Article13 January 2005free access XIAP inhibits caspase-3 and -7 using two binding sites: evolutionarily conserved mechanism of IAPs Fiona L Scott Fiona L Scott Program in Apoptosis and Cell Death Research, The Burnham Institute, La Jolla, CA, USA Search for more papers by this author Jean-Bernard Denault Jean-Bernard Denault Program in Apoptosis and Cell Death Research, The Burnham Institute, La Jolla, CA, USA Search for more papers by this author Stefan J Riedl Stefan J Riedl Program in Apoptosis and Cell Death Research, The Burnham Institute, La Jolla, CA, USAPresent address: Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, NJ 08544, USA Search for more papers by this author Hwain Shin Hwain Shin Program in Apoptosis and Cell Death Research, The Burnham Institute, La Jolla, CA, USAPresent address: Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland Search for more papers by this author Martin Renatus Martin Renatus Program in Apoptosis and Cell Death Research, The Burnham Institute, La Jolla, CA, USAPresent address: Novartis Pharma AG, Postfach, 4002 Basel, Switzerland Search for more papers by this author Guy S Salvesen Corresponding Author Guy S Salvesen Program in Apoptosis and Cell Death Research, The Burnham Institute, La Jolla, CA, USA Search for more papers by this author Fiona L Scott Fiona L Scott Program in Apoptosis and Cell Death Research, The Burnham Institute, La Jolla, CA, USA Search for more papers by this author Jean-Bernard Denault Jean-Bernard Denault Program in Apoptosis and Cell Death Research, The Burnham Institute, La Jolla, CA, USA Search for more papers by this author Stefan J Riedl Stefan J Riedl Program in Apoptosis and Cell Death Research, The Burnham Institute, La Jolla, CA, USAPresent address: Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, NJ 08544, USA Search for more papers by this author Hwain Shin Hwain Shin Program in Apoptosis and Cell Death Research, The Burnham Institute, La Jolla, CA, USAPresent address: Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland Search for more papers by this author Martin Renatus Martin Renatus Program in Apoptosis and Cell Death Research, The Burnham Institute, La Jolla, CA, USAPresent address: Novartis Pharma AG, Postfach, 4002 Basel, Switzerland Search for more papers by this author Guy S Salvesen Corresponding Author Guy S Salvesen Program in Apoptosis and Cell Death Research, The Burnham Institute, La Jolla, CA, USA Search for more papers by this author Author Information Fiona L Scott1, Jean-Bernard Denault1, Stefan J Riedl1, Hwain Shin1, Martin Renatus1 and Guy S Salvesen 1 1Program in Apoptosis and Cell Death Research, The Burnham Institute, La Jolla, CA, USA *Corresponding author. Program for Apoptosis & Cell Death, The Burnham Institute, 10901 N Torrey Pines Road, La Jolla, CA 92037, USA. Tel.: +1 858 646 3114; Fax: +1 858 713 6274; E-mail: [email protected] The EMBO Journal (2005)24:645-655https://doi.org/10.1038/sj.emboj.7600544 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction The importance of the caspase family of proteases in apoptosis is well documented by biochemical, cell biologic and genetic studies (reviewed in Nicholson, 1999; Fesik and Shi, 2001; Denault and Salvesen, 2002). Mammals have developed regulatory proteins, members of the inhibitor of apoptosis (IAP) family, which target a subset of these enzymes. The prototype member of the family, X-linked IAP (XIAP), contains three distinct baculovirus IAP repeat (BIR) domains and a C-terminal RING finger. This protein inhibits caspases at both the initiation phase (caspase-9) and the execution phase (caspase-3 and -7) of apoptosis. The second BIR domain (BIR2) inhibits caspase-3 and -7, while the third BIR domain (BIR3) inhibits caspase-9 (Deveraux et al, 1999). The RING domain is an E3 ubiquitin ligase, promoting proteasomal degradation of XIAP, caspase-3 and second mitochondrial activator of caspases (Smac) also known as direct IAP binding protein with low pI (DIABLO; Du et al, 2000; Verhagen et al, 2000; Yang et al, 2000; Suzuki et al, 2001b; MacFarlane et al, 2002; Shin et al, 2003). No function has been attributed to the first BIR domain of XIAP. Crystal structures of BIR–caspase complexes have elucidated much of what we understand about the mechanism of XIAP inhibition of caspases. Intriguingly, despite sharing 40% identity, the individual BIR domains seem to have evolved different strategies for inhibiting the same class of protease. BIR3 inhibits caspase-9 by taking advantage of the fact that active caspases are obligate dimers (Renatus et al, 2001; Shiozaki et al, 2003). The C-terminal helix of BIR3 enforces an interface with the newly revealed caspase-9 