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Disease‐causing mutations in the XIAP BIR 2 domain impair NOD 2‐dependent immune signalling

2013; Springer Nature; Volume: 5; Issue: 8 Linguagem: Inglês

10.1002/emmm.201303090

1757-4684

Rune Busk Damgaard, Berthe Katrine Fiil, Carsten Speckmann, Monica Yabal, Udo zur Stadt, Simon Bekker‐Jensen, Philipp J. Jost, Stephan Ehl, Niels Mailand, Mads Gyrd‐Hansen,

Immune Response and Inflammation

Research Article1 July 2013Open Access Disease-causing mutations in the XIAP BIR2 domain impair NOD2-dependent immune signalling Rune Busk Damgaard Rune Busk Damgaard Department of Disease Biology, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, DenmarkThese authors contributed equally to this work. Search for more papers by this author Berthe Katrine Fiil Berthe Katrine Fiil Department of Disease Biology, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, DenmarkThese authors contributed equally to this work. Search for more papers by this author Carsten Speckmann Carsten Speckmann CCI – Centre of Chronic Immunodeficiency, University Hospital Freiburg, Freiburg, Germany Search for more papers by this author Monica Yabal Monica Yabal III. Medizinische Klinik, Klinikum rechts der Isar, Technische Universität München, Munich, Germany Search for more papers by this author Udo zur Stadt Udo zur Stadt Center for Diagnostic, University Medical Center Hamburg Eppendorf, Hamburg, Germany Search for more papers by this author Simon Bekker-Jensen Simon Bekker-Jensen Department of Disease Biology, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Philipp J. Jost Philipp J. Jost III. Medizinische Klinik, Klinikum rechts der Isar, Technische Universität München, Munich, Germany Search for more papers by this author Stephan Ehl Stephan Ehl CCI – Centre of Chronic Immunodeficiency, University Hospital Freiburg, Freiburg, Germany Search for more papers by this author Niels Mailand Niels Mailand Department of Disease Biology, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Mads Gyrd-Hansen Corresponding Author Mads Gyrd-Hansen Department of Disease Biology, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Rune Busk Damgaard Rune Busk Damgaard Department of Disease Biology, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, DenmarkThese authors contributed equally to this work. Search for more papers by this author Berthe Katrine Fiil Berthe Katrine Fiil Department of Disease Biology, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, DenmarkThese authors contributed equally to this work. Search for more papers by this author Carsten Speckmann Carsten Speckmann CCI – Centre of Chronic Immunodeficiency, University Hospital Freiburg, Freiburg, Germany Search for more papers by this author Monica Yabal Monica Yabal III. Medizinische Klinik, Klinikum rechts der Isar, Technische Universität München, Munich, Germany Search for more papers by this author Udo zur Stadt Udo zur Stadt Center for Diagnostic, University Medical Center Hamburg Eppendorf, Hamburg, Germany Search for more papers by this author Simon Bekker-Jensen Simon Bekker-Jensen Department of Disease Biology, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Philipp J. Jost Philipp J. Jost III. Medizinische Klinik, Klinikum rechts der Isar, Technische Universität München, Munich, Germany Search for more papers by this author Stephan Ehl Stephan Ehl CCI – Centre of Chronic Immunodeficiency, University Hospital Freiburg, Freiburg, Germany Search for more papers by this author Niels Mailand Niels Mailand Department of Disease Biology, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Mads Gyrd-Hansen Corresponding Author Mads Gyrd-Hansen Department of Disease Biology, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Author Information