality of the structure was assessed using measured and backcalculated 1DNH residual dipolar coupling (RDC) data yielding a Pearson correlation coefficient of 0.97 and a Cornilescu Q-factor of 0.21,27 indicating a good agreement between structure and RDC data. The best-fit superposition of the structures demonstrates a well-defined tertiary structure of N-ASPP2 with rms deviation (rmsd) of 0.66 Å for the entire structured domain (residues 4–83) and 0.27 Å for all secondary structure elements (Figure 3(a)). Compared to the core of the protein, loops L1, L3, and L6, respectively, are less well defined. This is due to higher intrinsic flexibility of the backbone as verified by measurement of {1H}15N heteronuclear nuclear Overhauser effect (NOE) values, T1 and T2 times (Figure 3(b)). The structure of N-ASPP2 has a β-β-α-β-β-α-β secondary-structure organisation and consists of a mixed twisted β-sheet with a strand order 21534 and
α-helices that pack on one side of the β-sheet. Helix H1 (residues R29–C35) packs against strands β1 and β2 (residues F5–L10, and H16–P21) at an angle of ∼ 15°. The slightly longer helix H2 (residues M62– F69) packs against strands β3–β5 (residues H44– W49, S52–V56, R77–R81) at an angle of ∼ 15° (Figure 4(a)). While the strands form a mixed β-sheet, the helices are connected both to this sheet and to each other via the interacting loops L2 (P24–C28) and L5 (D58–R61). A stable hydrogen-bond network exists within the mixed sheet and helix H1 as detected by long-range HNCO connectivities28 and H/Dexchange experiments resulting in protection factors for these residues in the range of 2 × 105–4 × 106. Long-range NOEs were observed between loops L2 and L5 and between both helices and different strands within the sheet. The hydrophobic core is built by residues L6, V8, V22, V31, L45, V56, V65, F69, V76, and F78, which display extensive longrange NOE connectivities among each other. The overall fold of N-ASPP2 belongs to the widespread β-Grasp “superfold”29 that is found in
Solution Structure of ASPP2 N-terminal Domain
951
Table 1. Structure calculation statistics for the NMR structure of N-ASPP2 (60 conformers) Structural constraints Intra-residue Sequential Medium-range (2≤|i–j|≤4) Long-range (|i–j|N4) Dihedral TALOS constraints Hydrogen bonds constraints (each contributes 2 constraints) Total Statistics for accepted structures Statistics parameter (±SD) rms deviation for distance constraints (Å) rms deviation for dihedral constraints (°) Mean CNS energy term (kcal mol− 1±SD) E (overall) E (van der Waals) rms deviations from the ideal geometry (±SD) Bond lengths (Å) Bond angles (°) Improper angles (°) Coordinate precision Pairwise rmsd for backbone atoms (residues 4–83) (Å) Pairwise rmsd for heavy atoms (residues 4–83) (Å) rmsd from the mean (residues 4–83) (Å) rmsd from the mean for regular secondary structureb (Å) Ramachandran plot (% residues) Most favoured regions Additional allowed regions Generously allowed regions Disallowed regions
384 252 135 261 47 27 1106
0.024 ± 0.001 0.89 ± 0.04 302 ± 27 120 ± 13 0.0031 ± 0.0002 0.46 ± 0.02 0.41 ± 0.02 0.73 ± 0.15 1.65 ± 0.19 0.66/1.53a 0.27/1.12a
77.3 18.2 3.9 0.6
60 structures out off 90 were accepted where no distance violations were N0.5 Å and no angle violations were N5°. a Backbone atoms/heavy atoms. b Residues 5–10, 16–20, 29–35, 44–49, 52–56, 62–69, 77–81.
