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Structural Biology

The Structure and Interactions of the Proline-rich Domain

Profile image of Dmitry VeprintsevDmitry Veprintsev

2000

https://doi.org/10.1074/JBC.M708717200
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10 pages

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Abstract

ASPP2 is a pro-apoptotic protein that stimulates the p53-mediated apoptotic response. The C terminus of ASPP2 contains ankyrin (Ank) repeats and a SH3 domain, which mediate its interactions with numerous partner proteins such as p53, NFB, and Bcl-2. It also contains a proline-rich domain (ASPP2 Pro), whose structure and function are unclear. Here we used biophysical and biochemical methods to study the structure and the interactions of ASPP2 Pro, to gain insight into its biological role. We show, using biophysical and computational methods, that the ASPP2 Pro domain is natively unfolded. We found that the ASPP2 Pro domain interacts with the ASPP2 Ank-SH3 domains, and mapped the interaction sites in both domains. Using a combination of peptide array screening, biophysical and biochemical techniques, we found that ASPP2 Ank-SH3, but not ASPP2 Pro, mediates interactions of ASPP2 with peptides derived from its partner proteins. ASPP2 Pro-Ank-SH3 bound a peptide derived from its partner protein NFB weaker than ASPP2 Ank-SH3 bound this peptide. This suggested that the presence of the proline-rich domain inhibited the interactions mediated by the Ank-SH3 domains. Furthermore, a peptide from ASPP2 Pro competed with a peptide derived from NFB on binding to ASPP2 Ank-SH3. Based on our results, we propose a model in which the interaction between the ASPP2 domains regulates the intermolecular interactions of ASPP2 with its partner proteins.

