XIAP inhibits caspase-3 and -7 using two binding sites: evolutionarily conserved mechanism of IAPs

Abstract

The X-linked inhibitor of apoptosis protein (XIAP) uses its second baculovirus IAP repeat domain (BIR2) to inhibit the apoptotic executioner caspase-3 and -7. Structural studies have demonstrated that it is not the BIR2 domain itself but a segment N-terminal to it that directly targets the activity of these caspases. These studies failed to demonstrate a role of the BIR2 domain in inhibition. We used site-directed mutagenesis of BIR2 and its linker to determine the mechanism of executioner caspase inhibition by XIAP. We show that the BIR2 domain contributes substantially to inhibition of executioner caspases. A surface groove on BIR2, which also binds to Smac/DIABLO, interacts with a neoepitope generated at the N-terminus of the caspase small subunit following activation. Therefore, BIR2 uses a two-site interaction mechanism to achieve high specificity and potency for inhibition. Moreover, for caspase-7, the precise location of the activating cleavage is critical for subsequent inhibition. Since apical caspases utilize this cleavage site differently, we predict that the origin of the death stimulus should dictate the efficiency of inhibition by XIAP.

Figure 1

The BIR2 linker is a weak caspase-3 inhibitor. (A) Schematic diagram of XIAP and mutants used in this study. (B) Analysis of caspase-3 inhibition. A 100 pM portion of caspase-3 was preincubated with varying inhibitor concentrations at 37°C for 30 min and residual enzyme activity was analyzed by Ac-DEVD-afc hydrolysis. Relative activity is expressed as the ratio of inhibited to uninhibited enzyme activity (v_i/v_o). (C) Integrity of GST-linker-GFP and linker-GFP.

Figure 2

Caspase-7 must be activated by cleavage at the correct linker position for efficient inhibition by XIAP. (A) Schematic diagram of caspase-7 showing activating cleavage sites within the linker sequence (adapted from Zhou and Salvesen, 1997). (B) Procaspase-7 was activated by incubation with optimal amounts of the serine proteases GraB or CatG. A 20 μl portion of each reaction was analyzed by SDS–PAGE and stained with GELCODE Blue. (C) A 5 nM portion of active Casp7-D198 (○) or Casp7-D196 (•) was incubated with either BIR2 or XIAP for 15 min at 37°C in modified caspase buffer. Ac-DEVD-afc was added to each reaction and the relative activity expressed as the ratio of inhibited to uninhibited enzyme activity (v_i/v_o). (D) Samples from (C) containing the highest inhibitor concentration were analyzed by SDS–PAGE.

Figure 3

BIR2 is less efficient at inhibiting caspase-7 cleaved at D198 compared to D206. (A) Recombinant proteins were analyzed by SDS–PAGE. Casp7-D206: caspase-7 processed at both D198 and D206; Casp7-D198: caspase-7 cleaved at D198 with GraB. (B) A 5 nM portion of Casp7-D198 (○) or Casp7-D206 (•) was incubated with BIR2 in modified caspase buffer for 15 min at 37°C and 100 μM Ac-DEVD-afc was added to each reaction and the residual enzyme activity expressed as the ratio of inhibited to uninhibited enzyme activity (v_i/v_o). (C) A 5 nM portion of Casp7-D198 or Casp7-D206 was incubated with BIR2 for 15 min at 37°C. Recombinant p35 C2A (3 μM), a noninhibitory protein substrate, was added and incubated for a further 30 min at 37°C. Monitoring cleavage of p35 C2A by SDS–PAGE assessed residual enzyme activity. The data demonstrate that BIR2 is at least a 10-fold better inhibitor of Cas7-D206 than of Casp7-D198.

Figure 4

(A) As a crystal contact interaction between asymmetric units, the caspase-3 small subunit N-termini (SGVDDD₃₁₅; green sticks) docks into a conserved surface groove on the BIR2 domain (taken from PDB 1I3O; Riedl et al, 2001b). This binding mode is almost identical to (B) caspase-9 small subunit N-termini (green sticks) binding to the analogous surface groove on BIR3 of XIAP (taken from PDB 1NW9; Shiozaki et al, 2003). BIR domains are in gray trace with interacting residues in magenta. The BIR-coordinated zinc atom does not directly contact the ligands, but is included as reference.

Figure 5

Ablation of the IBM interacting groove of BIR2 reduces XIAP's potency as an executioner caspase inhibitor. (A) 293A cells were transfected with 0.5 μg of myc-XIAP wild-type or mutant constructs, treated with 100 ng/ml TRAIL for 2 h and lysed in mRIPA buffer. One-tenth of the lysate was added to 100 μM Ac-DEVD-afc. Initial rates were analyzed and normalized for protein amount. Lysates from duplicate, untreated transfectants were balanced for equal protein, electrophoresed by reducing SDS–PAGE and immunoblotted with mouse anti-XIAP or mouse anti-HSP90 antibody as a loading control. 293A cells were (B) transfected with 0.45, 0.35, 0.25 or 0.15 μg myc-XIAP variant plasmid and treated with 100 ng/ml TRAIL for 2 h; (C) cotransfected with 0.5 μg Fas and 0.5 μg myc-XIAP mutants; (D) cotransfected with 0.25 μg ΔN-caspase-7 and 2.75 μg myc-XIAP mutants. (B–D) FACS analysis of Annexin V-PE-stained cells was performed. (A, C, D) Data were analyzed with a two-tailed paired Student's t-test (n=3; ^*P<0.05; ^**P<0.01). For (D), P-values are derived from comparison with wild-type XIAP.

Figure 6

Specificity of the IBM interacting groove of BIR2. (A) A 5 μl portion of GST-BIR2 beads or GST-BIR2 E219R H223V beads was incubated with 100 nM AVPI-Smac or ANPR-Smac in binding buffer for 30 min at 4°C and bound proteins eluted by boiling in SDS sample buffer. Samples were analyzed by SDS–PAGE. ‘Input' corresponds to 20% of the total input prior to addition of beads. Top panel: immunoblot with rabbit anti-Smac antibody; bottom panel: GELCODE Blue stain of corresponding gel. (B) SDS–PAGE analysis of recombinant Smac mutants. (C) A 5 μl portion of GST or GST-BIR2 Sepharose beads was incubated with 100 nM AVPI-Smac, SGPI-Smac, ANPR-Smac or MVPI-Smac in binding buffer for 30 min at 4°C. Samples were processed as in (A). (D) A 300 pM portion of caspase-3 was incubated for 30 min with 5 nM wild-type BIR2 or 30 nM BIR2 E219R H223V in the presence of 0, 1, 10 or 100 μM Smac 7-mer (AVPIAQK). Ac-DEVD-afc (100 μM) was added and the residual enzyme activity expressed as the ratio of inhibited to uninhibited enzyme activity (v_i/v_o).

Figure 7

Proposed two-site binding model for caspase-3 and -7 inhibition by XIAP. The BIR2 domain is shown in surface representation (A), with the N-terminus of caspase-3 small subunit lining the IBM interacting groove in a ‘domain swapping' mode, as seen in the crystal structure of caspase-3/BIR2 (Riedl et al, 2001b). We propose that in solution the contact is made by the small subunit N-terminus of the domain that forms a partner to the inhibited domain (see white arrow in the inset). The model is more clearly seen in a cartoon (B), where the red circles represent the catalytic sites of a caspase dimer, and the yellow patch represents the IBM interacting groove on BIR2. Panel (C) highlights the degree of species conservation across the interdomain linker of caspase-7. The distant Fugu protein is probably close to the ancestor of both caspase-3 and -7.