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. 2013 Oct;41(18):8748-59.
doi: 10.1093/nar/gkt630. Epub 2013 Jul 17.

Structural studies of p53 inactivation by DNA-contact mutations and its rescue by suppressor mutations via alternative protein-DNA interactions

Amir Eldar  1 , Haim Rozenberg, Yael Diskin-Posner, Remo Rohs, Zippora Shakked
Affiliations

Structural studies of p53 inactivation by DNA-contact mutations and its rescue by suppressor mutations via alternative protein-DNA interactions

Amir Eldar et al. Nucleic Acids Res. 2013 Oct.

Abstract

A p53 hot-spot mutation found frequently in human cancer is the replacement of R273 by histidine or cysteine residues resulting in p53 loss of function as a tumor suppressor. These mutants can be reactivated by the incorporation of second-site suppressor mutations. Here, we present high-resolution crystal structures of the p53 core domains of the cancer-related proteins, the rescued proteins and their complexes with DNA. The structures show that inactivation of p53 results from the incapacity of the mutated residues to form stabilizing interactions with the DNA backbone, and that reactivation is achieved through alternative interactions formed by the suppressor mutations. Detailed structural and computational analysis demonstrates that the rescued p53 complexes are not fully restored in terms of DNA structure and its interface with p53. Contrary to our previously studied wild-type (wt) p53-DNA complexes showing non-canonical Hoogsteen A/T base pairs of the DNA helix that lead to local minor-groove narrowing and enhanced electrostatic interactions with p53, the current structures display Watson-Crick base pairs associated with direct or water-mediated hydrogen bonds with p53 at the minor groove. These findings highlight the pivotal role played by R273 residues in supporting the unique geometry of the DNA target and its sequence-specific complex with p53.