Figures (9)
FIGURE 2. Determination of the quaternary structure of ASPP2 Pro. Ana- lytical gel filtration studies of ASPP2 Pro compared with ASPP2 Ank-SH3 domains supports its lack of structure: A, ASPP2 Pro: The calculated molecular mass of ASPP2 Pro is 24,500 Da, but the experimental molecular mass esti- mated from the calibration curve corresponds to ~ 130,000 Da. B, ASPP2 Ank- SH3: ASPP2 Ank-SH3 eluted after 15 ml, as expected from its calculated molecular mass of around 27,000 Da.
FIGURE 2. Determination of the quaternary structure of ASPP2 Pro. Ana- lytical gel filtration studies of ASPP2 Pro compared with ASPP2 Ank-SH3 domains supports its lack of structure: A, ASPP2 Pro: The calculated molecular mass of ASPP2 Pro is 24,500 Da, but the experimental molecular mass esti- mated from the calibration curve corresponds to ~ 130,000 Da. B, ASPP2 Ank- SH3: ASPP2 Ank-SH3 eluted after 15 ml, as expected from its calculated molecular mass of around 27,000 Da.
— IPRPLSPTKLLPFLSNPYRNQSDADLEALRKKLSNAPRPLKKRSSTEPEGPNGPNIQ KLLYQRTTIAAMETISVPSYPSKSASVTASSESPVEIQNPYLHVEPEKEVVSLVPES LSPEDVGNASTENSDMPAPSPGLD YEPEGVPDNSPNLOQNNPEEPNPEAPHVLDVYLE EYPPYPPPPY PSGEPEGPGEDSVSMRPPEITGOVSLPPGKRINLRKTGSERIAH
— IPRPLSPTKLLPFLSNPYRNQSDADLEALRKKLSNAPRPLKKRSSTEPEGPNGPNIQ KLLYQRTTIAAMETISVPSYPSKSASVTASSESPVEIQNPYLHVEPEKEVVSLVPES LSPEDVGNASTENSDMPAPSPGLD YEPEGVPDNSPNLOQNNPEEPNPEAPHVLDVYLE EYPPYPPPPY PSGEPEGPGEDSVSMRPPEITGOVSLPPGKRINLRKTGSERIAH
FIGURE 4. Direct interaction between the ASPP2 Pro and ASPP2 Ank-SH3 domains. A, nickel-affinity pull- down assay of ASPP2 Ank-SH3 by N-terminal His-tagged ASPP2 Pro. ASPP2 Ank-SH3 was incubated with His-tagged ASPP2 Pro (upper panel) or with wash buffer (lower panel) for 1 h at 4°C and then incubated for another hour with nickel-NTA beads. The samples were centrifuged, and the suspension was kept (/ane 7). The beads were washed with wash buffer containing 10 mm imidazole for five times (Janes 2-6), and then the proteins were eluted by the elution buffer, containing 250 mm imidazole, four times (lanes 8-11). ASPP2 Ank-SH3 was found in the elution fractions (8-11) only when incubated with ASPP2 Pro, but not with the buffer, indicating binding between the proteins. LMW (Amersham Biosciences) marker was used in /ane 7. B, incubation of ASPP2 Pro-Ank-SH3 with different concentrations of the cross-linker BS3 resulted in some formation of dimers. Shown are the results for 25 equivalents of BS3 (/ane 2), 50 equivalents of BS3 (/ane 3), and 100 equivalents of BS3 (/ane 4). No dimer was observed in the absence of the cross-linker (/ane 1); C, incubation of ASPP2 Ank-SH3 with ASPP2 Pro-Ank-SH3 with or without BS3 (/anes 5 and 6), resulted also in some formation of a second type of dimer with a different molecular weight, corresponding to a dimer between ASPP2 Pro- Ank-SH3 and ASPP2 Ank-SH3. Incubation of ASPP2 Pro-Ank-SH3 with or without BS3 (/Janes 7 and 2) and incubation of ASPP2 Ank-SH3 with or without BS3 (/anes 3 and 4) are shown as controls. D, AUC analysis of ASPP2 Pro-Ank-SH3 (top panel). Sedimentation equilibrium experiments shows that the protein is monomeric with molecular mass = 43,126 + 2,047 Da. The expected molecular mass of the protein is 48,300 Da. The bottom panel represents the residuals for the fit.
FIGURE 4. Direct interaction between the ASPP2 Pro and ASPP2 Ank-SH3 domains. A, nickel-affinity pull- down assay of ASPP2 Ank-SH3 by N-terminal His-tagged ASPP2 Pro. ASPP2 Ank-SH3 was incubated with His-tagged ASPP2 Pro (upper panel) or with wash buffer (lower panel) for 1 h at 4°C and then incubated for another hour with nickel-NTA beads. The samples were centrifuged, and the suspension was kept (/ane 7). The beads were washed with wash buffer containing 10 mm imidazole for five times (Janes 2-6), and then the proteins were eluted by the elution buffer, containing 250 mm imidazole, four times (lanes 8-11). ASPP2 Ank-SH3 was found in the elution fractions (8-11) only when incubated with ASPP2 Pro, but not with the buffer, indicating binding between the proteins. LMW (Amersham Biosciences) marker was used in /ane 7. B, incubation of ASPP2 Pro-Ank-SH3 with different concentrations of the cross-linker BS3 resulted in some formation of dimers. Shown are the results for 25 equivalents of BS3 (/ane 2), 50 equivalents of BS3 (/ane 3), and 100 equivalents of