A Single BIR Domain of XIAP Sufficient for Inhibiting Caspases

Abstract

The inhibitor of apoptosis proteins (IAPs) constitute an evolutionarily conserved family of homologous proteins that suppress apoptosis induced by multiple stimuli. Some IAP family proteins, including XIAP, cIAP-1, and cIAP-2, can bind and directly inhibit selected caspases, a group of intracellular cell death proteases. These caspase-inhibiting IAP family proteins all contain three tandem BIR domains followed by a RING zinc finger domain. To determine the structural basis for caspase inhibition by XIAP, we analyzed the effects of various fragments of this IAP family protein on caspase activity in vitro and on apoptosis suppression in intact cells. The RING domain of XIAP failed to inhibit the activity of recombinant caspases-3 or -7, whereas a fragment of XIAP encompassing the three tandem BIR domains potently inhibited these caspases in vitro and blocked Fas (CD95)-induced apoptosis when expressed in cells. Further dissection of the XIAP protein demonstrated that only the second of the three BIR domains (BIR2) was capable of binding and inhibiting these caspases. The apparent inhibition constants (Ki) for BIR2-mediated inhibition of caspases-3 and -7 were 2–5 nm, indicating that this single BIR domain possesses potent anti-caspase activity. Expression of the BIR2 domain in cells also partially suppressed Fas-induced apoptosis and blocked cytochrome c-induced processing of caspase-9 in cytosolic extracts, whereas BIR1 and BIR3 did not. These findings identify BIR2 as the minimal caspase-inhibitory domain of XIAP and indicate that a single BIR domain can be sufficient for binding and inhibiting caspases. The inhibitor of apoptosis proteins (IAPs) constitute an evolutionarily conserved family of homologous proteins that suppress apoptosis induced by multiple stimuli. Some IAP family proteins, including XIAP, cIAP-1, and cIAP-2, can bind and directly inhibit selected caspases, a group of intracellular cell death proteases. These caspase-inhibiting IAP family proteins all contain three tandem BIR domains followed by a RING zinc finger domain. To determine the structural basis for caspase inhibition by XIAP, we analyzed the effects of various fragments of this IAP family protein on caspase activity in vitro and on apoptosis suppression in intact cells. The RING domain of XIAP failed to inhibit the activity of recombinant caspases-3 or -7, whereas a fragment of XIAP encompassing the three tandem BIR domains potently inhibited these caspases in vitro and blocked Fas (CD95)-induced apoptosis when expressed in cells. Further dissection of the XIAP protein demonstrated that only the second of the three BIR domains (BIR2) was capable of binding and inhibiting these caspases. The apparent inhibition constants (Ki) for BIR2-mediated inhibition of caspases-3 and -7 were 2–5 nm, indicating that this single BIR domain possesses potent anti-caspase activity. Expression of the BIR2 domain in cells also partially suppressed Fas-induced apoptosis and blocked cytochrome c-induced processing of caspase-9 in cytosolic extracts, whereas BIR1 and BIR3 did not. These findings identify BIR2 as the minimal caspase-inhibitory domain of XIAP and indicate that a single BIR domain can be sufficient for binding and inhibiting caspases. IAP 1The abbreviations used are: IAP, inhibitor of apoptosis protein; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; AFC, 7-amino-4-trifluoromethyl-coumarin; DEVD, Asp-Glu-Val-Asp; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GFP, green fluorescence