dimer interface, locking this initiator caspase in its inactive monomeric state (Shiozaki et al, 2003). This state is secured by binding a neoepitope (encompassing the N-terminus of the small subunit) to a surface groove on BIR3. This negatively charged surface groove is conserved in many BIR domains across species. We refer to this surface region as the ‘IBM interacting groove’, because of its ability to bind to the extreme N-terminal IAP binding motif (IBM) of IBM containing proteins. These include the mammalian IAP antagonists Smac/DIABLO and HtrA2, and the Drosophila Hid, Grim and Reaper proteins (reviewed in Salvesen and Duckett, 2002; Vaux and Silke, 2003). The two essential units of BIR3 in this interaction are the IBM interacting groove and the C-terminal helix. In contrast, the structures of BIR2 in complex with either caspase-3 or -7 reveal an inhibitory mechanism that seems to be unrelated to BIR3 and caspase-9 (Chai et al, 2001; Huang et al, 2001; Riedl et al, 2001b). Protease inhibition is achieved through binding the region immediately N-terminal to the BIR2 domain (linker) across the substrate groove in a reverse orientation with respect to substrate binding. This binding prevents caspase substrates from interacting with the catalytic machinery by steric occlusion. The essential unit in this interaction is the linker region preceding BIR2. Whereas the interaction between caspase-9 and the IBM interacting groove on BIR3 is essential for binding and inhibition, there is surprisingly very little contact between the BIR2 domain and caspase-3 or -7. In fact, no electron density for the entire BIR2 domain was observed in the two structures with caspase-7 (Chai et al, 2001; Huang et al, 2001). Despite the lack of structure-based evidence, there are mounting indications that the BIR2 domain itself contributes to inhibition. Recombinant caspase-7 binds to a linker-deleted BIR2, although with lower affinity than to linker-BIR2 (Huang et al, 2001; Suzuki et al, 2001a). Indeed, the only structure of a caspase/BIR2 complex in which the BIR domain is visible reveals two other potential interactions. Within the asymmetric crystal unit, methionine 182 of caspase-3 docks into a hydrophobic pocket formed by Y154 and F228 of BIR2. An additional interaction is reminiscent of caspase-9/BIR3 complex, with the N-terminus of a caspase-3 small subunit docked into an equivalent surface groove on BIR2 (Riedl et al, 2001b). Although this binding was not observed within a biologically functional unit but as a crystal contact between symmetry mates, NMR studies also show significant chemical-shift changes in this part of the BIR2 domain when bound to caspase-3 (Sun et al, 1999). In our report on the structure of the caspase-3/BIR2 complex, we predicted that the putative IBM interacting groove of BIR2 may play a role in complex stabilization (Riedl et al, 2001b). Based on conservation of mechanism, we hypothesize that the caspase-3 and -7 inhibitory mechanism of XIAP should include mechanistic components similar to those demonstrated in caspase-9 inhibition. If this is true, the IBM interacting groove of BIR2 should play a role in inhibition. This hypothesis would require a revision of the structure-based predictions for XIAP function, and will have consequences for therapeutic intervention in the caspase/IAP axis. To test this hypothesis, we dissected the inhibitory mechanism of caspase-3 and -7 with XIAP by focusing on the role of both the BIR2 domain and putative interaction sites on caspase-3 and -7. Results The BIR2 linker inhibits executioner caspases weakly Mutagenesis and structural studies indicate that caspase-3 and -7 inhibition by XIAP is achieved through binding the linker region preceding BIR2 (linker-BIR2) across the substrate binding cleft of the protease (Sun et al, 1999; Chai et al, 2001; Huang et al, 2001; Riedl et al, 2001b). To determine whether the capacity of XIAP to inhibit caspase-3 and -7 is confined to this region, a linker peptide corresponding to residues 124–168 of XIAP was synthesized and kinetics of caspase inhibition analyzed. Previous studies showed that D148 is crucial for inhibition, so a control peptide containing asparagine at this position was also tested. A similar peptide (residues 134–154) has been investigated by others and it was concluded that it was insufficient for caspase inhibition (Sun et al, 1999). In contrast, our peptide inhibited with a Ki of 9–11 μM, demonstrating that it is sufficient for weak inhibition (Figure 1B and Table I). HPLC confirmed that it was not a substrate because it was not significantly cleaved after 3 h with 1 μM caspase-3 (data not shown). As expected, a control peptide (Asp148Asn) was even less efficient at caspase inhibition (Table I). 