BS3 (/ane 4). No dimer was observed in the absence of the cross-linker (/ane 1); C, incubation of ASPP2 Ank-SH3 with ASPP2 Pro-Ank-SH3 with or without BS3 (/anes 5 and 6), resulted also in some formation of a second type of dimer with a different molecular weight, corresponding to a dimer between ASPP2 Pro- Ank-SH3 and ASPP2 Ank-SH3. Incubation of ASPP2 Pro-Ank-SH3 with or without BS3 (/Janes 7 and 2) and incubation of ASPP2 Ank-SH3 with or without BS3 (/anes 3 and 4) are shown as controls. D, AUC analysis of ASPP2 Pro-Ank-SH3 (top panel). Sedimentation equilibrium experiments shows that the protein is monomeric with molecular mass = 43,126 + 2,047 Da. The expected molecular mass of the protein is 48,300 Da. The bottom panel represents the residuals for the fit.
The peptides in this table, which were found to bind the ASPP2 Ank-SH3 domains of ASPP2, are all derived from the proline-rich domain of the protein. See Fig. 5C for screening results. TABLE 3
The peptides in this table, which were found to bind the ASPP2 Ank-SH3 domains of ASPP2, are all derived from the proline-rich domain of the protein. See Fig. 5C for screening results. TABLE 3
FIGURE 5. The specific binding sites between the ASPP2 Pro and ASPP2 Ank-SH3 domains. A, the sites in ASPP2 Ank-SH3 that bind ASPP2 Pro: An array consisting of peptides derived from ASPP2 was screened for binding ASPP2 Pro. Every dark spot represents binding between the corresponding peptide and ASPP2 Pro. For sequences of binding peptides see Table 1. For the complete peptide list see the supplemental data. B, the ASPP2 Pro bind- ing sites in the ankyrin repeats (green) and the SH3 domain (blue) (coordinates taken from PDB id: 1YCS). The binding peptides are marked in red. C, the sites in ASPP2 Pro that bind ASPP2 Ank-SH3: An array consisting of peptides derived from ASPP2 was screened for binding ASPP2 Ank-SH3. Every dark spot represents binding between the corresponding peptide and ASPP2 Ank-SH3. For sequences of binding peptides see Table 2. D, a schematic diagram show- ing the different peptides in ASPP2 693-1128 that are involved in the domain-domain interactions. £, quantification of the binding of ASPP2 Ank- SH3 to peptides derived from the ASPP2 Pro domain: Binding of ASPP2 Ank- SH3 to fluorescein-labeled ASPP2 723-737 was studied using fluorescence anisotropy. ASPP2 Ank-SH3 bound ASPP2 723-737 with K, = 16 + 1 um at physiological IS. Binding was dependent on the IS, with Kj = 5 wmat IS of 100 mn and K, = 0.6 mat IS of 50 mm.
FIGURE 5. The specific binding sites between the ASPP2 Pro and ASPP2 Ank-SH3 domains. A, the sites in ASPP2 Ank-SH3 that bind ASPP2 Pro: An array consisting of peptides derived from ASPP2 was screened for binding ASPP2 Pro. Every dark spot represents binding between the corresponding peptide and ASPP2 Pro. For sequences of binding peptides see Table 1. For the complete peptide list see the supplemental data. B, the ASPP2 Pro bind- ing sites in the ankyrin repeats (green) and the SH3 domain (blue) (coordinates taken from PDB id: 1YCS). The binding peptides are marked in red. C, the sites in ASPP2 Pro that bind ASPP2 Ank-SH3: An array consisting of peptides derived from ASPP2 was screened for binding ASPP2 Ank-SH3. Every dark spot represents binding between the corresponding peptide and ASPP2 Ank-SH3. For sequences of binding peptides see Table 2. D, a schematic diagram show- ing the different peptides in ASPP2 693-1128 that are involved in the domain-domain interactions. £, quantification of the binding of ASPP2 Ank- SH3 to peptides derived from the ASPP2 Pro domain: Binding of ASPP2 Ank- SH3 to fluorescein-labeled ASPP2 723-737 was studied using fluorescence anisotropy. ASPP2 Ank-SH3 bound ASPP2 723-737 with K, = 16 + 1 um at physiological IS. Binding was dependent on the IS, with Kj = 5 wmat IS of 100 mn and K, = 0.6 mat IS of 50 mm.
FIGURE 6. Binding of ASPP2 to peptides derived from its binding proteins is mediated by ASPP2 Ank-SH3 but not by ASPP2 Pro. A peptide array consisting of peptides derived from the ASPP2-binding proteins specified at Table 3 (Bcl-W in red, p65 in green, protein phosphatase 1 in pink, HCV core protein in orange, and YAP1 in blue) was screened for binding of ASPP2 Pro (A), which did not bind almost any of the peptides in the array and, ASPP2 Ank-SH3 (B). For sequences of binding peptides see Table 3. For the complete peptide list see the supplemental data. Binding of ASPP2 Ank-SH3 to ASPP2-binding proteins: screening of peptide array #3