functionally diverse proteins sharing low or no sequence similarity.30 This fold is characterized by a cradle-like shape of the β-sheet that “grasps” around the α-helix and the mixed β-sheet having the strand order 2143. Strand β4 in N-ASPP2 has no structural equivalent in the β-Grasp superfold. N-ASPP2 shows characteristics of Ras-binding (RB)/Ras-association (RA) domains Several members of the family of Ras binding (RB) or Ras association (RalGDS/AF6 Ras associating) (RA) domain proteins share a weak but statistically significant sequence similarity to N-ASPP2. These proteins are putative Ras effector proteins, i.e. they only bind to the activated GTP-bound form of Ras, which plays an important role in various signal transduction pathways such as apoptosis and growth control.31 It has been shown, however, that not all of them bind to Ras proteins, irrespective of whether they are called RA or RB domains, and that they require a key set of residues at the interface for binding.32 Binding of RBDs to Ras is mediated by
edge-to-edge β-sheet association involving a network of backbone H-bonds. Ras-binding is determined by the presence of positively charged, solvent-exposed residues in β-strands 1 and 2 and in α-helix 1, and mutation of these residues has a drastic effect on binding.33 These crucial positively charged residues present in other RDBs are not conserved in N-ASPP2 (Figure 5(a)) (most obvious for strand β1). Indeed, using NMR and ITC-binding experiments we could not detect binding between N-ASPP2 and human HRas (data not shown), confirming the importance of these residues. Members of the RBD-family being similar to N-ASPP2 include the Ras-binding domains of AF634 (Mueller, T. D. et al., DOI: 10.2210/pdb1rax/pdb), GRB7 (Li, H. et al., DOI: 10.2210/pdb1wgr/pdb), c-Raf1 kinase (RAFRBD)35 and RGL.36 The most similar structure to N-ASPP2 is the N-terminal domain of chromosome 12 open reading frame 2 (C12orf2) (Nagashima, T. et al., DOI: 10.2210/pdb2cs4/pdb), which was released during our structure determination. Both proteins superimpose with an rmsd of 2.38 Å (Figure 6). From 71 structurally equivalent residues 21 are identical. The molecular function of C12orf2 is unknown. Its gene maps close to the KRAS2 gene and encodes for RASSF8 (Ras association (RalGDS/AF-6) domain family 8), which was recently proposed to be a novel tumour suppressor for lung cancer.37 Several other proteins containing RA domains have also been described to act as tumour suppressor genes.38,39 N-ASPP2 belongs to the ubiquitin-like superfamily Ras-binding domains belong to the ubiquitin-like superfamily. This superfamily contains also a family of “ubiquitin-like modifiers” (UBLs) that can be conjugated to other proteins in a similar way to ubiquitin and “ubiquitin-domain proteins” (UDPs) that are related to ubiquitin but are not conjugated to other proteins.40 Structures of several UBL and UDP domains have been solved, e.g. ubiquitin,27,41 Sumo-1,42,43 PLCε,44 and HHR23A,45 etc. Other protein families of the ubiquitin-like superfamily that are structurally similar to N-ASPP2 include UBX domains,46,47 GABARAP-like domains,48,49 and FERM domains.50,51 In general, these proteins show a conserved pattern of hydrophobic residues that form the hydrophobic core and are likely to contribute to the fold stability. Most of them show characteristic patterns for amphipathic helices like N-ASPP2 (most obvious for helix α1 in Figure 5(b) and (e) and for helix α2 in Figure 5(b) and (d)). Differences occur in the length of the second helix, which is smaller in ubiquitin-related and FERM proteins, and in the length of the connecting loops (Figure 5). A superposition of the structures of N-ASPP2 with ubiquitin (Figure 6) reveals the conservation of the overall fold. A total of 60 residues are structurally equivalent and superimpose with an rmsd of 2.64 Å. Within the structurally equivalent regions ten residues are identical. The surface electrostatic
952
Solution Structure of ASPP2 N-terminal Domain
Figure 3. Solution structure and backbone dynamics of N-ASPP2. (a) Superposition of the 20 lowest energy conformers representing the NMR structure of the N-terminal domain of ASPP2 (PDB code 2uwq). (b) {1H}15N-heteronuclear NOE values (filled circles), T1 (open triangles) and T2 (filled diamonds) times measured at 500 MHz. Residues with NOE values of 0.7 or more are expected to be well-ordered, while values below 0.7 indicate increased backbone dynamics. All data indicate increased backbone dynamics at the N-terminus and in loops L1 (N12-Q15), L3 (P38-C43) and L6 (S71-E75).
