Interaction of the p53 DNA-Binding Domain with Its N-Terminal Extension Modulates the Stability of the p53 Tetramer

Natan, Eviatar; Baloğlu, Çetin; Pagel, Kevin; Freund, Stefan; Morgner, Nina; Robinson, Carol V.; Fersht, Alan R.; Joerger, A.C.

doi:10.1016/j.jmb.2011.03.047

Cited by 88 publications

(100 citation statements)

References 53 publications

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“…The boundaries of the p53 core domain were recently questioned by the finding that cation-π interaction of R174 and W91 stabilizes the domain (27). Methyl NMR spectra of the N-terminally extended core domain (89-296) spectra are significantly different from the shorter 94-296 construct (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…S2A) indicate that chemical exchange observed in the extended core domain is caused by an internal slow-conformational dynamics within the domain. The recent X-ray studies of an extended p53C domain (27) and the magnitudes of observed chemical shift perturbations suggest that the second set of NMR signals observed in the methyl NMR spectra results from breaking of the R174-W91 cation-π interaction, which allows the "opening" of the p53C-PRR hinge and increases the solvent exposure of W91 residue. This mechanism is corroborated by methyl NMR spectrum of a p53C (89-296) W91A mutant, which exhibits single set of cross-peaks resembling the "minor" conformation of the native p53C (89-296) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Proteins were expressed as His-lipoyl domain fusions [full-length p53, p53C (94-296) and (89-296), TET L348A (325-355), p53 TAD-PRR-C (1-312), p53C-TET (96-355), and alanine point mutants of the proteins] were purified by combination of Ni 2þ -, heparin-affinity, and gel-filtration chromatography (6,22,27,36). Briefly: Cell lysis was performed using high-pressure homogenizer in a lysis buffer containing 25 mM K-phosphate, 300 mM NaCl, 10 mM imidazole, 5 mM β-mercaptoethanol, pH ¼ 8.0.…”

Section: Methodsmentioning

confidence: 99%

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Domain–domain interactions in full-length p53 and a specific DNA complex probed by methyl NMR spectroscopy

Bista

Freund

Fersht

2012

Proc. Natl. Acad. Sci. U.S.A.

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The tumor suppressor p53 is a homotetramer of 4 × 393 residues. Its core domain and tetramerization domain are linked and flanked by intrinsically disordered sequences, which hinder its full structural characterization. There is an outstanding problem of the state of the tetramerization domain. Structural studies on the isolated tetramerization domain show it is in a folded tetrameric conformation, but there are conflicting models from electron microscopy of the full-length protein, one of which proposes that the domain is not tetramerically folded and the tetrameric protein is stabilized by interactions between the N and C termini. Here, we present methyl-transverse relaxation optimized NMR spectroscopy (methyl-TROSY) investigations on the full-length and separate domains of the protein with its methionine residues enriched with 13 C to probe its quaternary structure. We obtained high-quality spectra of both the full-length tetrameric p53 and its DNA complex, observing the environment at 11 specific methyl sites. The tetramerization domain was as tetramerically folded in the full-length constructs as in the isolated domain. The N and C termini were intrinsically disordered in both the full-length protein and its complex with a 20-residue specific DNA sequence. Additionally, we detected in the interface of the core (DNA-binding) and N-terminal parts of the protein a slow conformational exchange process that was modulated by specific recognition of DNA, indicating allosteric processes.transcription factor | intrinsically disordered protein I ntrinsically disordered domains are crucial, functional components of many proteins, especially those involved in cell signaling and regulation of the cell cycle (1, 2), p53 being an archetypical example of such a protein. p53 is a homotetramer of 4 × 393 residues ( Fig. S1): Residues 1-93 form the intrinsically disordered N-terminal domain (TAD), with a proline-rich region (PRR) 61-93; 94-292 form the folded core domain (p53C); 293-324 form a disordered linker; 325-353 associate to form the folded tetramerization domain (TET); and 354-393 are intrinsically disordered (CT). The large size of this protein and presence of disordered regions have so far prevented the full-length p53 from being crystallized or its high-resolution structure solved by NMR. The structures of isolated p53 domains have been extensively studied by high-resolution methods (for an overview see ref. 3; Fig. S1); however, their arrangement in the full-length protein has been a subject of controversy, and alternative models of quaternary structural arrangement have been postulated. One model of the human protein obtained from electron microscopy and small-angle X-ray scattering (SAXS) is consistent with structural studies on the isolated TET domain whereby the full-length tetramer and truncated constructs associate via the TET domain (4-6). But in a model of the murine protein, it is proposed that oligomerization occurs via contacts between N and C termini of the protein, as well as the core domains, and th...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Domain–domain interactions in full-length p53 and a specific DNA complex probed by methyl NMR spectroscopy

