Molecular basis of the interaction between the antiapoptotic Bcl-2 family proteins and the proapoptotic protein ASPP2.
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.
Katz
Chen
C
Institute of Chemistry, Hebrew University of Jerusalem, Safra Campus, Givat Ram, Jerusalem 91904, Israel.
Benyamini
Hadar
H
Rotem
Shahar
S
Lebendiker
Mario
M
Danieli
Tsafi
T
Iosub
Anat
A
Refaely
Hadar
H
Dines
Monica
M
Bronner
Vered
V
Bravman
Tsafrir
T
Shalev
Deborah E
DE
Rüdiger
Stefan
S
Friedler
Assaf
A
D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D003198:Computer Simulation; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*
D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; C090468:TP53BP2 protein, human