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 (vi/vo). (C) Integrity of GST-linker-GFP and linker-GFP. Download figure Download PowerPoint Table 1. Inhibition constant (Ki) for linker proteins/peptides with caspases Caspase-3 (nM) Caspase-7 (nM) Oligomer status BIR2 <0.4 <0.05 Monomer Linker peptide 9000 11 000 ND Control peptide D148N 319 000 >100 000 ND Linker-GFP 276 632 Monomer GST-linker-GFP 12 51 Trimer/oligomer ND: not determined. We also produced GST-linker-GFP and linker-GFP (Figure 1C). We used these to test the hypothesis that, to be available for active site binding, the linker needs to be stabilized by a structured protein at both N- and C-termini (similar to its environment within full-length XIAP) or at the C-terminus only (like BIR2 alone). Replacing BIR2 with GFP did not restore full inhibitory function (Figure 1B and Table I). Including GST at the N-terminus improved inhibition 12- to 23-fold, yet it still fell short by 30- to 1000-fold compared to linker-BIR2. These data are in line with other reports using GST-linker chimeras: IC50 of 100 nM (Chai et al, 2001) and Kd of 35 nM (Huang et al, 2001) with caspase-7. GST-linker fusions have artificially high binding affinities, which may be due to oligomerization of the linker, driven by GST and confirmed by gel filtration analysis (Table I; Vargo et al, 2004). Importantly, our results demonstrate that the BIR2 domain itself, not just any carrier protein, endows the linker with tight binding inhibition of caspase-3 and -7. Our data also show that GST chimeras should be avoided in kinetic studies of caspase inhibition by IAPs because the oligomeric status leads to overestimation of inhibitory constants. The cleavage position within the executioner caspase linker is important for efficient inhibition by BIR2 of XIAP Cathepsin G (CatG) can activate procaspase-7 by cleavage at Q196 (Casp7-Q196), two residues upstream from the canonical activation site (D198) that is cleaved by Granzyme B (GraB) and initiator caspase-8 and -9 (Figure 2A; Zhou and Salvesen, 1997). The resulting small subunit N-terminus is ADSG—as opposed to SGPI—for GraB-activated caspase-7 (Casp7-D198). Kinetic characterization of Casp7-Q196 and Casp7-D198 shows that they are essentially identical enzymes (Supplementary Table I; Zhou and Salvesen, 1997). However, they differ dramatically in their interaction with XIAP and BIR2, which were unable to inhibit efficiently caspase-7 activated with CatG compared to GraB (Figure 2C and Table II). Loss of inhibitory activity was not due to degradation of XIAP or BIR2 by residual protease activity (Figure 2D). Therefore, like caspase-9, the position of cleavage within the executioner caspase linker is important for downstream regulation by XIAP (Srinivasula et al, 2001). We hypothesize that cleavage not only activates the caspase but also reveals a motif that interacts with the IBM interacting groove on BIR2, contributing to inhibition. 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 (vi/vo). (D) Samples from (C) containing the highest inhibitor concentration were analyzed by SDS–PAGE. Download figure Download PowerPoint Table 2. Inhibition constant (Ki, nM) for BIR2 with caspase-7 variants Casp7-Q196 287 Casp7-D198 5.0 Casp7-D206 <0.3 Up to this point, our studies had been performed with recombinant caspase-7 that is processed at D198, with variable amounts of additional autocatalytic cleavage at D206 (Denault and Salvesen, 2003). The cleavage following D198 results in the liberation of SGPI—whereas the D206 cleavage results in ANPR—at the N-terminus of the small subunit (Figure 2A). We therefore asked whether processing at D198 or D206 differentially influences downstream regulation by BIR2 of XIAP. We produced two different variants of caspase-7. Casp7-D198 was made by processing procaspase-7 with GraB. Casp7-D206 was generated by expression of a linker mutant where NDTD206 was replaced with IEPD206. This caspase-7 is cleaved at D198 followed rapidly by cleavage at D206 during expression in Escherichia coli, with removal of the S199-D206 peptide. Both enzymes contain an identical large subunit but differ in length and sequence of the small subunit N-terminus (Figure 3A). Characterization of these enzymes shows that they are catalytically equivalent (Supplementary Table I). However, BIR2 inhibits Casp7-D206 at least 10 times more efficiently than Casp7-D198 (Figure 3B and Table II). To determine whether this was due to an artifact of using a small synthetic substrate, we used a large protein substrate—the C2A mutant of baculovirus protein p35—which converts this caspase inhibitor to a substrate (Riedl et al, 2001c; Xu et al, 2001). Again, Casp7-D206 was inhibited more efficiently by BIR2 than Casp7-D198 (Figure 3C). 