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.apoptosis ͉ peptides ͉ protein-protein interactions ͉ peptide arrays ͉ docking P rotein-protein interactions (PPI) are crucial for most processes critical for the proper function of living cells. Studying PPI is important for understanding the cell functionality and for designing drugs against diseases in which PPI are impaired. Peptides are excellent tools for studying PPI. They are good models for binding studies of protein domains because they often undergo induced fit upon ligand binding and gain their native structure. This was shown for peptides derived from the BH4 domain of Bcl-2 (1) and from Bak (2). Peptide arrays provide an efficient way for identifying binding sites (3). Peptide arrays were particularly valuable for epitope mapping and analyzing enzyme binding and PPI (4-7).Impairment of regulation of apoptosis pathways is a key event in malignant transformation. Understanding how apoptotic pathways are regulated is essential to understanding the basics for developing anticancer therapies. Two key apoptotic pathways are the p53 pathway and the mitochondrial death pathway. We used the peptide approach to characterize the interaction between two key players in these apoptotic pathways: ASPP2 and the Bcl-2 family proteins.Proteins from the Bcl-2 family play a major role in the mitochondrial death pathway (8). The Bcl-2 family consists of both proapoptotic (e.g., Bax and Bak) and antiapoptotic (e.g., Bcl-2, Bcl-X L , and Bcl-W...

Molecular Basis of the Interaction between Proapoptotic Truncated BID (tBID) Protein and Mitochondrial Carrier Homologue 2 (MTCH2) Protein

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Production of prone‐to‐aggregate proteins

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“…This family of fusion proteins includes the lipoyl domain from B. Stearothermophilus E2p [24], consisting of a short acidic 109 amino acid tag (pI: 4.53 and MW: 11 994.3 Da). The lipoyl domain fusion tag containing the N-terminus His tag and an optimized Tobacco Etch Virus (TEV) protease cleavage site (termed HLT-tag) is used at our facility as the preferable fusion protein for enhancing the solubility of IDR proteins [25][26][27]. This tag is best suited for NMR studies and is highly resistant to proteases (received from Dr. Mark Allen: MRC-CPE, Cambridge, England).…”

Low expression of MN1 associates with better treatment response in older patients with de novo cytogenetically normal acute myeloid leukemia

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