Molecular basis of the interaction between the antiapoptotic Bcl-2 family proteins and the proapoptotic protein ASPP2

Abstract

We have characterized the molecular basis of the interaction between ASPP2 and Bcl-2, which are key proteins in the apoptotic pathway. The C-terminal ankyrin repeats and SH3 domain of ASPP2 (ASPP2(Ank-SH3)) mediate its interactions with the antiapoptotic protein Bcl-2. We used biophysical and computational methods to identify the interaction sites of Bcl-2 and its homologues with ASPP2. Using peptide array screening, we found that ASPP2(Ank-SH3) binds two homologous sites in all three Bcl proteins tested: (i) the conserved BH4 motif, and (ii) a binding site for proapoptotic regulators. Quantitative binding studies revealed that binding of ASPP2(Ank-SH3) to the Bcl-2 family members is selective at two levels: (i) interaction with Bcl-2-derived peptides is the tightest compared to peptides from the other family members, and (ii) within Bcl-2, binding of ASPP2(Ank-SH3) to the BH4 domain is tightest. Sequence alignment of the ASPP2-binding peptides combined with binding studies of mutated peptides revealed that two nonconserved positions where only Bcl-2 contains positively charged residues account for its tighter binding. The experimental binding results served as a basis for docking analysis, by which we modeled the complexes of ASPP2(Ank-SH3) with the full-length Bcl proteins. Using peptide arrays and quantitative binding studies, we found that Bcl-2 binds three loops in ASPP2(Ank-SH3) with similar affinity, in agreement with our predicted model. Based on our results, we propose a mechanism in which ASPP2 induces apoptosis by inhibiting functional sites of the antiapoptotic Bcl-2 proteins.

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-X_L was screened for binding ASPP2_Ank-SH3. Each dark spot represents binding of ASPP2_Ank-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-X_L [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 ASPP2_Ank-SH3 and Bcl-2 BH4 peptide (–24) using three independent methods. (E) Fluorescence spectroscopy. ASPP2_Ank-SH3 was titrated into fluorescein-labeled Bcl-2 7–24, and the binding curve was fit to 1:1 binding model. K_d 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 ASPP2_Ank-SH3, simultaneously injected at concentrations of 40, 30, 15, 10, and 7.5 μM (from top to bottom). K_d was found to be 1.6 μM. (G) ELISA. ASPP2_Ank-SH3 was adsorbed on the ELISA plate, and binding of the peptide was studied as described in Materials and Methods. The apparent K_d is at the low micromolar range, in agreement with the two other methods.

Fig. 2.

Positively charged residues in Bcl-2 account for its tighter binding To ASPP2. (A) Sequence alignment of ASPP2 binding peptides: The ASPP2 binding peptides derived from the three Bcl proteins are bold and colored by amino acid type. (B and C) ELISA studies of ASPP2_Ank-SH3 binding to Bcl-2 and Bcl-X_L peptides modified at the nonconserved amino acid K15 and H18 of Bcl-2. (B) Peptides in which the positively charged residues from the Bcl-2 peptides were replaced by alanine or by the corresponding residues from Bcl-X_L. (C) Peptides in which positive charges were introduced into the relevant positions at the Bcl-X_L peptide.

Fig. 3.

Docking model for the interaction between the ASPP2_Ank-SH3 and Bcl-2/Bcl-X_L proteins. (A) Bcl-2 binding to ASPP2_Ank-SH3. Bcl-2 is colored gray, the BH4 site is in magenta, and the proapoptotic site is in green. ASPP2_Ank-SH3 is colored orange. (B) Two aligned models of Bcl-2 and Bcl-X_L complexes with ASPP2_Ank-SH3. Bcl-X_L is colored cyan, and Bcl-2 is in gray. ASPP2_Ank-SH3 is colored red and orange for its complex models with Bcl-X_L and Bcl-2_, respectively. Representative binding residues from both partners are depicted in sticks and spheres. The conserved interactions between Bcl-2 (PDB entry 1YSW) and ASPP2 (PDB entry 1ycs) are as follows: K15–D468 and E470; H18–D468 and E481; Y19–W467; K20–D468 and E481; R24–D352; R103–D358 and D359; Y105–P356; and R106–D333, D358, and E359. The homologous amino acids in Bcl-X_L show the same interactions with ASPP2. For consistency with the PDB file, numbering of the ASPP2 residues is according to the sequence of its truncated form 53BP2.

Fig. 4.

Bcl-2 binding sites in ASPP2. (A) An array consisting of overlapping peptides derived from ASPP2 was screened for binding Bcl-2. Each dark spot represents binding of Bcl-2 to a specific peptide (Table 2). (B) The Bcl-2 binding sites, as discovered in the peptide array screening, are colored blue on the 3D structure of ASPP2_Ank-SH3 (PDB entry 1YCS). (C–E) Quantitative analysis. Shown are representative binding curves of the interaction between Bcl-2 and the peptides derived from ASPP2_Ank-SH3 as indicated. Binding was quantified by using fluorescence spectroscopy as described in Materials and Methods. For binding affinity, see Table 2.

Fig. 5.

Proposed mechanism for induction of apoptosis after binding of ASPP2 to Bcl-2 family proteins. ASPP2 binds the antiapoptotic Bcl-2 family members at two functional sites and mediates apoptosis by inducing the release of other proapoptotic proteins that also bind Bcl-2 at those sites, which may enable their apoptotic activity, as specified in the text.