The tumor-suppressor protein p53 is among the most effective of the cell's natural defenses against cancer. In response to cellular stress, p53 binds as a tetramer to diverse DNA targets containing two decameric half-sites, thereby activating the expression of genes involved in cell-cycle arrest or apoptosis. Here we present high-resolution crystal structures of sequence-specific complexes between the core domain of human p53 and different DNA half-sites. In all structures, four p53 molecules self-assemble on two DNA half-sites to form a tetramer that is a dimer of dimers, stabilized by protein-protein and base-stacking interactions. The protein-DNA interface varies as a function of the specific base sequence in correlation with the measured binding affinities of the complexes. The new data establish a structural framework for understanding the mechanisms of specificity, affinity, and cooperativity of DNA binding by p53 and suggest a model for its regulation by regions outside the sequence-specific DNA binding domain.

p53 binds as a tetramer to DNA targets consisting of two decameric half-sites separated by a variable spacer. Here we present high-resolution crystal structures of complexes between p53 core-domain tetramers and DNA targets consisting of contiguous half-sites. In contrast to previously reported p53-DNA complexes that display standard Watson-Crick base pairs, the newly reported structures exhibit non-canonical Hoogsteen base-pairing geometry at the central A/T doublet of each half-site. Structural and computational analyses demonstrate that the Hoogsteen geometry distinctly modulates the B-DNA helix in terms of local shape and electrostatic potential which together with the contiguous DNA configuration results in enhanced protein-DNA and protein-protein interactions compared to non-contiguous half-sites. Our results suggest a mechanism, which relates spacer length to protein-DNA binding affinity. Our findings also expand the current understanding of protein-DNA recognition and establish the structural and chemical properties of Hoogsteen base pairs as the basis for a novel mode of sequence readout.

Runt-domain (RD) proteins are transcription factors that play fundamental roles in various developmental pathways. They bind specifically to DNA sequences of the general form PyGPyGGTPy (Py = pyrimidine), through which they regulate transcription of target genes. The DNA duplex TCTGCGGTC/TGACCGCAG, incorporating the binding site for the RD transcription factors (bold), was crystallized in space group P4(3). X-ray analysis of two crystals diffracting to 1.7 and 2.0 angstroms resolution, which had slight variations in their unit-cell parameters, revealed two distinct conformations of the A-DNA helix. The two crystal structures possessed several structure and hydration features that had previously been observed in A-DNA duplexes. A comparative analysis of the present A-DNA structures and those of previously reported B-DNA crystal structures of RD-binding sites in free and protein-bound states showed the various duplexes to display several common features. Within this series, the present A-DNA duplexes adopt two conformations along the pathway from the canonical A-DNA to the B-DNA forms and the protein-bound helices display conformational features that are intermediate between those of the current A-DNA structures and that of the B-DNA-type helix of the free RD target. Based on these data and energy considerations, it is likely that the propensity of the RD-binding site to adopt the A-DNA or B-DNA conformation in solution depends on the sequence context and environmental conditions, and that the transition from either DNA form to the protein-bound conformation involves a small energy barrier.

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