protein; DAPI, 4′,6-di- amidino-2-phenylindole. family proteins have been identified in baculoviruses, Drosophila, and humans, where they function as suppressors of apoptosis (1Clem R.J. Miller L.K. Mol. Cell. Biol. 1994; 14: 5212-5222Crossref PubMed Scopus (496) Google Scholar, 2Hay B.A. Wassarman D.A. Rubin G.M. Cell. 1995; 83: 1253-1262Abstract Full Text PDF PubMed Scopus (647) Google Scholar, 3Roy N. Mahadevan M.S. McLean M. Shutler G. Yaraghi Z. Farahani R. Baird S. Besner-Johnson A. Lefebvre C. Kang X. Salih M. Aubry H. Tamai K. Guan X. Ioannou P. Crawford T.O. de Jong P.J. Surh L. Ikeda J.-E. Korneluk R.G. MacKenzie A. 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In all cases examined thus far, apoptosis has been shown to depend upon the activation of a group of intracellular cysteine proteases with specificity for aspartic acid in the P1 position of substrates, called caspases (reviewed in Ref. 20Salvesen G.S. Dixit V.M. Cell. 1997; 91: 443-446Abstract Full Text Full Text PDF PubMed Scopus (1943) Google Scholar). The human XIAP, cIAP-1, and cIAP-2 proteins can bind to and directly inhibit selected caspases. The caspases identified as targets of these IAP family proteins thus far include caspases-3, -7, and -9 but not caspases-1, 2, 8, or 10 (9Roy N. Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1140) Google Scholar, 10Deveraux Q. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; 388: 300-303Crossref PubMed Scopus (1724) Google Scholar, 24Deveraux Q.L. Roy N. Stennicke H.R. Van Arsdale T. Zhou Q. Srinivasula S.M. Alnemri E.S. Salvesen G.S. Reed J.C. EMBO J. 1998; (in press)PubMed Google Scholar). 2R. Takahashi, Q. Deveraux, I. Tamm, K. Welsh, N. Assa-Munt, G. S. Salvesen, and J. C. Reed, submitted for publication. XIAP, cIAP-1, and cIAP-2 all contain three tandem copies of a ∼70-amino acid motif termed the BIR (baculovirusIAP repeat) domain, reflecting the historical discovery of IAP family proteins originally in baculoviruses by Miller and colleagues (1Clem R.J. Miller L.K. Mol. Cell. Biol. 1994; 14: 5212-5222Crossref PubMed Scopus (496) Google Scholar, 11Crook N.E. Clem R.J. Miller L.K. J. Virol. 1993; 67: 2168-2174Crossref PubMed Google Scholar). These IAP family proteins also contain a RING zinc finger domain near their C termini. The IAP family protein NAIP, in contrast, contains three tandem BIRs but lacks the RING domain (3Roy N. Mahadevan M.S. McLean M. Shutler G. Yaraghi Z. Farahani R. Baird S. Besner-Johnson A. Lefebvre C. Kang X. Salih M. Aubry H. Tamai K. Guan X. Ioannou P. Crawford T.O. de Jong P.J. Surh L. Ikeda J.-E. Korneluk R.G. MacKenzie A. Cell. 1995; 80: 167-178Abstract Full Text PDF PubMed Scopus (875) Google Scholar). An additional member of the human IAP family, survivin, contains only a single BIR domain but nevertheless can suppress apoptosis in cells (8Ambrosini G. Adida C. Altieri D. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (3025) Google Scholar). The IAP family proteins of Drosophila and the insect-infecting baculoviruses all contain two or three tandem copies of the BIR domain followed by a RING domain (1Clem R.J. Miller L.K. Mol. Cell. Biol. 1994; 14: 5212-5222Crossref PubMed Scopus (496) Google Scholar, 2Hay B.A. Wassarman D.A. Rubin G.M. Cell. 1995; 83: 1253-1262Abstract Full Text PDF PubMed Scopus (647) Google Scholar, 11Crook N.E. Clem R.J. Miller L.K. J. Virol. 