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

Eldar A, Rozenberg H, Diskin-Posner Y, Rohs R, Shakked Z - Nucleic Acids Res. (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel and Molecular and Computational Biology Program, University of Southern California, Los Angeles, CA 90089, USA.

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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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).
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gkt630-F2: 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).

Mentions: Significant variability, however, is observed in flexible regions such as the DNA-binding loop L1, which exhibits different conformations between the free and DNA-bound p53 as shown previously (12–17). A new conformational variant is observed here in the L2 loop of one of the two R273H crystal structures, referred to as R273H (form II) in Tables 1 and 3. The major changes are shown by the backbone conformation of residues 182–187 next to the H1 helix (Figure 2A). In addition to the primary Zn atom (Zn1), which is common to all core-domain structures, this structural variant contains a second Zn atom (Zn2), linking two adjacent molecules related by crystal symmetry. Whereas the first Zn atom is of functional importance, as it supports the core-domain integrity and dimerization on binding to DNA (13), the second Zn atom observed in R273H (form II) is coordinated to C182 of one molecule, H115 of the symmetry-related molecule and the thiol groups of a trapped DTT (dithiothreitol) molecule (Figure 2B). The interface between the two molecules is further stabilized by the side chain of H178 from one molecule positioned in a pocket created by three amino acids (H115, Y126 and P128) from the second molecule (shown in Figure 2B) and hence interferes with DNA binding. In this manner, a continuous chain of p53 molecules linked by zinc atoms is formed in the crystal via the crystallographic 61 axis (Supplementary Figure S2).Figure 2.


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

Eldar A, Rozenberg H, Diskin-Posner Y, Rohs R, Shakked Z - Nucleic Acids Res. (2013)

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).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3794590&req=5

gkt630-F2: 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).
Mentions: Significant variability, however, is observed in flexible regions such as the DNA-binding loop L1, which exhibits different conformations between the free and DNA-bound p53 as shown previously (12–17). A new conformational variant is observed here in the L2 loop of one of the two R273H crystal structures, referred to as R273H (form II) in Tables 1 and 3. The major changes are shown by the backbone conformation of residues 182–187 next to the H1 helix (Figure 2A). In addition to the primary Zn atom (Zn1), which is common to all core-domain structures, this structural variant contains a second Zn atom (Zn2), linking two adjacent molecules related by crystal symmetry. Whereas the first Zn atom is of functional importance, as it supports the core-domain integrity and dimerization on binding to DNA (13), the second Zn atom observed in R273H (form II) is coordinated to C182 of one molecule, H115 of the symmetry-related molecule and the thiol groups of a trapped DTT (dithiothreitol) molecule (Figure 2B). The interface between the two molecules is further stabilized by the side chain of H178 from one molecule positioned in a pocket created by three amino acids (H115, Y126 and P128) from the second molecule (shown in Figure 2B) and hence interferes with DNA binding. In this manner, a continuous chain of p53 molecules linked by zinc atoms is formed in the crystal via the crystallographic 61 axis (Supplementary Figure S2).Figure 2.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel and Molecular and Computational Biology Program, University of Southern California, Los Angeles, CA 90089, USA.

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.

Show MeSH
Related in: MedlinePlus