Academia.eduAcademia.edu

Figure 3 – uploaded by Dmitry Veprintsev

See full PDFdownloadDownload figure
— IPRPLSPTKLLPFLSNPYRNQSDADLEALRKKLSNAPRPLKKRSSTEPEGPNGPNIQ KLLYQRTTIAAMETISVPSYPSKSASVTASSESPVEIQNPYLHVEPEKEVVSLVPES LSPEDVGNASTENSDMPAPSPGLD YEPEGVPDNSPNLOQNNPEEPNPEAPHVLDVYLE EYPPYPPPPY PSGEPEGPGEDSVSMRPPEITGOVSLPPGKRINLRKTGSERIAH

Figure 3 — IPRPLSPTKLLPFLSNPYRNQSDADLEALRKKLSNAPRPLKKRSSTEPEGPNGPNIQ KLLYQRTTIAAMETISVPSYPSKSASVTASSESPVEIQNPYLHVEPEKEVVSLVPES LSPEDVGNASTENSDMPAPSPGLD YEPEGVPDNSPNLOQNNPEEPNPEAPHVLDVYLE EYPPYPPPPY PSGEPEGPGEDSVSMRPPEITGOVSLPPGKRINLRKTGSERIAH

Related Figures (8)

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.
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.
Related topics:
Protein Structure and FunctionIntrinsically disordered proteinsPeptide LibrariesIntermolecular InteractionComputational Method
580 California St., Suite 400
San Francisco, CA, 94104
© 2025 Academia. All rights reserved