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. 2013 Sep;41(17):8368-76.
doi: 10.1093/nar/gkt584. Epub 2013 Jul 8.

Structure of p53 binding to the BAX response element reveals DNA unwinding and compression to accommodate base-pair insertion

Yongheng Chen  1 , Xiaojun Zhang, Ana Carolina Dantas Machado, Yuan Ding, Zhuchu Chen, Peter Z Qin, Remo Rohs, Lin Chen
Affiliations

Structure of p53 binding to the BAX response element reveals DNA unwinding and compression to accommodate base-pair insertion

Yongheng Chen et al. Nucleic Acids Res. 2013 Sep.

Abstract

The p53 core domain binds to response elements (REs) that contain two continuous half-sites as a cooperative tetramer, but how p53 recognizes discontinuous REs is not well understood. Here we describe the crystal structure of the p53 core domain bound to a naturally occurring RE located at the promoter of the Bcl-2-associated X protein (BAX) gene, which contains a one base-pair insertion between the two half-sites. Surprisingly, p53 forms a tetramer on the BAX-RE that is nearly identical to what has been reported on other REs with a 0-bp spacer. Each p53 dimer of the tetramer binds in register to a half-site and maintains the same protein-DNA interactions as previously observed, and the two dimers retain all the protein-protein contacts without undergoing rotation or translation. To accommodate the additional base pair, the DNA is deformed and partially disordered around the spacer region, resulting in an apparent unwinding and compression, such that the interactions between the dimers are maintained. Furthermore, DNA deformation within the p53-bound BAX-RE is confirmed in solution by site-directed spin labeling measurements. Our results provide a structural insight into the mechanism by which p53 binds to discontinuous sites with one base-pair spacer.

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Figures

Figure 1.
Figure 1.
Structure of the p53 core domain bound to the BAX-RE with the two half-sites separated by a 1-bp spacer. (A) Overall structure of p53 core domain tetramer bound to the BAX-RE. The DNA sequence is shown with the two half-sites highlighted by red rectangles. (B) Structural comparison with p53 core domain tetramer bound to a continuous site (PDB ID 3KMD) shows that the two structures share the same overall architecture. (C) Structural comparison between the complexes BAX and 3KMD shows an almost identical intra-dimer interface between chains A and B. The similarity is also seen at the interface between chains C and D (not shown). (D) Structural comparison between the two complexes also shows an almost identical inter-dimer interface. The similarity is also seen at the interface between chains B and C (not shown). (E) Superimposition of the two complexes reveals that the p53 core domain tetramer binds DNA similarly in the two complexes. Here we use chains A and B as examples. Chains C and D also follow the same pattern (not shown).
Figure 2.
Figure 2.
DNA deformation in the p53/BAX-RE complex. (A) Electron densities of the DNA-binding sites in the p53/BAX-RE complex (left panel) and 3KMD structure (right panel). Both are shown within their respective electron density maps (2Fo − Fc at 1σ level). The central region of the phosphodiester backbones in the p53/BAX-RE complex shows poor and discontinuous electron densities. (B) Comparison of electron densities of base pairs in p53/BAX-RE complex (left panel) and 3KMD structure (right panel). Both are shown within their respective electron density maps (2Fo − Fc at 1σ level). The base pairs in the central region of the DNA in the p53/BAX-RE complex show weak electron density and do not match the canonical Watson–Crick base-pairing geometry.
Figure 3.
Figure 3.
DNA structure analyses. (A) Schematic representation of the DNA conformations in the BAX (green) and 3KMD (magenta) structures. The helix axes indicate a slight bending, while the backbones suggest an increase in helix diameter, due to deformations accommodating the additional base pair in the BAX-RE. (B) The increase in helix diameter in the center of the BAX (green) vs. the 3KMD (magenta) DNA target is illustrated based on a CURVES analysis. (C) The minor groove width comparison of the BAX (green) vs. the 3KMD (magenta) DNA conformation. (D) The difference in helix twist between an increasing number of base pairs centered around the interface of both half-sites of the BAX and 3KMD DNA structures demonstrates that 7 bp in the BAX-RE are accommodated in the same rotational space as 6 bp in the 3KMD structure. This arrangement places the CWWG core elements at a similar relative positioning, allowing for the formation of bidentate hydrogen bonds between the guanines of the CWWG core elements and Arg280 residues of an almost identical p53 core tetramer assembly.
Figure 4.
Figure 4.
Structural comparison of the L1 loop of chain B in the BAX (A) and 3KMD (B) structures. The electron density (2Fo − Fc at 1σ level) of the L1 loop of chain B is well-defined in the 3KMD structure, but is disordered in the BAX structure.
Figure 5.
Figure 5.
Assessment of p53/BAX-RE conformation in solution. (A) The R5 nitroxide probe. In this particular example, the label was designated as A14, as the R5 probe is attached at the phosphorothioate group sandwiched between G13 and A14. Note that following previously validated distance measurement protocols (35), all data reported here were acquired without separating the Rp and Sp phosphorothioate diastereomers present at each attachment site. (B) An example of measured DEER dipolar evolution data (top panel) and the resulting inter-nitroxide distance distribution (bottom panel). The pair of R5s were attached at A14 and C15’ (see Figure 1A). The shaded box indicates the major band in the distance distribution profile, from which the mean distance of 27 Å (r0, marked by the dotted line) and the width of the distance distribution (σ = 3.7 Å) were determined. Additional data sets are reported in Supplementary Figure S7. (C) Allowable R5 ensembles at A14 and C15’ within the p53/BAX-RE core tetramer predicted using NASNOX (see ‘Materials and Methods’ section). The NASNOX search parameters were t1 steps: 3; t2 steps: 6; t3 steps: 6; fine search: on; t1 starting value: 180°; t2 starting value: 180°; t3 starting value 180°. The average distance between these two nitroxide ensembles were found to be 27.5 Å.
Figure 6.
Figure 6.
A model for the p53 core domain tetramer bound to DNA. The model is based on the BAX and 3KMD structures. When individual p53 core domains bind to continuous half-sites, they will form a tetramer and lead to cooperative binding (lower right). When they bind to the discontinuous BAX site, the additional base pair (red), if in B-DNA conformation, will cause separation and rotation between the two p53 dimers, leading to a loss of binding cooperativity (upper right). To maintain the cooperativity, the interactions between the two p53 dimers override the DNA constraints, unwinding and compressing the central region (purple). This effect reestablishes the p53 tetramer (middle right) in almost identical conformation to the tetramer observed on continuous half-sites (lower right).
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