Rune Busk Damgaard1, Berthe Katrine Fiil1, Carsten Speckmann2, Monica Yabal3, Udo zur Stadt4, Simon Bekker-Jensen1, Philipp J. Jost3, Stephan Ehl2, Niels Mailand1 and Mads Gyrd-Hansen 1 1Department of Disease Biology, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark 2CCI – Centre of Chronic Immunodeficiency, University Hospital Freiburg, Freiburg, Germany 3III. Medizinische Klinik, Klinikum rechts der Isar, Technische Universität München, Munich, Germany 4Center for Diagnostic, University Medical Center Hamburg Eppendorf, Hamburg, Germany *Corresponding author: Tel: +45 3525 5010; Fax: +45 3525 5001E-mail: [email protected] EMBO Mol Med (2013)5:1278-1295https://doi.org/10.1002/emmm.201303090 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info X-linked Inhibitor of Apoptosis (XIAP) is an essential ubiquitin ligase for pro-inflammatory signalling downstream of the nucleotide-binding oligomerization domain containing (NOD)-1 and -2 pattern recognition receptors. Mutations in XIAP cause X-linked lymphoproliferative syndrome type-2 (XLP2), an immunodeficiency associated with a potentially fatal deregulation of the immune system, whose aetiology is not well understood. Here, we identify the XIAP baculovirus IAP repeat (BIR)2 domain as a hotspot for missense mutations in XLP2. We demonstrate that XLP2-BIR2 mutations severely impair NOD1/2-dependent immune signalling in primary cells from XLP2 patients and in reconstituted XIAP-deficient cell lines. XLP2-BIR2 mutations abolish the XIAP-RIPK2 interaction resulting in impaired ubiquitylation of RIPK2 and recruitment of linear ubiquitin chain assembly complex (LUBAC) to the NOD2-complex. We show that the RIPK2 binding site in XIAP overlaps with the BIR2 IBM-binding pocket and find that a bivalent Smac mimetic compound (SMC) potently antagonises XIAP function downstream of NOD2 to limit signalling. These findings suggest that impaired immune signalling in response to NOD1/2 stimulation is a general defect in XLP2 and demonstrate that the XIAP BIR2-RIPK2 interaction may be targeted pharmacologically to modulate inflammatory signalling. INTRODUCTION Disease-causing mutations in XIAP/BIRC4 were first described in 2006 in families with patients suffering from X-linked lymphoproliferative syndrome (XLP) with no mutations in the SH2D1A gene encoding SAP (Rigaud et al, 2006). Classical XLP due to SAP deficiency (XLP1) is characterized by susceptibility to fulminant Epstein-Barr virus (EBV) infection, frequently leading to haemophagocytic lymphohistiocytosis (HLH), development of lymphoma and hypogammaglobulinemia (Purtilo et al, 1975). XLP2 caused by mutation in XIAP shares the susceptibility to EBV with a high risk of HLH, but no patient with lymphoma has so far been reported (Filipovich et al, 2010; Pachlopnik Schmid et al, 2011; Yang et al, 2012). Moreover, severe chronic colitis, hepatitis or persistent splenomegaly are increasingly reported as initial and even as the only clinical manifestations of patients with XIAP mutations [(Worthey et al, 2011), Carsten Speckmann et al, in preparation]. The molecular basis of these inflammatory manifestations remains poorly characterized. The best described cellular function of XIAP is its role in limiting apoptosis through inhibition of apoptotic caspases (Gyrd-Hansen & Meier, 2010) and, as recently reported by us and others, its role in facilitating innate immune signalling downstream of the NOD1 and NOD2 bacterial sensors (Bauler et al, 2008; Damgaard et al, 2012; Krieg et al, 2009; Lipinski et al, 2012). Caspase regulation is mediated by the N-terminal part of XIAP composed of three baculovirus IAP repeat (BIR) domains. BIR domains mediate interactions with proteins that contain an IAP binding motif (IBM) as well as other non-IBM type protein