Journal of Molecular Biology
Volume 341, Issue 5, 27 August 2004, Pages 1145-1159
Journal home page for Journal of Molecular Biology

Cooperative Binding of Tetrameric p53 to DNA

https://doi.org/10.1016/j.jmb.2004.06.071Get rights and content

We analysed by analytical ultracentrifugation and fluorescence anisotropy the binding of p53 truncation mutants to sequence-specific DNA. The synthetic 30 base-pair DNA oligomers contained the 20 base-pair recognition elements for p53, consisting of four sites of five base-pairs per p53 monomer. We found that the binding at low ionic strengths was obscured by artifacts of non-specific binding and so made measurements at higher ionic strengths. Analytical ultracentrifugation of the construct p53CT (residues 94–360, containing the DNA-binding core and tetramerization domains) gave a dissociation constant of ∼3 μM for its dimer–tetramer equilibrium, similar to that of full-length protein. Analytical ultracentrifugation and fluorescence anisotropy showed that p53CT formed a complex with the DNA constructs with 2 : 1 stoichiometry (dimer:DNA). The binding of p53CT (1–100 nm range) to DNA was highly cooperative, with a Hill coefficient of 1.8 (dimer:DNA). The dimeric L344A mutant of p53CT has impaired tetramerization. It bound to full-length DNA p53 recognition sequence, but with sixfold less affinity than wild-type protein. It did not form a detectable complex with a 30-mer DNA construct containing two specific five base-pair sites and two random sites, emphasizing the high co-operativity of the binding. The fundamental active unit of p53 appears to be the tetramer, which is induced by DNA binding, although it is a dimer at low concentrations.

Introduction

The p53 tumour suppressor gene is the most frequently mutated gene in cancer known to date, with p53 mutations occurring in over half of all human tumours.1., 2. The vast majority of these mutations occur in the sequence-specific DNA-binding domain, underscoring the importance of DNA binding in the ability of p53 to maintain genomic stability.3 When cells are exposed to a variety of stresses such as UV light, hypoxia, DNA damage, and heat shock, p53 binds to a consensus sequence present in a variety of genes, which leads to p53-mediated transcriptional activation. Ultimately, cells then undergo cell cycle arrest, DNA repair, or apoptosis.4., 5.

Wild-type p53 is a sequence-specific transactivator,6 which binds to a double-stranded DNA consensus site containing two copies of the “half-site” decameric motif RRRC(A/T)|(T/A)GYYY separated by up to 13 base-pairs (bp).7., 8. In this sequence, R and Y represent purines and pyrimidines, respectively, and the vertical bar indicates the centre of symmetry within the half-site. The full-length consensus sequence contains two half-sites, which can alternatively be viewed as four inverted 5 bp quarter sites (→← →←). The crystal structure of p53 bound to DNA showed that the central p53 core binding domain can bind to each quarter site.9 Studies later verified that four molecules of p53 core domain bind to the full-length recognition element,10., 11. and that dimers of p53 bind to adjacent 10 bp half sites, and not alternating quarter sites.12

Even though the isolated p53 core domain can bind DNA in a sequence-specific manner, it is thought that tetramerization of p53 is a requirement for formation of a stable p53–DNA complex and subsequent transactivation of target genes.12., 13., 14. The oligomerization domain of p53 spans residues 320–360 and forms a tetramer composed of a dimer of dimers. Each dimeric subunit contains an antiparallel two-strand beta-sheet and two alpha-helices. The tetramer forms through hydrophobic packing between the helices, forming a four-helix bundle.15., 16., 17., 29. Mutation of this hydrophobic core has been shown to inhibit tetramerization.18 Stability studies on the tetramerization domain show that there is a transition between four monomers to the native tetramer,19., 20. while folding studies on the tetramerization domain uncovered the existence of a dimeric intermediate.21 This, along with in vitro transcription/translation experiments suggests that the formation of the dimeric subunit is faster than tetramer formation, and that tetramerization is concentration-dependent.13 These data raise the question as to whether the functional unit of p53 in DNA binding is the monomer, dimer, or tetramer and how this affects DNA binding.

The formation of a tetrameric p53–DNA complex is critical for the ability of p53 to function as a tumour suppressor. While individual core domains are able to bind to half-site recognition elements, it has been shown that this mode of binding is insufficient to activate transcription of p53 target genes.22 Studies have shown that the binding of monomeric p53 core domain to its full-length binding sequence is highly cooperative and is associated with the bending of DNA,11 which is believed to facilitate the architectural accommodation of four p53 molecules to a recognition element.23 Bending of the p53 DNA recognition element has also been shown to play a role in modulating p53 affinity to its target sequences and in stabilizing the nucleoprotein complex.24., 25., 26.