1993; 67: 2168-2174Crossref PubMed Google Scholar). Deletional analyses of the Drosophila and baculovirus IAP family proteins have produced conflicting results, with some studies suggesting a critical role for both the BIR and RING domains and others implying that BIRs are directly involved in apoptosis suppression with the RING motif functioning possibly as a negative regulatory domain (2Hay B.A. Wassarman D.A. Rubin G.M. Cell. 1995; 83: 1253-1262Abstract Full Text PDF PubMed Scopus (647) Google Scholar,12Harvey A.J. Soliman H. Kaiser W. Miller L.K. Cell Death Differ. 1998; (in press)Google Scholar). The structural similarities and differences among IAP family proteins prompted us to address the question of what constitutes the minimal domain necessary for inhibition of caspases and suppression of apoptosis. Using the human XIAP protein as a model, we determined that only one of the three BIR domains present within this protein was clearly capable of inhibiting caspases-3 and -7, as well as suppressing apoptosis when expressed in cells. These observations demonstrated that a single BIR domain can be sufficient for binding and inhibiting caspases and imply that all BIR domains may not be functionally equivalent, despite their extensive amino acid sequence identity. Plasmids encoding fragments of the XIAP protein, including BIR1+2+3 (residues 1–336), RING (residues 337–497), BIR1 (residues 1–123), BIR2 (residues 124–260), BIR3 (residues 261–336), BIR2+3 (residues 124–336), and BIR1+2 (residues 1–260) were created by a one-step polymerase chain reaction method employing a plasmid encoding a full-length XIAP as the template (10Deveraux Q. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; 388: 300-303Crossref PubMed Scopus (1724) Google Scholar) and using 5′-GGAATTCATGACTTTTAACAGTTTTGAAG-3′ (BIR1+2+3, BIR1 and BIR1+2), 5′-GGAATTCCAGCTGCAAGAGGAGAAGCTTTG-3′ (RING), 5′-GGAATTCAGAGATCATTTTGCCTTAGACA-3′ (BIR2 and BIR2+3), or 5′-GGATTCTCCATGGCAGATTATGAAGC-3′ (BIR3) as the forward primer and 5′-CTCTCGAGCATGCCTACTATAGAGTTAGA-3′ (RING), 5′-CTCTCGAGCTACTTGTCCCTTCTGTTCTAACAG-3′ (BIR1+2+3, BIR3 and BIR2+3), 5′-CTCTCGAGCTATGGATTTCTTGGAAGATTTGTTG-3′ (BIR2 and BIR1+2(, or 5′-CTCTCGAGTTACCCCATGGATCCCAGATAGTTTTCAACT-3′ (BIR1) as the reverse primer. The BIR1+3 mutant protein (see Fig. 1) was prepared by ligating the BIR1 and BIR3 polymerase chain reaction fragments digested with EcoRI+NcoI and NcoI+XhoI, respectively. All the fragments were subcloned into pGEX4T-1 (Pharmacia Biotech Inc.) and/or pcDNA3-myc (10Deveraux Q. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; 388: 300-303Crossref PubMed Scopus (1724) Google Scholar) by digestion with EcoRI and XhoI. The proper construction of all the plasmids was confirmed by DNA sequencing. For production of BIR1+2+3 and RING, pGEX4T-1-XIAP mutant plasmids were introduced into Escherichia coli strain BL21(DE3) containing the plasmid pT-Trx (13Yamakawa T. Kanei-Ishii C. Maekawa T. Fujimoto J. Yamamoto T. Ishii S. J. Biol. Chem. 1995; 270: 25328-25331Crossref PubMed Scopus (160) Google Scholar). BIR1, BIR2, and BIR3 were produced in XL-1 blue cells (Stratagene, Inc). The GST fusion proteins were prepared from the soluble fraction upon induction with 0.4 mmisopropyl-1-thio-β-d-galactopyranoside at 30 °C for 12 h, affinity purified using glutathione-Sepharose, and then dialyzed against phosphate-buffered saline. Coomassie staining analysis of GST fusion proteins following SDS-PAGE revealed ≥80% intact protein in all cases except BIR3 (see text). His6-tagged caspase-3 and -7 recombinant proteins are prepared as described (14Orth K. O'Rourke K. Salvesen G.S. Dixit V.M. J. Biol. Chem. 1996; 271: 20977-20980Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar,15Zhou Q. Snipas S. Orth K. Muzio M. Dixit V.M. Salvesen