FIGURE 6. Binding of ASPP2 to peptides derived from its binding proteins is mediated by ASPP2 Ank-SH3 but not by ASPP2 Pro. A peptide array consisting of peptides derived from the ASPP2-binding proteins specified at Table 3 (Bcl-W in red, p65 in green, protein phosphatase 1 in pink, HCV core protein in orange, and YAP1 in blue) was screened for binding of ASPP2 Pro (A), which did not bind almost any of the peptides in the array and, ASPP2 Ank-SH3 (B). For sequences of binding peptides see Table 3. For the complete peptide list see the supplemental data. Binding of ASPP2 Ank-SH3 to ASPP2-binding proteins: screening of peptide array #3
FIGURE 7. ASPP2 Pro and NFxB 303-332 compete for the same binding site in ASPP2 Ank-SH3. A, ASPP2 Ank-SH3 binds fluorescein-labeled NFKB 303-332 tighter than ASPP2 Pro-Ank-SH3: fluorescence anisotropy studies. ASPP2 Ank-SH3 bound NFx«B 303-332 with K, = 0.27 wm, whereas ASPP2 Pro-Ank-SH3 bound this peptide with Kj = 1.2 wm; B-D: ASPP2 723-737 and NF«B 303-332 compete for binding ASPP2 Ank-SH3: fluorescence anisotropy competition experiments. Left panel in B-D shows the binding curves for the titration of ASPP2 Ank-SH3 into a fluorescein-labeled peptide, and the right panel describes the competition, where unlabeled peptide was titrated into the fully formed complex. Shown are the following competition experiments: unlabeled ASPP2 723-737 and fluorescein labeled NF«B 303-332 (B), unla- beled NFxKB 303-332 and fluorescein labeled ASPP2 723-737 (C), and a con- trol experiment of competition between unlabeled ASPP2 723-737 and flu- orescein labeled ASPP2 723-737 (D).
FIGURE 7. ASPP2 Pro and NFxB 303-332 compete for the same binding site in ASPP2 Ank-SH3. A, ASPP2 Ank-SH3 binds fluorescein-labeled NFKB 303-332 tighter than ASPP2 Pro-Ank-SH3: fluorescence anisotropy studies. ASPP2 Ank-SH3 bound NFx«B 303-332 with K, = 0.27 wm, whereas ASPP2 Pro-Ank-SH3 bound this peptide with Kj = 1.2 wm; B-D: ASPP2 723-737 and NF«B 303-332 compete for binding ASPP2 Ank-SH3: fluorescence anisotropy competition experiments. Left panel in B-D shows the binding curves for the titration of ASPP2 Ank-SH3 into a fluorescein-labeled peptide, and the right panel describes the competition, where unlabeled peptide was titrated into the fully formed complex. Shown are the following competition experiments: unlabeled ASPP2 723-737 and fluorescein labeled NF«B 303-332 (B), unla- beled NFxKB 303-332 and fluorescein labeled ASPP2 723-737 (C), and a con- trol experiment of competition between unlabeled ASPP2 723-737 and flu- orescein labeled ASPP2 723-737 (D).
FIGURE 8. A proposed mechanism by which the ASPP2 Pro domain regu- lates the protein-protein interactions of ASPP2. The unstructured ASPP2 Pro domain binds the ASPP2 Ank-SH3 domains. This interaction could be either intramolecular within a given ASPP2 monomer, or intermolecular resulting in the formation of dimers. According to our results this equilibrium favors the intramolecular interaction and not dimerization. We propose that, by doing so, ASPP2 regulates its protein-protein interactions. The binding of the ASPP2 Pro domain to the ASPP2 Ank-SH3 domains blocks other protein- protein interactions that these domains mediate. Following a yet unknown control signal, the ASPP2 Pro domain is released from the ASPP2 Ank-SH3 domains, making them available to bind the partner proteins.
FIGURE 8. A proposed mechanism by which the ASPP2 Pro domain regu- lates the protein-protein interactions of ASPP2. The unstructured ASPP2 Pro domain binds the ASPP2 Ank-SH3 domains. This interaction could be either intramolecular within a given ASPP2 monomer, or intermolecular resulting in the formation of dimers. According to our results this equilibrium favors the intramolecular interaction and not dimerization. We propose that, by doing so, ASPP2 regulates its protein-protein interactions. The binding of the ASPP2 Pro domain to the ASPP2 Ank-SH3 domains blocks other protein- protein interactions that these domains mediate. Following a yet unknown control signal, the ASPP2 Pro domain is released from the ASPP2 Ank-SH3 domains, making them available to bind the partner proteins.