Here, we investigated the DNA binding of oligomeric p53 to specific DNA using fluorescence anisotropy and analytical ultracentrifugation (AUC). We demonstrate that the binding of oligomeric p53 to DNA in solution does have 4 : 1 stoichiometry. This interaction is highly cooperative, and relies on the recognition of DNA sequences by the core domain and on the tetramerization of the protein, which is heavily mediated by DNA. We also show that only 4 : 1 complexes of p53 to DNA are stable, suggesting that protein–protein interactions27., 28. and intrinsic properties of the full-length p53 DNA binding element, such as DNA bending, are critical in the formation of a biologically active tetrameric p53–DNA complex.

Section snippets

p53CT WT is in equilibrium between dimers and tetramers while p53CT L344A is only a dimer in solution

In order to analyse the DNA binding of p53, it was first necessary to determine the oligomerization states of p53CT and its L344A mutant in solution. p53CT (residues 94 to 360) is a truncation mutant of p53 containing the sequence-specific core DNA binding and oligomerization domains. Using sedimentation equilibrium AUC, we showed that p53CT exists as dimers and tetramers in solution while the L344A mutant only forms dimers under comparable concentrations. The data for p53CT was first fit to a

Specific binding of p53 to DNA is highly cooperative

We have demonstrated that p53CT, a truncation mutant containing the core DNA binding and oligomerization domains (residues 94 to 360), binds DNA in a highly cooperative manner. Wild-type p53CT is in equilibrium between its dimeric and tetrameric forms, with a dimer–tetramer equilibrium constant (KD–T) of 3.2(±1.4) μM. Thus, at sub-micromolar concentrations of p53, which are sufficient for high affinity specific DNA binding, the wild-type protein exists predominantly as a dimer, making it the

Conclusions

Using fluorescence anisotropy and analytical ultracentrifugation, we were able to show that specific p53 binding to DNA is a highly cooperative and specific interaction. p53 containing the oligomerization domain is in equilibrium between dimers and tetramers in solution, but the presence of a specific p53 DNA binding sequence dramatically increases the tetramerization of the protein. Tetrameric p53 binds specific DNA with nM affinity and has a Hill coefficient of 1.8, indicative of a highly

Cloning

The gene encoding for amino acid residues 94–360 of human p53 was amplified from pT7hp5341 using the primers 5′-CGCGGATCCTCATCTTCTGTCCCTTCCCAG AAAACCTACCAGGGC-3′ and 5′CCGGAATTCACCCTGGCTCCTTCCCAGCCTGGGC ATCCTTGAGTTCCA-3′. The gene encoding for amino acid residues 94–312 of human p53 was amplified using the primers 5′-CGCGGATCCTCATCTTCTGTCCCTTCCCAGAAAACCTACCAGGGC-3′ and 5′-CCGGAATTCAGGTGTTGTTGGGCAGTGCTCG-3′. The PCR products were digested with BamHI and HindIII, purified and inserted into a

Acknowledgements

R.L.W. is supported by a Howard Hughes Medical Institute Predoctoral Fellowship. We thank Mark Bycroft for his helpful discussions.

References (46)

  • B. Miroux et al.

    Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels

    J. Mol. Biol.

    (1996)
  • M. Hollstein et al.

    Database of p53 gene somatic mutations in human tumors and cell lines

    Nucl. Acids Res.

    (1994)
  • M. Hollenstein et al.

    Somatic point mutaions in the p53 gene of human tumors and cell lines: updated compilation

    Nucl. Acids Res.

    (1996)
  • J.A. Pietenpol et al.

    Sequence-specific transcriptional activation is essential for growth suppression by p53

    Proc. Natl Acad. Sci. USA

    (1994)
  • K.H. Vousden et al.

    Live or let die: the cell's response to p53

    Nature Rev. Cancer

    (2002)
  • S. Fields et al.

    Presence of a potent transcription activating sequence in the p53 protein

    Science

    (1990)
  • W.S. El-Deiry et al.

    Definition of a consensus binding site for p53

    Nature Genet.

    (1992)
  • W.D. Funk et al.

    A transcriptionally active DNA-binding site for human p53 protein complexes

    Mol. Cell. Biol.

    (1992)
  • Y. Cho et al.

    Crystal structure of a p53 tumor suppressor–DNA complex: understanding tumorigenic mutations

    Science

    (1994)
  • Y. Wang et al.

    Interaction of p53 with Its consensus DNA-binding site

    Mol. Cell. Biol.

    (1995)
  • P. Balagurumoorthy et al.

    Four p53 DNA-binding domain peptides bind natural p53-response elements and bend the DNA

    Proc. Natl Acad. Sci. USA

    (1995)
  • K.G. McLure et al.

    How p53 binds DNA as a tetramer

    EMBO J.

    (1998)
  • K.G. McLure et al.

    p53 DNA binding can be modulated by factors that alter the conformational equilibrium

    EMBO J.

    (1999)
  • Cited by (200)

    • The potential roles of p53 signaling reactivation in pancreatic cancer therapy

      2022, Biochimica et Biophysica Acta - Reviews on Cancer
    • Mechanistic roles of mutant p53 governing lipid metabolism

      2022, Advances in Biological Regulation
    View all citing articles on Scopus
    View full text