The structure and interactions of the proline-rich domain of ASPP2.
ASPP2 is a pro-apoptotic protein that stimulates the p53-mediated apoptotic response. The C terminus of ASPP2 contains ankyrin (Ank) repeats and a SH3 domain, which mediate its interactions with numerous partner proteins such as p53, NFkappaB, and Bcl-2. It also contains a proline-rich domain (ASPP2 Pro), whose structure and function are unclear. Here we used biophysical and biochemical methods to study the structure and the interactions of ASPP2 Pro, to gain insight into its biological role. We show, using biophysical and computational methods, that the ASPP2 Pro domain is natively unfolded. We found that the ASPP2 Pro domain interacts with the ASPP2 Ank-SH3 domains, and mapped the interaction sites in both domains. Using a combination of peptide array screening, biophysical and biochemical techniques, we found that ASPP2 Ank-SH3, but not ASPP2 Pro, mediates interactions of ASPP2 with peptides derived from its partner proteins. ASPP2 Pro-Ank-SH3 bound a peptide derived from its partner protein NFkappaB weaker than ASPP2 Ank-SH3 bound this peptide. This suggested that the presence of the proline-rich domain inhibited the interactions mediated by the Ank-SH3 domains. Furthermore, a peptide from ASPP2 Pro competed with a peptide derived from NFkappaB on binding to ASPP2 Ank-SH3. Based on our results, we propose a model in which the interaction between the ASPP2 domains regulates the intermolecular interactions of ASPP2 with its partner proteins.
Rotem
Shahar
S
Institute of Chemistry, The Hebrew University of Jerusalem, Safra Campus, Givat Ram, Jerusalem 91904, Israel.
Katz
Chen
C
Benyamini
Hadar
H
Lebendiker
Mario
M
Veprintsev
Dmitry
D
Rüdiger
Stefan
S
Danieli
Tsafi
T
Friedler
Assaf
A
D000818:Animals; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D016328:NF-kappa B / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods; D011392:Proline / Q000737:chemistry*; D011485:Protein Binding / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000378:metabolism; D018909:src Homology Domains / Q000502:physiology
D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; D016328:NF-kappa B; D019253:Proto-Oncogene Proteins c-bcl-2; C495901:TP53 protein, human; C090468:TP53BP2 protein, human; D016159:Tumor Suppressor Protein p53; D011392:Proline