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Figures

Figure 1.
Figure 1.
Stereo view of the R273-related mutants superposed on the wt core domain. The color code is wt (PDB ID 2AC0 molecule C) in gray, R273H (form I) in yellow, R273H (form II) in red, R273C in lime, R273H/S240R in orange, R273C/T284R in pink, R273H/T284R in blue, R273C/S240R-DNA in green, R273H/T284R-DNA in cyan and R273C/T284R-DNA in magenta (the color code is maintained throughout the figures).
Figure 2.
Figure 2.
Comparison of the two structural variants of R273H. (A) Stereo view of the structure of R273H (form II) (red) superposed on the structure of R273H (form I) (yellow), showing the conformational changes in the L2 region. Also shown are the primary zinc atoms (Zn1), and the second zinc atom (Zn2) in R273H (form II). This view is different than that of Figure 1 to highlight the changes in L2 and the location of the different Zn atoms. (B) Stereo view of the intermolecular interface formed by symmetry-related molecules in R273H (form II). Zn1 is the physiological zinc atom common to all p53 structures, bound to C176, H179, C238 and C242. Zn2 is the second ion bound to C182 of one molecule (red), to H115 from a symmetry-related molecule (pink) and to the thiol groups of a DTT molecule (red).
Figure 3.
Figure 3.
Interactions between the L2 loop and R175. (A) wt core-domain structure bound to DNA (PDB ID 2AC0). R175 forms a bidentate salt bridge with D184 supported by hydrogen bonds with C182 and R196. (B) The structure of the same region in R273H (form I) is similar to that of the wt protein. (C) The structure of the same region in R273H (form II) showing alternative supporting interactions between R175 and L2 loop formed by water-mediated hydrogen-bonded network. The second zinc is shown (yellow sphere).
Figure 4.
Figure 4.
Type I and type II complexes of the rescued proteins. (A) Type I complex shown by R273C/T284R-DNA. Here, the two DNA half sites (gray) are separated by two base pairs, and the two dimers (shown in cyan and green) are rotated relative to each other. (B) Type II complex is shown by both R273H/T284R-DNA and R273C/S240R-DNA structures. The figure is based on the structure of the former complex. Here, the two half sites are contiguous, and the two dimers are parallel to each other.
Figure 5.
Figure 5.
Close-up views of the mutation sites in the rescued p53 proteins bound to DNA, compared with the wt p53-DNA complex. (A) Wild-type p53-DNA interface showing the interaction site of R273 (PDB ID 2AC0). (B–D) The corresponding sites of the rescued proteins, indicating that both R273H and R273C side chains are too short to interact with the DNA backbone. The corresponding distances to the DNA are shown by gray dashed lines. Alternative hydrogen bonds to the DNA backbone are formed by the second-site suppressor mutations, T284R and S240R, shown by red dashed lines. Black labels denote wt residues. Red and blue labels denote primary and suppressor mutations, respectively.
Figure 6.
Figure 6.
The effect of base-pairing geometry on DNA shape in type II complexes. (A) Stereo view of the A/T dinucleotide pairs at the center of the DNA half-site, showing the local backbone shift and minor-groove narrowing caused by Hoogsteen base pairs in the wt complex (gray, PDB ID 3IGL) relative to Watson–Crick base pairs of the rescued complex R273H/T284R-DNA (cyan). (B) Comparison of three DNA helices bound to the wt and rescued p53. The color code is gray for wt-DNA (PDB ID 3IGL), cyan for R273H/T284R-DNA and green for R273C/S240R-DNA. Also shown is a superposition of the three helices. The continuous 20 bp long helices were obtained by modeling the missing phosphate groups in the full-length binding site (see scheme B in Supplementary Figure S3). The overall shape of the three DNA helices is similar except for the constriction shown by the wt DNA at the center of each half site, caused by the Hoogsteen base-pairing geometry (highlighted in red and indicated by arrows). (C) Close-up stereo view of the interaction modes of R273, T284R and S240R with their DNA-binding sites, showing the shift in the DNA backbone of the wt complex (gray) relative to those of the rescued complexes (cyan and green). The red arrow points to the position of the oxygen atom interacting with R273. Only one interaction site is shown for each complex. The other three sites are equivalent by symmetry.
Figure 7.
Figure 7.
DNA helix parameters and electrostatic potential in type II complexes. (A) Helix diameter as a function of the base sequence showing the compression of the DNA helix at the center of each half-site. A larger effect is displayed by the DNA helix of the wt complex (shown in gray) as a result of the Hoogsteen geometry of the A/T base pairs at each half-site. (B) Minor-groove width and the corresponding electrostatic potential as a function of the base sequence. The four minor-groove minima are aligned with the electrostatic potential minima. The positions of the four R248 residues are indicated by arrows.
Figure 8.
Figure 8.
The A/T dinucleotide pairs at the center of the DNA half-site and their interactions with the wt and rescued p53. (A) In type I complex of wt-DNA (PDB ID 2AC0), the A/T base pairs display the Watson–Crick geometry. R273 side chains interact with the DNA phosphate groups (also shown in Figure 5A). The R248 residues interact occasionally with the backbone phosphates (shown here) or via water molecules (not shown). (B and C) In type I complex of R273C/T284R-DNA, the two dinucleotide pairs display the Watson–Crick geometry. T284R side chains interact with the backbone phosphate (also shown in Figure 5B). The R248 side chains at the minor groove are occasionally disordered and form water-mediated hydrogen bonds with the N3 atoms of the adenine bases. (D) In type II complex of wt-DNA, the A/T base pairs display the Hoogsteen geometry. R273 side chains interact with the phosphate groups. R248 residues do not interact directly with DNA, but rather with the minor-groove hydration shell described previously (16). Only one dinucleotide pair is shown for type II complexes, as the other pair is related by crystal symmetry. (E) In type II complex of R273H/T284R-DNA, the A/T base pairs display the Watson–Crick geometry. T284R side chains interact with the backbone phosphate (also shown in Figure 5C). The R248 side chains form direct interactions with the N3 atoms of the adenine bases. (F) In type II complex of R273C/S240R-DNA, the A/T base pairs display the Watson–Crick geometry. S240R side chains interact with the backbone phosphate (also shown in Figure 5D). The R248 side chains form water-mediated interactions with the N3 atoms of the adenine bases.
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