Bista

Freund

Fersht

2012

Proc. Natl. Acad. Sci. U.S.A.

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“…The interaction between Trp91 and Arg174 in the p53 DNA-binding domain increases the melting temperature by several degrees and decreases the rate of aggregation compared with constructs that begin just a few residues away, such as at Ser94 (Natan et al 2011). In the DNA-binding domain structure, this interaction may hold the N-terminal region in an orientation that counteracts the exposure of the aggregation-prone region (Fig.…”

Section: Mechanisms Underlying the Prion-like Aggregation Of Tumor Sumentioning

confidence: 99%

Aggregation and Prion-Like Properties of Misfolded Tumor Suppressors: Is Cancer a Prion Disease?

Costa

Oliveira

Cino

et al. 2016

Cold Spring Harb Perspect Biol

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Prion diseases are disorders that share several characteristics that are typical of many neurodegenerative diseases. Recently, several studies have extended the prion concept to pathological aggregation in malignant tumors involving misfolded p53, a tumor-suppressor protein. The aggregation of p53 and its coaggregation with p53 family members, p63 and p73, have been shown. Certain p53 mutants exert a dominant-negative regulatory effect on wild-type (WT) p53. The basis for this dominant-negative effect is that amyloid-like mutant p53 converts WT p53 into an aggregated species, leading to a gain-of-function (GoF) phenotype and the loss of its tumor-suppressor function. Recently, it was shown that p53 aggregates can be internalized by cells and can coaggregate with endogenous p53, corroborating the prion-like properties of p53 aggregates. The prion-like behavior of oncogenic p53 mutants provides an explanation for its dominant-negative and GoF properties, including the high metastatic potential of cancer cells carrying p53 mutations. The inhibition of p53 aggregation appears to represent a promising target for therapeutic intervention in patients with malignant tumors.

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“…Although the behavior of individual domains may be modified by domain interactions in vivo, the properties of major domains within multidomain proteins frequently have a strong impact on the full-length protein. For example, the stability and levels of folded p53 in vivo are dictated by the p53CD (43,44) with additional modulation by domain interactions and phosphorylation (45,46). In this work, we present the first report on the conformational stability and folding of the RbAB pocket domain, which presents the main interaction sites with both cellular and viral proteins and is the site of most tumorigenic point mutations.…”

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confidence: 88%

Folding of a Cyclin Box

Chemes

Noval

Sánchez

et al. 2013

Journal of Biological Chemistry

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Background: Conformational stability and folding of retinoblastoma tumor suppressor are unknown to date. Results: The metastable RbAB domain undergoes oligomerization and aggregation, which are suppressed by natural ligands. Conclusion: RbAB requires binding to ligands or is prone to inactivation. Significance: Despite a different sequence and fold, Rb shares tumor suppression and multiligand binding with p53 where marginal stability and oligomerization may lead to inactivation.

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Interaction of the p53 DNA-Binding Domain with Its N-Terminal Extension Modulates the Stability of the p53 Tetramer

Cited by 88 publications

References 53 publications

Domain–domain interactions in full-length p53 and a specific DNA complex probed by methyl NMR spectroscopy

Domain–domain interactions in full-length p53 and a specific DNA complex probed by methyl NMR spectroscopy

Aggregation and Prion-Like Properties of Misfolded Tumor Suppressors: Is Cancer a Prion Disease?

Folding of a Cyclin Box

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