potential of N-ASPP2 shows a distinct arrangement into a cluster of positively charged epitopes (R54, R68, R73, R81) and negatively charged epitopes (E19, E25, D30, D33, E37, E40) (Figure 4(b)). While a division into a positive and a negative “face” also occurs in ubiquitin, SUMO, and NEDD8,42,52–54 the presence of an acidic patch comprising strand β2 and helix α1 is unique to N-ASPP2 (Figure 4(b) and (c)). Functional residues are also not conserved. The C-terminal GG-motif in ubiquitin that acts as a recognition site for a protease during conjugation for example is not present in N-ASPP2. Conclusion The present study reports the previously unknown solution structure of the N-terminal domain of ASPP2 and revises the current idea that the N-termini of ASPP proteins are either α-helical or unstructured.9,55 As this domain is the main differentiator between proapoptotic (ASPP1 and ASPP2) and anti-apoptotic (iASPP) members of the ASPP family, it might be possible that its ubiquitin-like structure described here is responsible for the selective pro-apoptotic function of ASPP2. As the N-terminus of ASPP2 is required for maximal transactivation activity12 and most structurally related protein domains are involved in protein–protein interactions we anticipate
that N-ASPP2 is responsible for binding to other proteins in the apoptotic response network and thus plays an important role in tumour suppression. Further analysis may reveal the role of N-ASPP2 within the complex functions of ASPP proteins in cell-cycle control.
Materials and Methods Molecular cloning The genes encoding for amino acid residues 1–83 and 1–135, respectively, of human ASPP2 were extracted from a human cDNA library and subcloned into a pRSET derived plasmid containing an N-terminal fusion of 6xHis/lipoamyl domain/TEV protease cleavage site. Protein expression and purification N-ASPP2 and N-ASPP2 (1–135) were expressed in Escherichia coli BL21 at 25 °C for 4 h and purified using standard His-tag purification protocols followed by TEV protease digestion, a second Ni-affinity chromatography step to separate the HisLipoTEV tag, and a final gel filtration step. 5% (v/v) glycerol was used throughout the purification. The protein was kept in storage buffer (25 mM sodium phosphate (pH 7.2), 150 mM NaCl, 5 mM DTT),
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Figure 4. Solution structure and surface electrostatic potential of N-ASPP2. (a) Cartoon representation of N-ASPP2 indicating elements of regular secondary structure. The structure is rainbow-coloured from N-terminus (blue) to Cterminus (red). Surface electrostatic potential of N-ASPP2 (b) and ubiquitin (c). The orientation of the molecule is identical in (a) and (b) and derived from superimposition in (c). The surface colour reflects the magnitude of the electrostatic potential: red, negative; blue, positive; white, neutral. Selected electronegative and electropositive residues are labelled. Two views rotated by 180° around a horizontal axis are presented. The charge topology was calculated and displayed using PyMOL.67 and flash frozen in liquid nitrogen. Isotopically labelled (15N, 13C) samples were expressed in E.coli C41 cells56 using M9 minimal media supplemented with vitamin mix and the appropriate nitrogen (1 g/l of 15NH4Cl) and carbon (4 g/l of [13C]glucose) sources. The mass and identity of samples and the completeness of labelling were confirmed by matrix-assisted laser desorption ionization (MALDI) mass-spectrometry. GMPPNP-loaded human H-Ras G12V (residues 1–166) was purified as described.57,58 Circular dichroism spectroscopy Far-UV CD measurements were made using a Jasco J-815 spectropolarimeter (Easton, MD). Spectra were recorded from 260–190 nm (far-UV) using a 1 mm path length cell (Quartz-Suprasil, Hellma UK Ltd) and 20 μM protein. Four scans were acquired using a scan rate of 20 nm/min. Buffer conditions were 25 mM sodium phosphate (pH 7.2), 1 mM DTE. Ellipticity was calculated from the instrument units as described elsewhere.59 Fluorescence spectroscopy Measurements were performed on a Perkin Elmer LS55 luminescence spectrofluorimeter equipped with a temperature device controlled by laboratory software. Intrinsic tryptophan fluorescence anisotropy was measured with excitation at 270 nm and emission range from 300 nm to 400 nm. Individual spectra and thermal denaturations from 20 to 90 °C were carried out using 2 μM protein in a 25 mM sodium phosphate buffer (pH 7.2), 150 mM NaCl, 5 mM DTT.