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The structure and Interactions of the proline-rich domain of ASPP2
Stefan Rüdiger

Journal of Biological Chemistry, 2008

ASPP2 is a pro-apoptotic protein that stimulates the p53-mediated apoptotic response. The C terminus of ASPP2 contains ankyrin (Ank) repeats and a SH3 domain, which mediate its interactions with numerous partner proteins such as p53, NFB, and Bcl-2. It also contains a proline-rich domain (ASPP2 Pro), whose structure and function are unclear. Here we used biophysical and biochemical methods to study the structure and the interactions of ASPP2 Pro, to gain insight into its biological role. We show, using biophysical and computational methods, that the ASPP2 Pro domain is natively unfolded. We found that the ASPP2 Pro domain interacts with the ASPP2 Ank-SH3 domains, and mapped the interaction sites in both domains. Using a combination of peptide array screening, biophysical and biochemical techniques, we found that ASPP2 Ank-SH3, but not ASPP2 Pro, mediates interactions of ASPP2 with peptides derived from its partner proteins. ASPP2 Pro-Ank-SH3 bound a peptide derived from its partner protein NFB weaker than ASPP2 Ank-SH3 bound this peptide. This suggested that the presence of the proline-rich domain inhibited the interactions mediated by the Ank-SH3 domains. Furthermore, a peptide from ASPP2 Pro competed with a peptide derived from NFB on binding to ASPP2 Ank-SH3. Based on our results, we propose a model in which the interaction between the ASPP2 domains regulates the intermolecular interactions of ASPP2 with its partner proteins.

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Regulation of ASPP2 Interaction with p53 Core Domain by an Intramolecular Autoinhibitory Mechanism
Chen Katz

PLoS ONE, 2013

ASPP2 is a key protein in regulating apoptosis both in p53-dependent and-independent pathways. The C-terminal part of ASPP2 contains four ankyrin repeats and an SH3 domain (Ank-SH3) that mediate the interactions of ASPP2 with apoptosis related proteins such as p53, Bcl-2 and the p65 subunit of NFkB. p53 core domain (p53CD) binds the n-src loop and the RT loop of ASPP2 SH3. ASPP2 contains a disordered proline rich domain (ASPP2 Pro) that forms an intramolecular autoinhibitory interaction with the Ank-SH3 domains. Here we show how this intramolecular interaction affects the intermolecular interactions of ASPP2 with p53, Bcl-2 and NFkB. We used biophysical methods to obtain better understanding of the relationship between ASPP2 and its partners for getting a comprehensive view on ASPP2 pathways. Fluorescence anisotropy competition experiments revealed that both ASPP2 Pro and p53CD competed for binding the n-src loop of the ASPP2 SH3, indicating regulation of p53CD binding to this loop by ASPP2 Pro. Peptides derived from the ASPP2-binding interface of Bcl-2 did not compete with p53CD or NFkB peptides for binding the ASPP2 n-src loop. However, p53CD displaced the NFkB peptide (residues 303-332) from its complex with ASPP2 Ank-SH3, indicating that NFkB 303-332 and p53CD bind a partly overlapping site in ASPP2 SH3, mostly in the RT loop. These results are in agreement with previous docking studies, which showed that ASPP2 Ank-SH3 binds Bcl-2 and NFkB mostly via distinct sites from p53. However they show some overlap between the binding sites of p53CD and NFkB in ASPP2 Ank-SH3. Our results provide experimental evidence that the intramolecular interaction in ASPP2 regulates its binding to p53CD and that ASPP2 Ank-SH3 binds Bcl-2 and NFkB via distinct sites. Citation: Rotem-Bamberger S, Katz C, Friedler A (2013) Regulation of ASPP2 Interaction with p53 Core Domain by an Intramolecular Autoinhibitory Mechanism. PLoS ONE 8(3): e58470. PLOS ONE | www.plosone.org March 2013 | Volume 8 | Issue 3 | e58470