G.S. J. Biol. Chem. 1997; 272: 7797-7800Crossref PubMed Scopus (486) Google Scholar). Caspase-3 and -7 activities were assayed by release of 7-amino-4-trifluoromethyl-coumarin (AFC) from Asp-Glu-Val-Asp (DEVD)-AFC using continuous reading instruments as described (15Zhou Q. Snipas S. Orth K. Muzio M. Dixit V.M. Salvesen G.S. J. Biol. Chem. 1997; 272: 7797-7800Crossref PubMed Scopus (486) Google Scholar). Apparent Ki values were determined using 0.1–0.3 nm caspases and a range of concentrations of recombinant XIAP or XIAP deletion mutants, as described previously (9Roy N. Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1140) Google Scholar,10Deveraux Q. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; 388: 300-303Crossref PubMed Scopus (1724) Google Scholar). 2 μg of His6-tagged caspases-3 and -7 were immobilized on 4 μl of Ni2+-Sepharose and incubated with 5 μl of in vitro translated, [35S]l-methionine-labeled BIR1, BIR2, BIR2+3, BIR1+2, or BIR1+3 proteins that were prepared from pcDNA3-myc (XIAP mutants) templates using a coupled transcription/translation system involving rabbit reticulocyte lysates (TNT-lysate, Promega, Inc.) and T7 RNA polymerase. Incubations were performed in 0.1 ml of caspase buffer, consisting of 50 mm Tris-Cl (pH 7.5), 100 mm KCl, 10% sucrose, 0.1% CHAPS, and 5 mmdithiothreitol at 4 °C for 1 h. The beads were washed with 1 ml of caspase buffer three times before resuspending samples in boiling Laemmli buffer and analyzing by SDS-PAGE using 750 mmTris/15% polyacrylamide gels and autoradiography. Cytosolic extracts were prepared using 293 cells as described (10Deveraux Q. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; 388: 300-303Crossref PubMed Scopus (1724) Google Scholar). 35S-Labeled in vitro translated pro-caspase-9 (1 μl) was added to 5 μl of extracts, and caspase activation was initiated by addition of 10 μm horse heart cytochrome c (Sigma) and 1 mm dATP at 30 °C. Subconfluent 293 cells were transfected in 3-cm-diameter wells using a calcium phosphate method with 0.5 μg of the green fluorescence protein (GFP) marker plasmid pEGFP (CLONTECH), 1 μg of pCMV-Fas expression plasmid (16Sato T. Irie S. Kitada S. Reed J.C. Science. 1995; 268: 411-415Crossref PubMed Scopus (694) Google Scholar), and 5 μg of either pcDNA3-myc-XIAP mutant plasmids or the control plasmid pcDNA3-myc tag. After culturing ∼1 day, both floating and attached cells were collected, fixed in 3.7% formaldehyde in phosphate-buffered saline for 10 min, and stained with 10 μg/ml of 4′,6-diamidino-2-phenylindole (DAPI). The percentage of apoptotic cells revealed by DAPI staining was counted among the GFP-positive cells by fluorescence microscopy. Transfected 293 cells were lysed in 10 mm HEPES (pH 7.5), 142 mm KCl, 1 mmEGTA, 1 mm dithiothreitol, 0.2% Nonidet P-40, 0.1 mm phenylmethylsulfonyl fluoride and used for immunoblot analysis (9Roy N. Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1140) Google Scholar, 10Deveraux Q. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; 388: 300-303Crossref PubMed Scopus (1724) Google Scholar). Samples were normalized for total protein content (25 μg), and immunoblot analysis was performed using 750 mmTris/12–15% polyacrylamide gels and anti-Myc tag monoclonal antibody 9E10 (Santa Cruz, Inc.) with emission chemiluminescence-based detection (9Roy N. Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1140) Google Scholar, 10Deveraux Q. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; 388: 300-303Crossref PubMed Scopus (1724) Google Scholar). Fig. 1 depicts the fragments of XIAP that were expressed as GST fusion proteins in bacteria and affinity purified. The roles of the BIR and RING domains in inhibition of caspases were examined using a C-terminal truncation mutant of XIAP that contained the three tandem BIRs but lacked the downstream linker region and RING domain (BIR1+2+3) and a XIAP fragment lacking the N-terminal BIR domain but retaining the linker and RING regions (RING). Comparison were made of the effects of the BIR1+2+3 (residues 1–336) and RING (residues 337–497) fragments on recombinant caspase-3- or caspase-7-mediated cleavage of a fluorigenic tetrapeptide substrate, DEVD-AFC, in vitro. As shown in Fig.2 (A and B), the BIR1+2+3 fragment of XIAP inhibited DEVD-AFC substrate cleavage by caspases-3 and -7, whereas the C-terminal fragment of XIAP containing the RING domain did not. Next, fragments of XIAP containing each of the individual BIR domains (Fig. 1) were expressed in bacteria, purified, and tested for inhibition of recombinant caspases-3 and -7 in vitro. Of the three recombinant BIR domains, only BIR2 prevented DEVD-AFC substrate hydrolysis by caspases-3 and -7 (Fig. 2, C and D). However, because less than half of the purified BIR3 domain appeared to be intact, an additional BIR3-containing fragment of XIAP, which proved to be more stable and which combined BIR1 and BIR3 (Fig. 1), was also produced and tested. This BIR1+3 fragment of XIAP was ineffective at inhibiting caspases-3 or -7 in vitro, whereas BIR1+2 and BIR2+3 fragments that contained BIR2 completely suppressed hydrolysis of DEVD-AFC under the conditions of these in vitro protease assays (not shown). Determination of the apparent Ki for inhibition of recombinant caspases-3 and -7 by the BIR2 fragment of XIAP (residues 124–260) produced values of 1–5 nm, thus demonstrating potent suppression of these proteases (TableI). Similarly, the BIR1+2 and BIR2+3 fragments of XIAP that contained the BIR2 domain also inhibited caspases-3 and -7 with Ki values of 1–3 nm. Thus, although not as potent as the full-length XIAP molecule that has been reported to inhibit these proteases with Ki values of 0.2–0.7 nm (10Deveraux Q. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; 388: 300-303Crossref PubMed Scopus (1724) Google Scholar), the BIR2 domain nevertheless retains substantial anti-caspase activity. By comparison, the commonly used tetrapeptide inhibitor DEVD-aldehyde has been reported to inhibit caspases-3 and -7 with Kivalues of 0.5–3.5 nm (17Fernandes-Alnemri T. Takahashi A. Armstrong R. Krebs J. Fritz L. Tomaselli K.J. Wang L. Yu Z. Croce C.M. Salvesen G. Earnshaw W.C. Llitwack G. Alnemri E.S. Cancer Res. 1995; 55: 6045-6052PubMed Google Scholar, 18Margolin N. Raybuck S.A. Wilson K.P. Chen W. Fox T. Gu Y. Livingston D.J. J. Biol. Chem. 1997; 272: 7223-7228Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 19Mittl P.R.E. Marco S.D. Krebs J.F. Bai X. Karanewsky D.S. Priestle J.P. Tomaselli K.J. Grutter M.G. J. Biol. Chem. 1997; 10: 6539-6547Abstract Full Text Full Text PDF Scopus (223) Google Scholar).Table IApparent inhibition constants (Ki app (nm)) for GST-XIAP fragments measured against caspases-3 and -7KiBIR2 (124–260)BIR1+2 (1–260)BIR2+3 (124–336)BIR1 (1–123)BIR3 (261–336)nmCaspase-3533>1000>1000Caspase-7211>1000>1000 Open table in a new tab In addition to exploring their ability to inhibit the activity of caspases, the binding of recombinant caspases-3 and -7 to BIR2 and other fragments of XIAP was examined using His6-tagged caspases immobilized on Ni2+ resin and35S-labeled in vitro translated XIAP fragments. All BIR2-containing