Recognition of Smac-mimetic compounds by the BIR domain of cIAP1

Abstract

Inhibitor of apoptosis proteins (IAPs) are negative regulators of apoptosis. As IAPs are overexpressed in many tumors, where they confer chemoresistance, small molecules inactivating IAPs have been proposed as anticancer agents. Accordingly, a number of IAP-binding pro-apoptotic compounds that mimic the sequence corresponding to the N-terminal tetrapeptide of Smac/DIABLO, the natural endogenous IAPs inhibitor, have been developed. Here, we report the crystal structures of the BIR3 domain of cIAP1 in complex with Smac037, a Smac-mimetic known to bind potently to the XIAP-BIR3 domain and to induce degradation of cIAP1, and in complex with the novel Smac-mimetic compound Smac066. Thermal stability and fluorescence polarization assays show the stabilizing effect and the high affinity of both Smac037 and Smac066 for cIAP1- and cIAP2-BIR3 domains.

Figure 1

Chemical structures of Smac005, Smac010, Smac037, and Smac066. Note the four different substitutions in the fourth position of the central ring. In brackets the chemical structure of Smac001.

Figure 2

Fluorescence Polarization Assays. (A) Fluorescence polarization saturation curves of the probe used (2 nM) mixed with increasing concentrations of recombinant cIAP1- (left) and cIAP2-BIR3 (right). (B) Displacement of the fluorescent probe from cIAP1- (left) and cIAP2-BIR3 (right) using the unsubstituted Smac001 as matched up-control and the three 4-substituted azabicyclo[5.3.0]alkane Smac-mimetics (Smac005, Smac010, and Smac037). The symbols represent Smac001(▪), Smac005 (▴), Smac010 (▾), and Smac037 (♦). The curve for Smac066 is not reported because its affinity exceeds the sensitivity limit of the probe.

Figure 3

Overall architecture of cIAP1-BIR3/Smac037 and /Smac066 complexes (A, B) and details of the cIAP1-BIR3 interaction with Smac037 and Smac066 (C, D). (A, B) The cIAP1-BIR3 molecule is drawn according to its secondary structure: the six α-helices building BIR3 are shown in purple, the three anti-parallel β-strands in yellow, the two β-turns in pink, and, finally, coiled regions are represented in grey. The core Zn-atom is represented as an orange sphere. The magenta cage shows the Fo-Fc difference Fourier map (contoured at 7.5 σ), calculated after few refinement cycles of the protein structure alone. The difference electron density falls in the IBM groove, comprised between the β3 strand and the α3 helix, where the Smac-mimetics bind (panel A, Smac037; panel B, Smac066) (represented in fat bonds coloured by atom with green carbons, blue nitrogens, and red oxygens). (C, D) The main residues involved in stabilizing interactions with the Smac-mimetics are labelled and shown in gold. The Smac-mimetics are represented in fat bonds with magenta carbons, nitrogen atoms in blue, and oxygens in red; the Smac-mimetics interacting atoms are labelled in magenta; cIAP1-BIR3 overall structure is shown in light green. The main hydrogen bonds linking BIR3 and the Smac-mimetics are shown as red dashed lines (drawn with PyMOL41).

Figure 4

cIAP1-BIR3/SmacAVPI and cIAP1-BIR3/Smac037 superposition (A), XIAP-BIR3/Smac037 and cIAP1-BIR3/Smac037 structure superposition (B), cIAP1-BIR3, cIAP2-BIR3, and XIAP-BIR3 alignment (C), and cIAP2 degradation assay (D). (A) The superposition of cIAP1-BIR3/SmacAVPI and cIAP1-BIR3/Smac037 highlights the relative locations of Smac AVPI (light blue) and Smac037 (magenta) once bound to cIAP1-BIR3 (green ribbons). The interacting residues in cIAP1-BIR3 are represented in gold. The figure also reports the detail of the negative patch composed by the negatively charged residues Glu311, Asp314 and Glu319, that produces a Smac037 displacement of 0.5 Å relative to Smac AVPI towards the negative patch. The hydrophobic region designed by Trp310 side chain is also shown in grey, to evidence its role in the Smac037 shift of 0.8 Å relative to Smac AVPI, away from the β3 strand (structure and surfaces drawn with Pymol41). (B) XIAP-BIR3/Smac037 and cIAP1-BIR3/Smac037 superposition: cIAP1-BIR3 (green ribbons, 60% transparency) binding groove residues are in gold sticks, corresponding XIAP-BIR3 (cyan ribbons, 60% transparency) residues are highlighted in cyan. Substituted residues are labeled as follows (cIAP-BIR3/XIAP-BIR3). Smac037 in complex with cIAP1-BIR3 is reported in magenta, whereas the ligand in complex with XIAP-BIR3 is shown in cyan sticks. (C) The cIAP1-BIR3 and XIAP-BIR3 IBM-specific groove sequence alignment: the nonconservatively substituted residues are reported in magenta, whereas the residues that are conservatively substituted are reported in light blue. (D) cIAPs degradation in presence of Smac037 and Smac066 in MDA-MB231 cell line. The blot was normalized for the levels of b-Actin. The DMSO lane refers to untreated cells.