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Insight into the Structural Basis of Pro- and Antiapoptotic p53 Modulation by ASPP Proteins
Chang Byeon

Journal of Biological Chemistry, 2009

p53-dependent apoptosis is modulated by the ASPP family of proteins (apoptosis-stimulating proteins of p53; also called ankyrin repeat-, Src homology 3 domain-, and Pro-rich regioncontaining proteins). Its three known members, ASPP1, ASPP2, and iASPP, were previously found to interact with p53, influencing the apoptotic response of cells without affecting p53-induced cell cycle arrest. More specifically, the bona fide tumor suppressors, ASPP1 and ASPP2, bind to the core domain of p53 and stimulate transcription of apoptotic genes, whereas oncogenic iASPP also binds to the p53 core domain but inhibits p53dependent apoptosis. Although the general interaction regions are known, details of the interfaces for each p53-ASPP complex have not been evaluated. We undertook a comprehensive biophysical characterization of ASPP-p53 complex formation and mapped the binding interfaces by NMR. We found that the interaction interface on p53 for the proapoptotic protein ASPP2 is distinct from that for the antiapoptotic iASPP. ASPP2 primarily binds to the core domain of p53, whereas iASPP predominantly interacts with a linker region adjacent to the core domain. Our detailed structural analyses of the ASPP-p53 interactions provide insight into the structural basis of the differential behavior of pro-and antiapoptotic ASPP family members.

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ASPP proteins discriminate between PP1 catalytic subunits through their SH3 domain and the PP1 C-tail
Matthias Gstaiger

Nature Communications, 2019

Serine/threonine phosphatases such as PP1 lack substrate specificity and associate with a large array of targeting subunits to achieve the requisite selectivity. The tumour suppressor ASPP (apoptosis-stimulating protein of p53) proteins associate with PP1 catalytic subunits and are implicated in multiple functions from transcriptional regulation to cell junction remodelling. Here we show that Drosophila ASPP is part of a multiprotein PP1 complex and that PP1 association is necessary for several in vivo functions of Drosophila ASPP. We solve the crystal structure of the human ASPP2/PP1 complex and show that ASPP2 recruits PP1 using both its canonical RVxF motif, which binds the PP1 catalytic domain, and its SH3 domain, which engages the PP1 C-terminal tail. The ASPP2 SH3 domain can discriminate between PP1 isoforms using an acidic specificity pocket in the n-Src domain, providing an exquisite mechanism where multiple motifs are used combinatorially to tune binding affinity to PP1.

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Molecular interactions of ASPP1 and ASPP2 with the p53 protein family and the apoptotic promoters PUMA and Bax
M-am Retras

Nucleic Acids Research, 2008

The apoptosis stimulating p53 proteins, ASPP1 and ASPP2, are the first two common activators of the p53 protein family that selectively enable the latter to regulate specific apoptotic target genes, which facilitates yes yet unknown mechanisms for discrimination between cell cycle arrest and apoptosis. To better understand the interplay between ASPP-and p53-family of proteins we investigated the molecular interactions between them using biochemical methods and structure-based homology modelling. The data demonstrate that: (i) the binding of ASPP1 and ASPP2 to p53, p63 and p73 is direct; (ii) the C-termini of ASPP1 and ASPP2 interact with the DNA-binding domains of p53 protein family with dissociation constants, K d , in the lower micro-molar range; (iii) the stoichiometry of binding is 1:1; (iv) the DNA-binding domains of p53 family members are sufficient for these protein-protein interactions; (v) EMSA titrations revealed that while tri-complex formation between ASPPs, p53 family of proteins and PUMA/ Bax is mutually exclusive, ASPP2 (but not ASPP1) formed a complex with PUMA (but not Bax) and displaced p53 and p73. The structure-based homology modelling revealed subtle differences between ASPP2 and ASPP1 and together with the experimental data provide novel mechanistic insights.