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 (vi/vo). (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. Download figure Download PowerPoint These data demonstrate that the position of cleavage within the caspase-7 interdomain linker is important for efficient inhibition by XIAP. In turn, this suggests that XIAP inhibition of executioner caspases involves a two-site binding mechanism. Within XIAP, the BIR2 linker binds directly across the active site of the caspase but with low affinity. We hypothesize that the BIR2 domain provides a second binding site that stabilizes the inhibitory interaction. A crystal structure of the caspase-3/BIR2 complex reveals two other potential interactions involving the BIR2 domain: (a) M182 of caspase-3 docks into a hydrophobic pocket formed by Y154 and F228 of BIR2 and (b) the caspase-3 small subunit N-terminus and the IBM interacting groove of BIR2 (Riedl et al, 2001b). We produced XIAP and BIR2 variants to test biochemically the contribution these interactions make to inhibition of caspase-3 and -7 (Figure 1A). Caspase-3 M182 binding to BIR2 hydrophobic pocket does not contribute to inhibition Methionine 182 of capase-3 forms interactions with Y154 and F228 of BIR2 (Riedl et al, 2001b). Considering there is no electron density for this region of BIR2 when it is bound to caspase-7, the significance of this interaction for executioner caspase inhibition remains unclear. Mutating either the caspase-3 interacting residue (M182A) or the BIR2 interacting residues (Y154N and F228P, converting them to the residues found in the BIR2 domain of cIAP1 to conserve structure) had no significant effect on caspase-3 and -7 inhibition, ruling out this interaction as significant in generating tight inhibition (Table III and Supplementary Figure 1). Table 3. Inhibition constant (Ki, nM) for XIAP mutants with caspases BIR2 (124–237) XIAP (1–497) Casp3 Casp7 Casp3 Casp7 Wild-type <0.4 <0.05 <0.8 <0.07 D148A >1000 1.7 >1000 1.1 E219R H223V 2.3 2.2 3.1 1.1 D148A E219R H223V >1000 >1000 >1000 >1000 Y154N <0.4 <0.05 ND ND Y154N E219R H223V F228P 2.8 3.5 ND ND ND: not determined. The IBM interacting groove of BIR2 is important for efficient inhibition of caspase-3 and -7 Based on the crystal contact interactions, single point mutations were introduced to ablate the putative IBM interacting groove on BIR2 (Figure 4A). To maintain structural integrity within the BIR domain, residues were mutated to corresponding residues found in other BIR domains (BIR1 of XIAP, or BIR2 of cIAP1). Unfolding studies with guanidinium chloride confirmed that BIR2 (124–237) mutants were folded and had conformational stabilities comparable to wild-type BIR2 (data not shown). Against caspase-3 and -7, all proteins containing wild-type BIR2 domain showed comparable inhibition constants: Ki<1 nM for caspase-3 and <0.1 nM for caspase-7 (Table III). These inhibition constants are comparable to previous reports (Deveraux et al, 1997; Takahashi et al, 1998; Sun et al, 1999; Riedl et al, 2001b; Silke et al, 2001; Suzuki et al, 2001a). It was not possible to measure the Ki more accurately because the interaction is too tight for the sensitivity of our detection system. XIAP and BIR2 possessing mutations in the IBM interacting groove were consistently weaker inhibitors of caspase-3 and -7 (Table III). In agreement with previous reports, mutation of D148A within the N-terminal BIR2 linker results in complete loss of caspase-3 inhibition (Sun et al, 1999; Suzuki et al, 2001a). By comparison, mutation of D148 had less impact on caspase-7 inhibition. This is consistent with GST pull-down experiments where caspase-7 co-precipitates with GST-XIAP D148A and GST-BIR2 D148A (Suzuki et al, 2001a). Strikingly, when D148A is combined with the E219R H223V surface groove mutations, complete loss of caspase-7 inhibition occurs. These data show that the IBM interacting groove on BIR2 is a secondary binding site contributing to the overall efficiency of inhibition, and may be more important for caspase-7 than caspase-3. 