Fig. 1.
Analysis of the ASPP2 binding sites in the Bcl-2 family proteins. (A) An array consisting of overlapping peptides derived from the Bcl-2 family proteins Bcl-2, Bcl-W, and Bcl-XL was screened for binding ASPP2Ank-SH3. Each dark spot represents binding of ASPP2Ank-SH3 to a specific peptide (Table 1). (B–D) The ASPP2 binding sites, as discovered in the peptide array screening, are highlighted on the known 3D structures of the Bcl family proteins. The BH4 site is in magenta and the proapoptotic site is in green. See Table 1 for peptides details. (B) Bcl-2 [PDB entry 1YSW (23)]. (C) Bcl-XL [PDB entry 1G5J (20)]. (D) Bcl-W [PDB entry 1O0l (31)]. Figures were generated by using PyMOL (32). (E–G) Quantitative analysis of the interaction between ASPP2Ank-SH3 and Bcl-2 BH4 peptide (7–24) using three independent methods. (E) Fluorescence spectroscopy. ASPP2Ank-SH3 was titrated into fluorescein-labeled Bcl-2 7–24, and the binding curve was fit to 1:1 binding model. Kd was found to be 4.7 ± 0.2 μM. (F) SPR. Biotinylated Bcl-2 7–24 peptide was captured on a ProteOn NLC sensor chip, followed by the association of 150 μl of ASPP2Ank-SH3, simultaneously injected at concentrations of 40, 30, 15, 10, and 7.5 μM (from top to bottom). Kd was found to be 1.6 μM. (G) ELISA. ASPP2Ank-SH3 was adsorbed on the ELISA plate, and binding of the peptide was studied as described in Materials and Methods. The apparent Kd is at the low micromolar range, in agreement with the two other methods.