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Structural basis for ASPP2 recognition by the tumor suppressor p73.

Canning P, von Delft F, Bullock AN - J. Mol. Biol. (2012)

Bottom Line: The loop in p73 is changed by a two-residue insertion that also induces repacking around the site of the p53 mutational hotspot R175.Importantly, the binding of ASPP2 is preserved by conformational changes in both the ankyrin repeat and SH3 domains.These results further highlight the structural variation that impacts p53 family interactions within the p53 interactome.

View Article: PubMed Central - PubMed

Affiliation: Structural Genomics Consortium, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, UK.

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Structural overview of the p73–ASPP2 complex. Stereo view of the p73–ASPP2 complex (green/blue) superimposed on the equivalent p53 structure (gray). Arrows highlight a subtle shift in the ASPP2 position. The single zinc ion associated with p73 is shown as a green sphere.
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f0035: Structural overview of the p73–ASPP2 complex. Stereo view of the p73–ASPP2 complex (green/blue) superimposed on the equivalent p53 structure (gray). Arrows highlight a subtle shift in the ASPP2 position. The single zinc ion associated with p73 is shown as a green sphere.

Mentions: The H1 helix and surrounding L2 loop do not participate directly in DNA interaction but contribute instead to dimer contacts in the DNA-bound tetramer (Supplementary Fig. S4a), as well as other protein–protein interactions (Supplementary Fig. S4b). Superposition of the free p73 structure with the p53–ASPP2 complex suggests a potential steric clash from the p73 H1 helix (Supplementary Fig. S4b). Binding of ASPP2 to p53 and p73 DBD was confirmed in the low micromolar range by native-gel mobility shift assay (Fig. 5). Interestingly, the binding of p73 appeared slightly less stable than that of the p53 complex. To investigate the p73 interaction further, we tested a variety of expression constructs in co-crystallization. Due to dissociation of p73–ASPP2 complexes during preparative gel filtration (Supplementary Fig. S5), the two proteins were purified separately and then mixed at a 1:1 molar ratio prior to crystallization. Crystals of the complex were obtained in space group I121 when combining the p73 DBD with the ankyrin repeat and SH3 domains (residues 891–1128) of human ASPP2. The structure of the complex (Fig. 6) was solved using molecular replacement and refined at a resolution of 2.6 Å (see Table 1 for data collection and refinement statistics). The six p73 chains in the asymmetric unit were traceable between residues 114 and 311. Bound ASPP2 chains were traceable between residues 920 and 1121 and showed minor structural deviations in the SH3 RT and n-Src loops, as well as the protein termini. A full description of the crystallized ASPP2 fold (also known as 53BP2) has been reported previously and comprises four ankyrin repeats and a C-terminal SH3 domain.25


Structural basis for ASPP2 recognition by the tumor suppressor p73.

Canning P, von Delft F, Bullock AN - J. Mol. Biol. (2012)

Structural overview of the p73–ASPP2 complex. Stereo view of the p73–ASPP2 complex (green/blue) superimposed on the equivalent p53 structure (gray). Arrows highlight a subtle shift in the ASPP2 position. The single zinc ion associated with p73 is shown as a green sphere.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472557&req=5

f0035: Structural overview of the p73–ASPP2 complex. Stereo view of the p73–ASPP2 complex (green/blue) superimposed on the equivalent p53 structure (gray). Arrows highlight a subtle shift in the ASPP2 position. The single zinc ion associated with p73 is shown as a green sphere.
Mentions: The H1 helix and surrounding L2 loop do not participate directly in DNA interaction but contribute instead to dimer contacts in the DNA-bound tetramer (Supplementary Fig. S4a), as well as other protein–protein interactions (Supplementary Fig. S4b). Superposition of the free p73 structure with the p53–ASPP2 complex suggests a potential steric clash from the p73 H1 helix (Supplementary Fig. S4b). Binding of ASPP2 to p53 and p73 DBD was confirmed in the low micromolar range by native-gel mobility shift assay (Fig. 5). Interestingly, the binding of p73 appeared slightly less stable than that of the p53 complex. To investigate the p73 interaction further, we tested a variety of expression constructs in co-crystallization. Due to dissociation of p73–ASPP2 complexes during preparative gel filtration (Supplementary Fig. S5), the two proteins were purified separately and then mixed at a 1:1 molar ratio prior to crystallization. Crystals of the complex were obtained in space group I121 when combining the p73 DBD with the ankyrin repeat and SH3 domains (residues 891–1128) of human ASPP2. The structure of the complex (Fig. 6) was solved using molecular replacement and refined at a resolution of 2.6 Å (see Table 1 for data collection and refinement statistics). The six p73 chains in the asymmetric unit were traceable between residues 114 and 311. Bound ASPP2 chains were traceable between residues 920 and 1121 and showed minor structural deviations in the SH3 RT and n-Src loops, as well as the protein termini. A full description of the crystallized ASPP2 fold (also known as 53BP2) has been reported previously and comprises four ankyrin repeats and a C-terminal SH3 domain.25

Bottom Line: The loop in p73 is changed by a two-residue insertion that also induces repacking around the site of the p53 mutational hotspot R175.Importantly, the binding of ASPP2 is preserved by conformational changes in both the ankyrin repeat and SH3 domains.These results further highlight the structural variation that impacts p53 family interactions within the p53 interactome.

View Article: PubMed Central - PubMed

Affiliation: Structural Genomics Consortium, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, UK.

Show MeSH
Related in: MedlinePlus