Mechanism of the Interaction between the Intrinsically Disordered C-Terminus of the Pro-Apoptotic ARTS Protein and the Bir3 Domain of XIAP

ARTS (Sept4_i2) is a mitochondrial pro-apoptotic protein that functions as a tumor suppressor. Its expression is significantly reduced in leukemia and lymphoma patients. ARTS binds and inhibits XIAP (X-linked Inhibitor of Apoptosis protein) by interacting with its Bir3 domain. ARTS promotes degradation of XIAP through the proteasome pathway. By doing so, ARTS removes XIAP inhibition of caspases and enables apoptosis to proceed. ARTS contains 27 unique residues in its C-terminal domain (CTD, residues 248–274) which are important for XIAP binding. Here we characterized the molecular details of this interaction. Biophysical and computational methods were used to show that the ARTS CTD is intrinsically disordered under physiological conditions. Direct binding of ARTS CTD to Bir3 was demonstrated using NMR and fluorescence spectroscopy. The Bir3 interacting region in ARTS CTD was mapped to ARTS residues 266–274, which are the nine C-terminal residues in the protein. Alanine scan of ARTS 266–274 showed the importance of several residues for Bir3 binding, with His268 and Cys273 contributing the most. Adding a reducing agent prevented binding to Bir3. A dimer of ARTS 266–274 formed by oxidation of the Cys residues into a disulfide bond bound with similar affinity and was probably required for the interaction with Bir3. The detailed analysis of the ARTS – Bir3 interaction provides the basis for setting it as a target for anti cancer drug design: It will enable the development of compounds that mimic ARTS CTD, remove IAPs inhibition of caspases, and thereby induce apoptosis

Regulation of ASPP2 interaction with p53 core domain by an intramolecular autoinhibitory mechanism.

ASPP2 is a key protein in regulating apoptosis both in p53-dependent and-independent pathways. The C-terminal part of ASPP2 contains four ankyrin repeats and an SH3 domain (Ank-SH3) that mediate the interactions of ASPP2 with apoptosis related proteins such as p53, Bcl-2 and the p65 subunit of NFκB. p53 core domain (p53CD) binds the n-src loop and the RT loop of ASPP2 SH3. ASPP2 contains a disordered proline rich domain (ASPP2 Pro) that forms an intramolecular autoinhibitory interaction with the Ank-SH3 domains. Here we show how this intramolecular interaction affects the intermolecular interactions of ASPP2 with p53, Bcl-2 and NFkB. We used biophysical methods to obtain better understanding of the relationship between ASPP2 and its partners for getting a comprehensive view on ASPP2 pathways. Fluorescence anisotropy competition experiments revealed that both ASPP2 Pro and p53CD competed for binding the n-src loop of the ASPP2 SH3, indicating regulation of p53CD binding to this loop by ASPP2 Pro. Peptides derived from the ASPP2-binding interface of Bcl-2 did not compete with p53CD or NFkB peptides for binding the ASPP2 n-src loop. However, p53CD displaced the NFκB peptide (residues 303-332) from its complex with ASPP2 Ank-SH3, indicating that NFκB 303-332 and p53CD bind a partly overlapping site in ASPP2 SH3, mostly in the RT loop. These results are in agreement with previous docking studies, which showed that ASPP2 Ank-SH3 binds Bcl-2 and NFκB mostly via distinct sites from p53. However they show some overlap between the binding sites of p53CD and NFkB in ASPP2 Ank-SH3. Our results provide experimental evidence that the intramolecular interaction in ASPP2 regulates its binding to p53CD and that ASPP2 Ank-SH3 binds Bcl-2 and NFκB via distinct sites

p53CD and the Fl-ASPP2 Pro 722–737 peptide compete for binding ASPp2 Ank-SH3.

<p>(A) ASPP2 Ank-SH3 binds ASPP2 Pro 722–737 with <i>K</i>_d = 0.68±0.07 µM. (B) p53CD was titrated into the fully formed complex between ASPP2 Ank-SH3 and Fl-ASPP2 722–737 and displaced the Fl-ASPP2 722–737 peptide.</p

The ASPP2 Ank-SH3 binding peptides used in this study.