fragments of XIAP, including BIR2, BIR1+2, and BIR2+3, bound to immobilized caspases-3 and -7 (Fig.3 A). In contrast, fragments of XIAP lacking BIR2, including BIR1 and BIR1+3, failed to bind these caspases. Note that under these in vitro conditions where high concentrations of proteases are involved, caspase-3 cleaved the BIR2-containing fragments of XIAP, causing slightly faster migration of the 35S-labeled XIAP protein fragments in whereas did not these XIAP fragments (Fig.3 A). Thus, cleavage not with has been to indicating that XIAP cleavage by caspases under in intact cells. The specificity of the in vitro binding was confirmed by in which other recombinant proteins, including and were tested and not to bind the BIR2-containing fragment of XIAP (Fig. has been demonstrated that release of cytochrome c from apoptosis and in caspase activation by of a protein containing and pro-caspase-9 (reviewed in Ref. J.C. Cell. 1997; 91: Full Text Full Text PDF PubMed Scopus Google Scholar). we have that XIAP, cIAP-1, and cIAP-2 can bind and inhibit from when to cytochrome c and Q.L. Roy N. Stennicke H.R. Van Arsdale T. Zhou Q. Srinivasula S.M. Alnemri E.S. Salvesen G.S. Reed J.C. EMBO J. 1998; (in press)PubMed Google Scholar). tested BIR2 was sufficient for cytochrome c-induced processing of pro-caspase-9 in cytosolic As shown in full-length XIAP and the BIR2-containing fragment of XIAP completely blocked cytochrome c-induced processing of 35S-labeled in vitro translated pro-caspase-9 in cytosolic extracts, whereas BIR1, and fragments of XIAP were Thus, the BIR2 domain of XIAP to be sufficient for the in the apoptotic by cytochrome processing of The effects of the BIR2 of XIAP on apoptosis in cells were examined by this or other regions of XIAP in 293 which were induced to apoptosis by of of 293 cells in half the cells as determined by DAPI staining and analysis A). In contrast, Fas with plasmids full-length XIAP, or BIR2 the percentage of transfected cells Fas-induced apoptosis. the BIR2 fragment was less potent at suppressing Fas-induced apoptosis than full-length XIAP or the BIR1+2+3 fragment of XIAP, also may have been produced at in 293 cells (Fig. 5 In contrast, of Fas with plasmids fragments of XIAP that lacked BIR2, including and failed to suppress apoptosis (not shown). These XIAP mutants, were produced at with or than of on control using 293 cells that were not induced to apoptosis by Fas not these findings the that the BIR2 region of XIAP necessary and sufficient for inhibiting caspases and apoptosis. IAP family proteins contain two or three copies of the BIR domain, the that not all BIRs are in their ability to bind and inhibit caspases. In XIAP, that only the second BIR domain capable of inhibiting caspases-3 and their extensive amino acid sequence to BIR2, the BIR1 and BIR3 domains did not inhibit these caspases. the of protein when with a these findings nevertheless that BIR domains within IAP family protein may roles in the suppression of cell An that BIRs may of caspases, although for this has been that BIR domains a of protein domain. Miller and have of the baculovirus IAP family proteins with the Drosophila cell death proteins and D. Kaiser W.J. A.J. Miller L.K. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, A.J. Miller L.K. Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar), of which are caspases. to identify the targets of each of the individual BIR domains within IAP family proteins and to the structural basis for their inhibition of caspases additional into the by which suppress apoptosis. H. and K. for