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. 2012;7(11):e49527.
doi: 10.1371/journal.pone.0049527. Epub 2012 Nov 15.

Structural insight into inhibitor of apoptosis proteins recognition by a potent divalent smac-mimetic

Federica Cossu  1 , Mario Milani, Patrice Vachette, Francesca Malvezzi, Serena Grassi, Daniele Lecis, Domenico Delia, Carmelo Drago, Pierfausto Seneci, Martino Bolognesi, Eloise Mastrangelo
Affiliations

Structural insight into inhibitor of apoptosis proteins recognition by a potent divalent smac-mimetic

Federica Cossu et al. PLoS One. 2012.

Abstract

Genetic alterations enhancing cell survival and suppressing apoptosis are hallmarks of cancer that significantly reduce the efficacy of chemotherapy or radiotherapy. The Inhibitor of Apoptosis Protein (IAP) family hosts conserved proteins in the apoptotic pathway whose over-expression, frequently found in tumours, potentiates survival and resistance to anticancer agents. In humans, IAPs comprise eight members hosting one or more structural Baculoviral IAP Repeat (BIR) domains. Cellular IAPs (cIAP1 and 2) indirectly inhibit caspase-8 activation, and regulate both the canonical and the non-canonical NF-κB signaling pathways. In contrast to cIAPs, XIAP (X chromosome-linked Inhibitor of Apoptosis Protein) inhibits directly the effector caspases-3 and -7 through its BIR2 domain, and initiator caspase-9 through its BIR3 domain; molecular docking studies suggested that Smac/DIABLO antagonizes XIAP by simultaneously targeting both BIR2 and BIR3 domains. Here we report analytical gel filtration, crystallographic and SAXS experiments on cIAP1-BIR3, XIAP-BIR3 and XIAP-BIR2BIR3 domains, alone and in the presence of compound 9a, a divalent homodimeric Smac mimetic. 9a is shown to bind two BIR domains inter- (in the case of two BIR3) and intra-molecularly (in the case of XIAP-BIR2BIR3), with higher affinity for cIAP1-BIR3, relative to XIAP-BIR3. Despite the different crystal lattice packing, 9a maintains a right handed helical conformation in both cIAP1-BIR3 and XIAP-BIR3 crystals, that is likely conserved in solution as shown by SAXS data. Our structural results demonstrate that the 9a linker length, its conformational degrees of freedom and its hydrophobicity, warrant an overall compact structure with optimal solvent exposure of its two active moieties for IAPs binding. Our results show that 9a is a good candidate for pre-clinical and clinical studies, worth of further investigations in the field of cancer therapy.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Chemical structure of tail-tail dimer 9a.
The left inset shows a framed structure of the divalent Smac-mimetics based on the 1-aza-2-oxobicyclo[5.3.0]decane scaffold.
Figure 2
Figure 2. Western blot of MDA-MB-231 cells untreated or treated with 9a.
Upper part: cIAP1 and cIAP2 degradation in the MDA-MB-231 cell line after 30 min and 6h of treatment with 9a. Proteins were revealed by Western blot using polyclonal antibodies specific for cIAP1 and cIAP2. Lower part: activation of caspase-8 (arrows indicate p55, p43/41 and p18 forms), -9 (p37) and -3 (p17 and p19) by 9a (p89). Proteins were revealed by Western blot using rabbit polyclonal antibodies specific for cleaved Parp, and cleaved caspase-8, -9 and -3. Prestained Protein SHARPMASS V (11−250 kDa; EuroClone) was used as molecular weight marker.
Figure 3
Figure 3. Analytical Gel Filtration Chromatograms.
A) XIAP-BIR3 (33 µM) in absence/presence of an excess (1 mM) of 9a. B) XIAP-BIR2BIR3 in absence/presence of an excess (1 mM) of 9a. C) cIAP-BIR3 in absence/presence of an equal amount (33 µM) of 9a.F
Figure 4
Figure 4. Dimeric assemblies of cIAP1- and XIAP1-BIR3 bound to 9a.
A) X-Ray structure of cIAP1-BIR3 dimer (cartoon in blue and pale blue) in complex with 9a (green sticks). B) X-Ray structure of XIAP-BIR3 dimer in complex with 9a: the A and F molecules are in orange and pale yellow, respectively, 9a is represented as green sticks (drawn with Pymol).
Figure 5
Figure 5. cIAP1- and XIAP1-BIR3 bound to one head of 9a.
A) Left panel, XIAP-BIR3 (orange cartoon) with 9a (green sticks). The main residues involved in ligand interaction are shown in orange sticks; right-panel, cIAP-BIR3 (blue cartoon) with 9a (green sticks) and the main residues involved in ligand interaction (blue sticks). B) Left and right panels as A) with protein surface coloured by electrostatic potential (calculated using APBS2; drawn with Pymol).
Figure 6
Figure 6. SAXS study of XIAP-BIR2BIR3.
A) experimental scattering patterns with associated error bars; blue line: free XIAP-BIR2BIR3; red line: XIAP-BIR2BIR3 complexed with 9a. B) distance distribution functions p(r); color code as in panel A. C) distribution of Rg values of free XIAP-BIR2BIR3; green: random pool; orange: selected ensembles fitting the data; D) distribution of Dmax values; color code as in panel C.
Figure 7
Figure 7. Scattering patterns and high resolution model of XIAP-BIR2BIR3 in presence of 9a.
A) Experimental data with associated error bars is reported in black; green line: fit using Bunch and fixed domains with χ = 3.04; red line: fit using Coral and mobile domains with distant restraints between the two heads of 9a (χ = 1.30). B) Coral model: XIAP-BIR2BIR3 is represented in white/orange surface for BIR2BIR3/missing parts build by BUNCH, respectively. 9a is in blue stick and its conformation in XIAP- (shorter helical pitch) and cIAP1-BIR2BIR3 (longer helical pitch) crystal structures are in green/purple, respectively. The dimer that fit best SAXS data has somehow an intermediate structure with respect to the two observed crystal structures.
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This work was supported by the CARIPLO Foundation Project “Inhibitors of Apoptosis Proteins (IAPs) as anticancer therapeutic targets” (2010–2012; No. 2009-2534)to DD, MB and PS, which is gratefully acknowledged. FC, MB and PS are grateful to Progetto NEPENTE (15-03-3002010-3) – Regione Lombardia for financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.