*<p>Trp was added at the N terminus of the peptide for UV spectroscopy.</p

The ASPP2 Ank-SH3 – p53CD complex PDB ID: 1YCS [14].

<p>(A) Crystal structure of the complex between p53CD (black) and ASPP2 927–1119 (gray). The RT loop (residues 1067–1080, blue) and the n-Src loop (residues 1089–1096, purple) in the ASPP2 SH3 domain bind the L3 loop of p53 (residues 241–249, cyan), while the fourth ankyrin repeat (residues 1020–1027, red) binds the L2 loop (residues 178–183, cyan) of p53CD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058470#pone.0058470-Gorina1" target="_blank">[14]</a>; (B) The structure of the four ankyrin repeats (light gray) and the SH3 domain (dark gray) of ASPP2. The p53CD binding sites in the ASPP2 SH3 domain of ASPP2 are shown with the RT loop in blue and the n-src loop in purple. (C) The binding sites of ASPP2 Ank-SH3 to p53CD (cyan), Bcl2 (orange) and NFκB (green) are all on the same face of the molecule but with minimal overlap between the binding sites <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058470#pone.0058470-Benyamini1" target="_blank">[21]</a>; (D) The ASPP2 Pro (residues 693–918) binding sites in ASPP2 Ank-SH3 (magenta) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058470#pone.0058470-Rotem1" target="_blank">[13]</a>.</p

Bcl-2 and NFκB peptides bind the ASPP2 n-src loop 1089–1097 peptide in a different site than p53CD.

<p>(A) p53CD binds Fl-ASPP2 1089–1097 ASPP2 SH3 derived peptide In fluorescence anisotropy experiments with <i>K</i>_d = 0.63±0.02 µM (B) Non-labeled Bcl-2 or NFκB peptides were titrated into the fully formed complex between Fl-ASPP2 1089–1097 and p53CD. None of the peptides displaced p53CD from the complex.</p

ASPP2 Ank-SH3 binds NFκB 303–332 and is not displaced by Bcl-2 peptides.

<p>(A) ASPP2 Ank-SH3 binds Fl-NFκB 303–332 with K<i>_d</i> = 0.13±0.01 µM; (B) Non-labeled Bcl-2 103–120 (orange square) or Bcl-2 7–24 (orange triangle) peptides were titrated into the fully formed complex between Fl-NFκB 303–332 and ASPP2 Ank-SH3 but did not compete with ASPP2 Ank-SH3 on peptide binding.</p

ASPP2 Ank-SH3 binds ASPP2 722–737 and is not displaced by Bcl-2 peptides.

<p>(A) ASPP2 Ank-SH3 binds Fl-ASPP2 722–737 with K<i>_d</i> = 0.82±0.08 µM (magenta); (B) Non-labeled Bcl-2 103–120 (orange square) or Bcl-2 7–24 (orange triangle) peptides were titrated into the fully formed complex between Fl- ASPP2 722–737 and ASPP2 Ank-SH3 but did not compete with ASPP2 Ank-SH3 on peptide binding.</p

p53CD and ASPP2 Pro 723–737 compete for binding the Fl-ASPP2 n-src loop peptide residues 1089–1097: fluorescence anisotropy experiments.

<p>(A) A peptide derived from the proline rich domain of ASPP2 residues 722–737 binds the ASPP2 n-src peptide with <i>K</i>_d = 0.50±0.03 µM (magenta); Following titration of p53CD (cyan) into to the fully formed complex between Fl-ASPP2 1089–1097 and ASPP2 722–737, the anisotropy increased (B) p53CD residues 94–312 bind the ASPP2 n-src peptide with <i>K</i>_d = 0.63±0.02 µM (cyan). Unlabeled ASPP2 722–737 (magenta) displaced p53CD form its fully formed complex with ASPP2 1089–1097.</p