interactions (Gyrd-Hansen & Meier, 2010). IBMs are four-amino acid motifs starting with an N-terminal alanine and are present in several proteins including the processed, mature form of the mitochondrial factor Second mitochondria-derived activator of caspases (Smac; also known as direct IAP binding protein with low pI) and in cleavage-activated caspases. The XIAP BIR2 binds to the IBM in active caspase-3 and -7, and this aids the inhibition of the caspases through the linker region immediately N-terminal to the BIR2 domain (Scott et al, 2005). XIAP's role in NOD1/2 signalling relies on its ubiquitin (Ub) ligase activity provided by the C-terminal RING domain (Damgaard et al, 2012). NOD2 is a member of the NOD-like receptor family, which also includes NOD1 and NLRPs, and is particularly important for immune regulation at mucosal surfaces (Casanova & Abel, 2009; Chen et al, 2009). Accordingly, NOD2 was the first identified susceptibility gene for the inflammatory bowel disease termed Crohn's disease (Van Limbergen et al, 2009). Activation of NOD2 by the peptidoglycan component muramyl dipeptide (MDP) in the bacterial cell wall leads to recruitment of RIPK2 and the Ub ligases XIAP, cIAP1 and cIAP2 (Bertrand et al, 2009; Damgaard et al, 2012). This triggers Ub-dependent signalling events that activate mitogen-activated protein (MAP) kinases and the NF-κB-activating IκB kinase (IKK) complex composed of IKKα, IKKβ and NEMO (also termed IKKγ) (Beug et al, 2012; Damgaard & Gyrd-Hansen, 2011). XIAP conjugates Ub chains on RIPK2 together with cIAP1/2 to recruit and enable the activation of the TAK1-TAB1/2/3 and IKK kinase complexes. Full activation of the IKK complex, additionally, requires the presence of Ub chains linked via methionine 1 (M1-linked; also termed linear Ub chains) that are conjugated by the linear ubiquitin chain assembly complex (LUBAC) (Haas et al, 2009; Rahighi et al, 2009; Tokunaga et al, 2009). In turn, IKK phosphorylates IκBα to enable nuclear translocation of NF-κB transcription factors, transcription of NF-κB target genes and production of pro-inflammatory cytokines and chemokines (Bonizzi & Karin, 2004). LUBAC is a trimeric complex composed of the catalytic subunit HOIP and two adaptors HOIL-1 and SHARPIN (Gerlach et al, 2011; Ikeda et al, 2011; Tokunaga et al, 2011), and it is recruited to the NOD2 signalling complex by Ub chains conjugated by XIAP (Damgaard et al, 2012). Smac mimetic compounds (SMCs) are potent antagonists of IAP proteins and sensitize cancer cells to cell death induced by cytotoxic compounds and by TNF-receptor super family receptor ligands, including TNF. Recently, SMCs have additionally been demonstrated to deregulate inflammatory signalling downstream of TNF-receptor 1 (TNF-R1) and toll-like receptors, and to cause inappropriate activation of the NLRP1/3-inflammasome (Bertrand et al, 2008; Tseng et al, 2010; Vince et al, 2012; Wang et al, 2008). SMCs are designed to bind type-III BIR domains (BIR3 domain in cIAP1/cIAP2/XIAP), and they induce rapid auto-ubiquitylation and proteasomal degradation of cIAP1/2 by activating their Ub ligase activity (Dueber et al, 2011; Feltham et al, 2011; Gaither et al, 2007; Petersen et al, 2007; Varfolomeev et al, 2007; Vince et al, 2007). Contrary to this, SMCs do not activate XIAP's Ub ligase activity or cause its proteasomal degradation (Nakatani et al, 2013; Varfolomeev et al, 2007; Vince et al, 2007). How SMCs may affect XIAP's function in cellular signalling is currently not well understood although an SMC recently was reported to interfere with RIPK2 binding in vitro (Krieg et al, 2009). Here, we provide evidence that XLP2-causing mutations in the XIAP BIR2 domain, similar to RING domain mutations, impair NOD1/2-dependent immune signalling. We show that XLP2-BIR2 