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Related topics

  • Protein Structure and Function
  • Intrinsically disordered proteins
  • Peptide Libraries
  • Intermolecular Interaction
  • Computational Method

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    The C terminus of p53 binds the N-terminal domain of MDM2
    rachel beckerman

    Nature Structural & Molecular Biology, 2010

    The p53 tumor suppressor interacts with its negative regulator Mdm2 via the former's N-terminal region and core domain. Yet the extreme p53 C-terminal region contains lysine residues ubiquitinated by Mdm2 and can bear post-translational modifications that inhibit Mdm2-p53 association. We show that, the Mdm2-p53 interaction is decreased upon deletion, mutation or acetylation of the p53 C-terminus. Mdm2 decreases the association of full-length but not Cterminally deleted p53 with a DNA target sequence in vitro and in cells. Further, using multiple approaches we demonstrate that a peptide from p53 C-terminus directly binds Mdm2 N-terminus in vitro. We also show that p300-acetylated p53 binds inefficiently to Mdm2 in vitro, and Nutlin-3 treatment induces C-terminal modification(s) of p53 in cells, explaining the low efficiency of Nutlin-3 in dissociating p53-MDM2 in vitro.

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    Integrated strategy reveals the protein interface between cancer targets Bcl-2 and NAF-1
    Chen Katz, Sagi Tamir

    Proceedings of the National Academy of Sciences, 2014

    Life requires orchestrated control of cell proliferation, cell maintenance, and cell death. Involved in these decisions are protein complexes that assimilate a variety of inputs that report on the status of the cell and lead to an output response. Among the proteins involved in this response are nutrient-deprivation autophagy factor-1 (NAF-1)- and Bcl-2. NAF-1 is a homodimeric member of the novel Fe-S protein NEET family, which binds two 2Fe-2S clusters. NAF-1 is an important partner for Bcl-2 at the endoplasmic reticulum to functionally antagonize Beclin 1-dependent autophagy [Chang NC, Nguyen M, Germain M, Shore GC (2010) EMBO J 29(3):606-618]. We used an integrated approach involving peptide array, deuterium exchange mass spectrometry (DXMS), and functional studies aided by the power of sufficient constraints from direct coupling analysis (DCA) to determine the dominant docked conformation of the NAF-1-Bcl-2 complex. NAF-1 binds to both the pro- and antiapoptotic regions (BH3 and BH4) of Bcl-2, as demonstrated by a nested protein fragment analysis in a peptide array and DXMS analysis. A combination of the solution studies together with a new application of DCA to the eukaryotic proteins NAF-1 and Bcl-2 provided sufficient constraints at amino acid resolution to predict the interaction surfaces and orientation of the protein-protein interactions involved in the docked structure. The specific integrated approach described in this paper provides the first structural information, to our knowledge, for future targeting of the NAF-1-Bcl-2 complex in the regulation of apoptosis/autophagy in cancer biology.

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    Studying protein–protein interactions using peptide arrays
    Chen Katz

    Chemical Society Reviews, 2011

    Screening of arrays and libraries of compounds is well-established as a high-throughput method for detecting and analyzing interactions in both biological and chemical systems. Arrays and libraries can be composed from various types of molecules, ranging from small organic compounds to DNA, proteins and peptides. The applications of libraries for detecting and characterizing biological interactions are wide and diverse, including for example epitope mapping, carbohydrate arrays, enzyme binding and protein-protein interactions. Here, we will focus on the use of peptide arrays to study protein-protein interactions. Characterization of protein-protein interactions is crucial for understanding cell functionality. Using peptides, it is possible to map the precise binding sites in such complexes. Peptide array libraries usually contain partly overlapping peptides derived from the sequence of one protein from the complex of interest. The peptides are attached to a solid support using various techniques such as SPOT-synthesis and photolithography. Then, the array is incubated with the partner protein from the complex of interest. Finally, the detection of the protein-bound peptides is carried out by using immunodetection assays. Peptide array screening is semi-quantitative, and quantitative studies with selected peptides in solution are required to validate and complement the screening results. These studies can improve our fundamental understanding of cellular processes by characterizing amino acid patterns of protein-protein interactions, which may even develop into prediction algorithms. The binding peptides can then serve as a basis for the design of drugs that inhibit or activate the target protein-protein interactions. In the current review, we will introduce the recent work on this subject performed in our and in other laboratories. We will discuss the applications, advantages and disadvantages of using peptide arrays as a tool to study protein-protein interactions.

