The C-terminal domain of p53 influences target DNA binding and complex stability by altering p53 str

May 2015

The transcription factor p53 is a structurally complex protein at the hub of many signaling pathways that regulate the cell cycle and maintain the integrity of the genome. The cell fate decisions of p53 range from blocking proliferation to inducing cell-cycle arrest to differentiation or apoptosis. The role of p53 in cell fate control is underscored by the fact that an estimated 50% of malignancies harbor a mutation within the p53 locus. Because of this ability to control life and death, p53 has been dubbed ‘the guardian of the genome’ and has been studied extensively; however its complex structure has been limiting.

p53 functions primarily as a transcription factor and tumor suppressor. It is biologically active as a homotetramer and consists of 4 domains: the N-terminal transactivation domain (NTD), a DNA-binding Domain (DBD), an oligomerization domain (OD), and the C-terminal domain (CTD). While the DBD and OD consist of a modular domain structure, the NTD and CTD are intrinsically disordered regions. Structure and function have been mostly elucidated for the DBD and OD, however the disordered chameleon nature of the C-terminus has made the structure and function more difficult to pinpoint.

In a recent paper published in Molecular Cell, Laptenko and colleagues seek to unravel the role of the CTD on DNA binding specificity. The p53 tetramer cooperatively binds to its target duplex DNA in a sequence-specific manner. The target binding motif consists of two decameric motifs (half-sites) of the general form RRRCWWGYYY separated by 0–13 base pairs (R = A, G; W = A, T; Y = C, T). In this paper, they employ a number of techniques and compare the properties of wild-type (wt) p53 to those containing various mutations in the CTD.  Among these were p53 that lacked the final 30 amino acids of the CTD and one that had six CTD lysines converted to glutamine.  This mimics the acetylation that is associated with DNA damage.

Through a series of experiments including ChIP-on-Chip and SELEX, the authors found that when compared to wt p53, the C-terminal mutants bound to fewer target sequences. Each appeared to have a particular subset of binding partners that were sequence dependent. This suggests that the C-terminal domain functions to modulate the ability of p53 to bind target DNA and regulate specific genes. 

The authors then tested whether the CTD had an effect on the relative affinity between the p53 protein and specific target DNA sequences. The sequences represented three types of bona fide p53 binding sites with high, moderate and weak affinities. DNase I and exonuclease III footprinting indicated that while all the mutants maintained relatively strong interactions with the high affinity target, most significantly affected the binding of the p53 tetramer to the moderate and low affinity sequences. Furthermore, they utilized a crosslinking experiment using 4-Thio-2-deoxyuridine-modified oligos from TriLink to determine that the CTD affects the complex stability through protein-DNA interactions.

Finally, the authors postulate that the mechanism for stabilization could arise from either non-specific p53-DNA interactions or that the CTD induces a conformational change of the p53 tetramer upon binding to a target site, i.e. induced fit model. Evidence for the latter was provided through a series of biochemical assays. As a final note, the authors postulate that damage-induced modifications function as a type of filter, limiting the interaction of p53 and specific targets and thereby directing the scope of p53-mediated cellular pathways.

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