mutations abolish RIPK2 binding and that this impairs XIAP-mediated ubiquitylation of RIPK2 and NOD2-dependent induction of NF-κB target genes. Consistently, the SMC Compound A antagonized RIPK2 binding, RIPK2 ubiquitylation and NOD1/2-dependent activation of NF-κB. We conclude that defective NOD1/2 signalling is a common immune defect in XLP2 and thus may contribute to the pathogenesis, and propose that certain SMCs may be used to modulate NOD2-mediated inflammation. RESULTS XLP2-derived BIR2 mutations abrogate NOD2-dependent signalling Most XIAP mutations identified in XLP2 patients are nonsense mutations, frameshift mutations or deletions that cause severe aberrations in the encoded protein or loss of expression (Filipovich et al, 2010; Marsh et al, 2010; Pachlopnik Schmid et al, 2011; Yang et al, 2012). These mutations are positioned throughout XIAP and almost invariably interfere with the integrity of the C-terminal RING domain (Fig 1A). Several missense mutations have also been identified in XIAP, locating either to the RING or to the BIR2 domain. We report here three novel missense mutations identified in XLP2 patients, c.497G > T (p.R166I), c.620T > C (p.L207P) and c.592G > A (p.V198M), all locating to the BIR2 domain of XIAP [Fig 1A; during preparation of this manuscript the R166I mutations has also been reported by others (Marsh et al, 2013)]. The BIR2-mutated patients presented with EBV-induced HLH including pronounced splenomegaly (XLP phenotype) at age 9 years (p.R166I), 17 years (p.L207P) and 11 years (p.V198M), respectively. Clinical data from one patient (p.R166I) was recently published (P7 in Marsh et al, 2013). He died from acute Graft versus Host Disease (GvHD) and multi-organ failure after peripheral blood stem cell (PBSC) transplantation from an unrelated donor at 14 years. The other patients (p.L207P and p.V198M) had acute EBV-induced HLH and are currently alive and well after the initial treatment with immunosuppressants and rituximab. Clinical details of these patients will be described elsewhere (Carsten Speckmann et al, in preparation). Thus, of nine reported missense mutations, six cause single-amino acid substitutions in the BIR2 domain. Cross-species alignment of the amino acid sequence of the XIAP BIR2 domain and other type-II BIR domains (Eckelman et al, 2008) show that all of the XLP2-mutated residues are highly conserved between IAP proteins and through evolution (Fig 1B). This suggests that the mutated residues are important for the function of the BIR2 domain. Consistently, none of the BIR2 mutations have been reported as single-nucleotide polymorphisms [SNPs; source: Database of SNPs at the National Centre of Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/snp)]. Figure 1.XLP2-causing BIR2 mutations abrogate NOD1/2 signalling Schematic showing position and type of XIAP mutations identified in XLP2 patients. Superscript letters refer to the original report of the mutation: a, (Rigaud et al, 2006); b, (Marsh et al, 2009); c, (Marsh et al, 2010); d, (Zhao et al, 2010); e, (Filipovich et al, 2010); f, (Pachlopnik Schmid et al, 2011); g, (Worthey et al, 2011); h, (Yang et al, 2012). (*) Denotes that the mutation is listed as an SNP. Number sign (#) indicates that the mutation was incorrectly annotated in the original report. Amino acid sequences of type-II BIR domains of IAPs were aligned using ClustalX. Graph below aligned sequences shows conservation of amino acid residues. Filled circles denote the E219 and H223 (XIAP numbering) residues that define type-II BIR domains. Open circles indicate residues involved in coordinating the Zn2+ ion. XLP2-BIR2 mutations studied here are indicated in colour. Schematic view of XIAP's role in NOD1/2 signalling. In response to NOD1/2 activation by peptidoglycans (PGN), XIAP is recruited to the signalling complex by RIPK2 where it ubiquitylates RIPK2. This enhances recruitment of LUBAC and facilitates activation of NF-κB. NF-κB activity in lysates of WT and reconstituted XIAP-deficient HCT-116 cells. Cells were transfected as indicated and stimulated with L18-MDP (200 ng/mL) for 24 h. Data represent mean + s.e.m. (n = 4–6). (*) Indicates P = 0.0001 for G188E, P = 0.0001 for C203Y, P = 0.0001 for L207P, P = 0.0002 for R166I, P = 0.0009 for W173G, P = 0.0001 for V198M, all vs. WT. Expression of XIAP variants in XIAP-deficient HCT-116 cells were analysed by immunoblotting. NF-κB activity in lysates of WT and XIAP-deficient HCT-116 cells co-transfected with XIAP and NOD1 or NOD2 plasmids. Data represent mean + s.e.m. (n = 3–4). In NOD1 transfections (*) indicates P = 0.006 for G188E, P = 0.006 for C203Y, P = 0.0001 for L207P, P = 0.008 for R166I, P = 8.9E-05 for W173G, P = 2.9E-05 for V198M, all vs. WT. In NOD2 transfections (*) indicates P = 0.0002 for G188E, P = 4.6E-05 for C203Y, P = 4.5E-05 for L207P, P = 0.008 for R166I, P = 0.004 for W173G, P = 0.001 for V198M, all vs. WT. The two-tailed Student's t-test was used to determine statistical significance. Download figure Download PowerPoint XLP2-causing mutations that affect the RING domain (collectively referred to as XIAPXLP2-RING) abrogate XIAP's Ub ligase activity and cause impaired NOD2-dependent immune signalling (Damgaard et al, 2012). This prompted us to investigate if the XLP2-causing BIR2 mutations (collectively referred to as XIAPXLP2-BIR2) also affect NOD2 signalling (Fig 1C). XIAP-deficient HCT-116 cells were reconstituted with XIAPWT or XIAPXLP2-BIR2 variants and were stimulated with the NOD2 ligand L18-MDP (a lipidated form of MDP with increased potency). As expected, expression of XIAPWT in the XIAP-deficient cells fully restored L18-MDP-induced activation of an NF-κB reporter to the level measured in wild type cells (Fig 1D). Remarkably, none of the six XIAPXLP2-BIR2 variants were able to restore activation of the reporter in response to NOD2 activation although expressed at levels comparable to XIAPWT (Fig 1D). XIAPXLP2-BIR2 variants were also unable to facilitate NF-κB activation induced by ectopic expression of NOD1 or NOD2 (Fig 1E). This demonstrates that BIR2 mutations, like mutations that affect the XIAP RING domain, severely impair NOD1/2-induced NF-κB activation. To further establish that XLP2-BIR2 mutations impair NOD2-dependent signalling, we obtained peripheral blood mononuclear cells (PBMCs) isolated from two patients with either an XIAPL207P or an XIAPV198M mutation. The expression level of XIAP in PBMCs isolated from the L207P patient was comparable to that of a healthy donor, whereas XIAP levels appeared to be reduced in cells from the V198M patient (Fig 2A). L18-MDP failed to induce transcription of TNF and IL6 in PBMCs from the XLP2 patients (IL6 was not reliably detectable in the V198M patient cells), whereas transcription was readily induced in cells isolated from five different healthy donors (Fig 2B). Consistently, phosphorylation of IκBα and p38 MAP kinase after NOD2 stimulation was increased only in PBMCs from a healthy donor and not the XIAPL207P patient cells (Fig 2C). This was not due to a general signalling defect in the patient PBMCs because stimulation of Toll-like receptor 4 (TLR4) with lipopolysaccharide (LPS) induced transcription of TNF and IL6 in the patient cells although induction might be slightly reduced compared with healthy donor cells (Fig 2D). Figure 2.NOD2 signalling is defective in cells from two XLP2 patients with BIR2 mutations A.. XIAP levels in XLP2 patient cells. Whole cell lysates of PBMCs from healthy donor and two patients with BIR2 mutations were examined for XIAP levels by