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. Download figure Download PowerPoint Biological significance of the two proposed interaction sites We tested the significance of the IBM interacting groove in cells triggered to undergo apoptosis via the extrinsic apoptosis pathway or by a direct executioner caspase route. A caspase activity assay (DEVDase) and morphology assay (Annexin V binding) were used to assess apoptosis, and equivalent expression of each XIAP mutant was confirmed by Western blot (Figure 5A). The XIAP IBM interacting groove mutant E219R H223V is less effective than wild-type XIAP at inhibiting endogenously activated executioner caspases (primarily, caspase-3) as assessed by hydrolysis of Ac-DEVD-afc (Figure 5A). However, there was no significant difference in cell death protection between wild-type and E219R H223V mutant in response to exogenous TRAIL or Fas/CD95 transfection (Figure 5B and C). In contrast, the D148A linker mutation completely abrogates caspase inhibition and reduces protection from apoptosis. Significantly, when both binding sites are abrogated (D148A E219R H223V), there is an enhancement of caspase activity and cell death in response to TRAIL (Figure 5A and B). To address specifically caspase-7 regulation, we transfected caspase-7 minus its regulatory N-peptide into 293A cells. This enzyme induces apoptosis without activating any other caspases (Denault and Salvesen, 2003). In response to ectopic expression of active caspase-7, compared to wild-type XIAP the E219R H223V mutant was as defective as the D148A mutant in protecting from cell death (P>0.05; Figure 5D). In summary, the importance of both the linker and IBM interacting groove of BIR2 for executioner caspase inhibition was confirmed in the context of dying cells. 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. Download figure Download PowerPoint The IBM interacting grooves on BIR2 and BIR3 are similar in surface charge and topography. Consequently, there is a formal possibility that the BIR3 groove may also contribute to caspase-3 or -7 inhibition in the context of full-length XIAP. Mutation of this groove (Q319R W323V) had no effect on protection from TRAIL-induced apoptosis (Figure 5A) or on caspase-3 and -7 inhibition in kinetic assays with recombinant protein, while losing all affinity for caspase-9 (data not shown). This is in agreement with the finding that caspase-3 and -9 can bind XIAP simultaneously (Bratton et al, 2002). BIR2 binds ANPR-Smac with higher affinity than SGPI-Smac We propose that the N-terminus of the inhibited caspase small subunit occupies the IBM interacting groove of BIR2 and that this interaction stabilizes binding of the BIR2 linker across the catalytic binding site. We used wild-type Smac, and mutants containing various N-termini, to assess the binding specificity of BIR2. To confirm that Smac binds at this site, we performed a co-precipitation assay with GST-BIR2 mutated at its IBM interacting groove (E219R H223V) and demonstrated that Smac no longer binds (Figure 6A). We then asked whether the IBM interacting groove in BIR2 could bind sequences corresponding to the N-terminus of the two alternatively cleaved caspase-7 small subunits (ANPR and SGPI). MVPI-Smac was used as a negative control (Chai et al, 2000). ANPR-Smac binds to GST-BIR2 as efficiently as wild-type Smac under these conditions (AVPI; Figure 6C). SGPI-Smac bound less efficiently, which agrees with the observation that Ala and Ser are tolerated at the N-terminus of BIR2 binding peptides from combinatorial libraries, but Ser is less represented than Ala (BE Turk and LM Martins, personal communication). These results also explain why Casp7-D198 is more poorly inhibited by BIR2 compared to Casp7-D206 (Figure 3). Finally, we asked whether a Smac IBM peptide could antagonize caspase-3 inhibition by BIR2 E219R H223V. Smac 7-mer efficiently interferes with enzyme inhibition (Figure 6D). This indicates that Smac peptide and caspase-3 compete for a shared binding site on BIR2. Strikingly, caspase inhibition by BIR2 E219R H223V was unaffected. We conclude that Smac/DIABLO directly competes with the executioner caspase small subunit for binding to the IBM interacting groove on BIR2, again demonstrating the significance of this second binding component for caspase-3 and -7 inhibition. 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 (vi/vo). Download figure Download PowerPoint Discussion The fundamental mechanism of specific protein interactions is usually conserved during protein evolution. According to conservation of mechanism, the two units of XIAP that inhibit caspases should preserve a fundamental interaction strategy. On the basis of structural studies, this concept could be questioned. The key elements of caspase-9 inhibition by BIR3 are the IBM interacting groove and the C-terminal helix (Shiozaki et al, 2003). In contrast, the key element of caspase-3 and -7 inhibition by BIR2 seems to be the completely nonconserved N-terminal linker region (Chai et al, 2001; Huang et al, 2001; Riedl et al, 2001b). The most conserved surface structure of BIR domains is the IBM interacting groove. It is found on many BIR domains including the BIR2 and BIR3 of XIAP, and th
