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Structure of the p53 Tumor Suppressor Bound to the Ankyrin and SH3 Domains of 53BP2

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Science  08 Nov 1996:
Vol. 274, Issue 5289, pp. 1001-1005
DOI: 10.1126/science.274.5289.1001

Figures

  • Fig. 1.

    Electron density at the p53-SH3 domain interface, contoured at 1.2σ (25). The (2|Fobs| − |Fcalc|) Fourier synthesis was calculated at 2.2 Å resolution using phases calculated after omitting the interface residues shown, and subjecting the model to simulated annealing refinement from 3000 K. Met243 and Arg248 of p53 and Trp498 of 53BP2 are labeled.

  • Fig. 2.

    Comparison of the p53-53BP2 and p53-DNA (15) complexes in two orthogonal views (rotated by 90° about the x-axis). The six most frequently mutated amino acids of p53, highlighted in yellow, are at or near both the 53BP2 and DNA interfaces. (A and B) The 53BP2 SH3 domain (red) binds the L3 loop, while the fourth ankyrin repeat (magenta) binds the L2 loop of the p53 core domain (cyan). The zinc atom of p53 is shown as a green sphere. (C and D) Comparison with the p53-DNA (blue) complex (15) in the same p53 orientations as (A and B). Abbreviations for the amino acid residues are: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr.

  • Fig. 3.

    The 53BP2 SH3 domain uses its peptide binding groove, characterized in SH3-PXXP peptide complexes, to bind the rigid L3 loop of p53. (A) Comparison of the 53BP2 SH3-p53 interface with that of the c-Crk SH3 bound to a PXXP peptide derived from the C3G protein (17). 53BP2 is in red; p53 in blue; C-crk in green; the C3G PXXP peptide in yellow; and the some of the side chains discussed in the text are colored in brighter tones. (B) Side chains and backbone groups of the L3 loop of p53 (blue) make multiple van der Waals and hydrogen bond contacts (yellow dotted lines) to the peptide binding groove of the 53BP2 SH3 domain (red). Only interacting side chains are shown; orientation is similar to that of Fig. 3A.

  • Fig. 4.

    An ankyrin repeat forms an L-shaped structure that consists of a β hairpin and two α helices. (A) Topological diagram of the secondary structure elements of the 53BP2 ankyrin repeats. Circles indicate the α helices with their helix axes perpendicular to the plane of the figure. Residue numbers at the start and the end of each secondary structure element are indicated. For the third repeat, solid arrows indicate backbone hydrogen bonds, whereas dashed arrows indicate side-chain hydrogen bonds. A conserved histidine on the α helix that makes a pair of hydrogen bonds to the β-hairpin backbone of the next repeat, contributing to inter-repeat stabilization, is indicated for the second repeat (His365). The first repeat does not have a β hairpin, in part because there is no previous repeat to stabilize it, and the last repeat has an extended helix which is packing against the SH3 domain. (B) The four ankyrin repeats of 53BP2 are aligned according to their structure. Arrows and rectangles indicate the approximate positions of the β strands and α helices, respectively. Their exact positions are indicated by underlining. Residues conserved in two, or more, repeats are highlighted in yellow. (C) p53's L2 loop (blue), which is interrupted by the short H1 helix, and 53BP2's fourth ankyrin repeat (purple) interact through van der Waals contacts, a backbone-backbone hydrogen, and two backbone-side chain hydrogen bonds (yellow dotted lines). Interacting residues are labeled.

  • Fig. 5.

    The six p53 mutants most frequently observed in tumors fail to bind to 53BP2 as determined by the native gel mobility shift assay. Binding reactions contained 30 μM 53BP2 protein and 1.5 times molar excess of either the wild type or the indicated mutants of the p53 core domain. The binding was performed in 300 mM NaCl at 37°C for 5 min., except for V143A binding at the permissive temperature, which was done at 25°C. Free and p53 bound 53BP2 were separated on a 4.5% polyacrylamide gel in a buffer of 89 mM tris-borate, pH 8.4, and were visualized with Coomassie staining. The p53 core domain does not enter the gel, presumably because of its net positive charge. The mutant p53 core domains were constructed by PCR mutagenesis and were sequenced.

Tables

  • Table 1.

    Statistics from the crystallographic analysis.

    Data collection
    Data setNative-1Native-2HgPb
    Resolution (Å)2.52.22.83.0
    Observations1150052206543366016576
    Unique reflections1742330230140749717
    Data coverage (%)82.399.191.678.2
    Rsym* (%)3.75.76.27.9
    MIR analysis (20.0 to 3.2 Å)
    Isomorphous difference0.200.21
    Phasing power2.20.9
    Refinement statistics
    Atoms
    ResolutionReflections§ProteinWaterRRfree
    7.0-2.2 Å2543530662750.2050.286
    rmsd#Bond lengths: 0.013 ÅBond angles: 1.64°B factors: 3.17 Å2
    • * Rsym = ΣhΣi|Ih,i − Ih|/ΣhΣiIh,i for the intensity (I) of i observations of reflection h.

    • † Mean isomorphous difference = Σ|FPHFPFPH, where FPH and FP are the derivative and native structurefactors, respectively.

    • ‡ Phasing power = [FHc2/(FPHoFPHc)2]1/2.

    • § Reflections with |F| > 2σ.

    • R factor = Σ|FoFc|/Σ|Fo|, where Fo and Fc are the observed and calculated structure factors, respectively.

    • Rfree calculated with 10 percent of the data chosen randomly and omitted from simulated annealing refinement from 3000 K.

    • # Root-mean-square deviations from ideal geometry, and root-mean-square variation in the B factors of bonded atoms.