immunoblotting. Two separate vials of patient cells were examined and are indicated with 1 and 2. B, D.. Transcriptional response to NOD2 (B) and TLR4 (D) stimulation in PBMCs. Relative levels of TNF and IL6 mRNA in PBMCs stimulated with L18-MDP (200 ng/mL) or LPS (10 ng/ml) as indicated. Transcription of TNF and IL6 after L18-MDP was impaired in the patient PBMCs compared to cells from healthy donors. Data represent means of one to six experiments in healthy donor cells and two (V198M) or four to six (L207P) experiments in the XLP2 patient cells. IL6 mRNA was not reliably amplified in cells isolated from the V198M patient. All experiments were performed in duplicate. C.. NOD2 signalling in PBMCs from patient and healthy donor. Cells were examined by immunoblotting for phosphorylation of IκBα and p38 in response to stimulation with L18-MDP (200 ng/mL) as indicated. Asterisk (*) denotes p-p38 signal detectable after re-blotting with anti-IκBα. Download figure Download PowerPoint XLP2-derived XIAP variants partially retain the anti-apoptotic activity Peripheral T cells from XLP2 patients are often sensitized to activation-induced cell death in vitro compared to healthy donor cells (Filipovich et al, 2010; Pachlopnik Schmid et al, 2011; Yang et al, 2012). In line with this, we observed that T cell cultures from the XIAPL207P patient displayed increased rates of apoptosis after treatment with anti-CD3 to cells from a healthy donor (Carsten Speckmann et al, in preparation). The BIR2 domain and the immediate upstream linker contribute to XIAP's anti-apoptotic potential by binding to, and inhibiting, active caspase-3 and caspase-7 (Eckelman et al, 2006; Scott et al, 2005). To investigate if XLP2-BIR2 mutations impact directly on XIAP's ability to inhibit caspases and to protect against apoptosis, we evaluated the ability of XIAP variants to protect XIAP−/y HCT-116 cells against apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL). The XIAP-deficient cells were highly sensitive to treatment with TRAIL when compared to wild type HCT-116 cells as previously reported (Cummins et al, 2004), but were rescued by expression of XIAPWT (Fig 3A–C). Expression of XIAPXLP2-BIR2 or the IBM-binding pocket mutants XIAPD214S and XIAPE219R protected the cells comparably from TRAIL-induced apoptosis, but slightly less than XIAPWT (Fig 3B and C). Accordingly, the BIR2-mutated XIAP variants reduced caspase-3/-7 activity to a similar level as induced in wild type cells whereas ectopic expression of XIAPWT almost completely blocked TRAIL-induced caspase-3/-7 activity (Fig 3D and E). Together, this suggests that XLP-BIR2 mutations cause severe impairment of NOD1/2-mediated immune signalling, whereas the mutations have less severe consequences for XIAP's anti-apoptotic potential. Figure 3.XIAP BIR2 mutations have minor effect on TRAIL-induced cell death A.. Viability of WT and XIAP-deficient HCT-116 cells after TRAIL treatment. Cells were treated with indicated concentrations of TRAIL for 24 h and viability was determined by the MTT assay. Data are shown as percentage of vehicle-treated cells and represent mean ± s.e.m. (n = 3). Double arrow indicates the difference in sensitivity between WT and XIAP-deficient cells at the concentration used in the following experiments. B.. Expression of XIAP variants in XIAP-deficient HCT-116 cells. Cell lysates were analysed by immunoblotting. C.. XIAP variants protect against TRAIL-induced cell death. Viability of WT and XIAP-deficient HCT-116 cells transfected as indicated and treated with TRAIL (75 ng/mL) for 24 h was determined by the MTT assay. Data are shown as percentage of vehicle-treated cells and represent mean + s.e.m. (n = 4–6). (*) Indicates P = 2.2E-07 for WT, P = 0.0007 for G188E, P = 0.0004 for C203Y, P = 0.0003 for