Differential scanning calorimetry (DSC) DSC experiments were performed using a MicroCal™ VP-Capillary DSC instrument with auto sampler (MicroCal, Northampton, MA) with an active cell volume of 141 μl. Temperatures from 10 to 110 °C were scanned at a rate of 60 °C/h, 125 °C/h, and 250 °C/h, respectively. Protein concentrations ranging from 50 μM to 300 μM were used to exclude aggregation effects. Buffer conditions were 25 mM sodium phosphate (pH 7.2), 150 mM NaCl, 5 mM β-mercaptoethanol. This buffer was also used for baseline scans. Data analysis included subtraction of instrumental and chemical baselines and normalization for concentration and was performed using Origin™ software. Analytical ultracentrifugation (AUC) Equilibrium sedimentation experiments were performed with a Beckman XL-I ultracentrifuge equipped with a Ti-60 rotor and six-sector cells at speeds of 30,000 and 40,000 rpm at 10 °C. Data were collected in interference mode using a 200 μM NMR-sample. Buffer conditions were 25 mM sodium phosphate (pH 7.2), 150 mM NaCl, 5 mM DTT. Sample volume was 110 μl. Samples were considered to be at equilibrium as judged by a comparison of the several scans of each speed. Data were processed and analysed using UltraSpin software† and Kaleidagraph (Abelbeck Software, Reading, PA) for graph plotting. † http://www.mrc-cpe.cam.ac.uk/ultraspin
954 Solution Structure of ASPP2 N-terminal Domain
Figure 5. Structure-based sequence alignment of N-ASPP2 with members of the RBD family (a), ubiquitin family (b), UBX family (c), GABARAP-like family (d), and FERM family (e). The secondary structure elements of N-ASPP2 are indicated at the top. Residues with upper case are structurally equivalent. Residues are colour-coded according to their biophysical properties using ClustalX77 colouring scheme. The numbering is based on the sequence of N-ASPP2. RBD family: C12orf2 (2cs4.A), Grb7 (1wgr.A), Raf-1 (1c1y.B), Ral guanosine-nucleotide dissociation factor (1rax.A), RGL (1ef5.A)/ubiquitin family: ubiquitin (1ubi), NEDD8 (1ndd.B), HHR23A (1p98.A), SMT3C (1tgz.B), hHR23B (1uel.A)/ UBX family: FAF1 (1h8c.A), p47 (1jru.A)/GABARAP-like family: ATG12 (1wz3.A), GATE-16 (1eo6.A), LC3 (1ugm.A), GABARAP (1gnu.A), GABARAP (1kjt.A)/FERM family: ezrin (1ni2.A), radixin (1j19.A), merlin (1h4r.A).