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    A model for the interaction between NF-kappa-B and ASPP2 suggests an I-kappa-B-like binding mechanism
    Chen Katz

    Proteins, 2009

    We used computational methods to study the interaction between two key proteins in apoptosis regulation: the transcription factor NF-kappa-B (NFkappaB) and the proapoptotic protein ASPP2. The C-terminus of ASPP2 contains ankyrin repeats and SH3 domains (ASPP2(ANK-SH3)) that mediate interactions with numerous apoptosis-related proteins, including the p65 subunit of NFkappaB (NFkappaB(p65)). Using peptide-based methods, we have recently identified the interaction sites between NFkappaB(p65) and ASPP2(ANK-SH3) (Rotem et al., J Biol Chem 283, 18990-18999). Here we conducted a computational study of protein docking and molecular dynamics to obtain a structural model of the complex between the full length proteins and propose a mechanism for the interaction. We found that ASPP2(ANK-SH3) binds two sites in NFkappaB(p65), at residues 236-253 and 293-313 that contain the nuclear localization signal (NLS). These sites also mediate the binding of NFkappaB to its natural inhibitor IkappaB, whic...

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    Regulation of ASPP2 Interaction with p53 Core Domain by an Intramolecular Autoinhibitory Mechanism
    Chen Katz

    PLoS ONE, 2013

    ASPP2 is a key protein in regulating apoptosis both in p53-dependent and-independent pathways. The C-terminal part of ASPP2 contains four ankyrin repeats and an SH3 domain (Ank-SH3) that mediate the interactions of ASPP2 with apoptosis related proteins such as p53, Bcl-2 and the p65 subunit of NFkB. p53 core domain (p53CD) binds the n-src loop and the RT loop of ASPP2 SH3. ASPP2 contains a disordered proline rich domain (ASPP2 Pro) that forms an intramolecular autoinhibitory interaction with the Ank-SH3 domains. Here we show how this intramolecular interaction affects the intermolecular interactions of ASPP2 with p53, Bcl-2 and NFkB. We used biophysical methods to obtain better understanding of the relationship between ASPP2 and its partners for getting a comprehensive view on ASPP2 pathways. Fluorescence anisotropy competition experiments revealed that both ASPP2 Pro and p53CD competed for binding the n-src loop of the ASPP2 SH3, indicating regulation of p53CD binding to this loop by ASPP2 Pro. Peptides derived from the ASPP2-binding interface of Bcl-2 did not compete with p53CD or NFkB peptides for binding the ASPP2 n-src loop. However, p53CD displaced the NFkB peptide (residues 303-332) from its complex with ASPP2 Ank-SH3, indicating that NFkB 303-332 and p53CD bind a partly overlapping site in ASPP2 SH3, mostly in the RT loop. These results are in agreement with previous docking studies, which showed that ASPP2 Ank-SH3 binds Bcl-2 and NFkB mostly via distinct sites from p53. However they show some overlap between the binding sites of p53CD and NFkB in ASPP2 Ank-SH3. Our results provide experimental evidence that the intramolecular interaction in ASPP2 regulates its binding to p53CD and that ASPP2 Ank-SH3 binds Bcl-2 and NFkB via distinct sites. Citation: Rotem-Bamberger S, Katz C, Friedler A (2013) Regulation of ASPP2 Interaction with p53 Core Domain by an Intramolecular Autoinhibitory Mechanism. PLoS ONE 8(3): e58470. PLOS ONE | www.plosone.org March 2013 | Volume 8 | Issue 3 | e58470

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