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Figure 6. Backbone superposition of N-ASPP2 (green) with ubiquitin (cyan) (PDB code: 1ubi) and C12orf2 (magenta) (PDB code: 2cs4). Figures were generated with PyMOL67 and are based on the structure-based sequence alignment as shown in Figure 5. NMR spectroscopy All NMR experiments were performed at 298 K in a 25 mM sodium phosphate buffer (pH 7.2), 150 mM NaCl, 5 mM DTT on Bruker DRX500 and Avance800 spectrometers equipped with a cryogenic probe and single-axis gradients. Protein concentrations were 600 μM for triple resonance experiments, 700 μM for nuclear Overhauser effect spectroscopy (NOESY) experiments, 200 μM for RDC measurements, and 400 μM for hydrogen/deuterium-exchange (HDX) experiments. Backbone assignments were obtained using standard HNCA, HNCO, HN(CA) CO, CBCA(CO)NH, and HN(CO)CA triple-resonance experiments.25,60 Two-dimensional [1H, 1H]-DQF-COSY and [1H, 1H]-TOCSY spectra, three-dimensional [15N, 1 H, 1H]-TOCSY spectra, (H)CC(CO)NH, HBHA(CO)NH, CBCA(CO)NH and HNHB spectra enabled almost complete side-chain assignment with some of the Hβ resonances being stereospecifically assigned. Structural NOEs were obtained from two-dimensional [1H, 1H]NOESY spectra. The mixing times chosen were 54 ms (in 8.7 kHz B1) for TOCSY and 100 ms for NOESY spectra at 800 MHz. H/D-exchange fHSQC61 and long-range HNCO28 experiments were used to assign H-bonds. One-bond 1H-15N RDCs were measured using 8% (w/v) C12E6/hexanol bicelles62 as alignment medium to validate the structure of the backbone with 3 s 1H saturation following a 7 s recycle delay. Peak splittings were measured with {1 H, 15 N} IPAP-HSQC, and 1DNH were obtained by subtracting 1JNH values measured for a nonaligned sample. Backbone dynamics were examined by measuring {1H}15N-heteronuclear NOEs.63 Amide proton-
exchange experiments were performed as described.64 Protection factors for backbone amides were extracted according the formula P = kint/kexobs where kint was obtained from elsewhere.65 Time domain NMR data were processed in NMRPipe66 and Figures were generated using PyMOL.67 Structure calculation Distance restraints were obtained from two-dimensional [1H, 1H]-NOESY spectra with interproton distance restraints being classified into four categories corresponding to 1.8–2.8, 1.8–3.6, 1.8–4.7, and 1.8–6.0 Å, respectively, based on their NOESY peak volumes. Backbone ϕ and ψ angular constraints were determined from 13C chemical shifts using the program TALOS.68 The final ensemble of structures was calculated using the program CNS.69 A total of 60 structures out of 90 were accepted with no distance violation N0.5 Å and no angle violation N 5°. Structure calculation statistics are summarized in Table 1. The final structure was validated against measured RDCs using the program PALES.70 Geometries of all structures, as well as elements of secondary structure, were analyzed using MOLMOL71 and PROCHECK.26 PyMOL67 was used for visualization of the structures. Bioinformatics Sequence database search for identification of ASPP2 homologues was performed using PSI-BLAST).72,73 Multiple sequence alignments were generated with
Solution Structure of ASPP2 N-terminal Domain
956 ClustalW.74 Structure-based sequence alignments were produced as described elsewhere.75 Pairwise structure superimpositions were generated using ProSup.76 16. Protein Data Bank accession codes The data are deposited in the RCSB Protein Data Bank and are available under accession code 2uwq.
17.
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Acknowledgements We thank Drs S. Freund, C. Johnson, D. Veprintsev, A. Joerger, M. Bycroft and T. Religa for valuable discussions and help on the experimental setup, and C. Blair for TEV protease. We also thank Drs R. Williams and O. Perisic for the kind gift of H-Ras protein. H.T. was supported by a fellowship from the Boehringer Ingelheim Fonds.
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Edited by M. F. Summers (Received 24 March 2007; received in revised form 7 May 2007; accepted 7 May 2007) Available online 13 May 2007