L207P, P = 0.0002 for R166I, P = 0.0002 for D214S, P = 0.0001 for E219R, all vs. vector in XIAP-deficient cells. D, E.. Measurement of TRAIL-induced caspase activity. The cleavage of the fluorogenic caspase-3/-7 substrate (DEVD-AFC) was measured in total cell lysates from cells transfected as in (B) and treated with TRAIL (75 ng/mL) for 6 h. Values were corrected for the MTT reduction activity in parallel cultures of untreated cells. (D) Shows linear increase in AFC fluorescence throughout the assay. (E) Shows linear regression of the slope of measurements in (D). Data represent mean + s.e.m. (n = 4–6). (*) Indicates P = 5.0E-07 for WT, P = 0.0004 for G188E, P = 0.001 for C203Y, P = 0.001 for L207P, P = 0.003 for R166I, P = 0.006 for D214S, P = 0.0008 for E219R, all vs. vector. The two-tailed Student's t-test was used to determine statistical significance. Download figure Download PowerPoint XLP2-BIR2 mutations interfere with XIAP function at the NOD2 signalling complex Next, we investigated how XLP2-BIR2 mutations affect XIAP function. XIAP is recruited to the NOD2 signalling complex via RIPK2 (Damgaard et al, 2012), suggestively by an interaction between the BIR2 domain in XIAP and RIPK2's kinase domain (Krieg et al, 2009). Accordingly, the interaction between XIAP and RIPK2 required the XIAP BIR2 domain and did not involve other domains of XIAP [Fig 4A; (Krieg et al, 2009)]. Remarkably, all six XLP2-BIR2 mutations abrogated the co-purification of endogenous RIPK2 with XIAP, whereas the binding to the TAK1 adaptor protein TAB1, which binds to the XIAP BIR1 domain, was unaffected by the mutations [Fig 4B; (Lu et al, 2007)]. Figure 4.XLP2-BIR2 mutations interfere with XIAP function at the NOD2 signalling complex A, B.. Analysis of RIPK2 binding by XIAP variants and isolated domains. Tagged XIAP was immunoprecipitated from lysates of HEK293T cells transfected with plasmids encoding the indicated FLAG-XIAP fragments (A) or HA-XIAP variants (B). Immunoprecipitates were examined for co-purification of endogenous RIPK2 by immunoblotting. Asterisk by the TAB1 blot indicates non-specific band, possibly cross-reactivity with RIPK2, in the IP. C.. Analysis of XIAP-mediated ubiquitylation of RIPK2. Ub conjugates were purified with StrepTactin-Agarose resin from lysates of HEK293T cells transfected as indicated. Purified material was examined by immunoblotting. D.. Ubiquitin ligase activity of XIAP variants. Purified recombinant XIAP variants were analysed for their Ub ligase activity in vitro. Formation of Ub-conjugates was detected by immunoblotting and coomassie staining. Note, that the signal corresponding to GST-XIAP (WT, G188E, C203Y, L207P and R166I) in the coomassie stained gel is lost at 30 min, indicating extensive self-ubiquitylation. E.. Recruitment of LUBAC subunits to NOD2 by XIAP variants. HA-NOD2 was immunoprecipitated from lysates of WT or XIAP-deficient HCT-116 cells expressing HA-NOD2 and FLAG-XIAP (WT or XLP2-BIR2 variants). Immunoprecipitates were examined for co-purification of LUBAC subunits. Download figure Download PowerPoint We reasoned that if XIAP is unable to bind RIPK2 then it is unlikely that XIAP can facilitate the ubiquitylation of RIPK2. Indeed, all tested XLP2-derived BIR2 mutations strongly impaired XIAP-mediated ubiquitylation of endogenous RIPK2 similar to XIAP with a substitution of phenylalanine 495 to alanine (F495A), a mutation previously shown to specifically abrogate XIAP's Ub ligase activity [Fig 4C; (Gyrd-Hansen et al, 2008)]. In contrast to the F495A mutation, the BIR2 mutations did not interfere with XIAP auto-ubiquitylation suggesting that the XIAPXLP2-BIR2 variants retain normal Ub ligase activity (Fig 4C). Accordingly, recombinant XIAPXLP2-BIR2 variants conjugated Ub chains in vitro similar to XIAPWT whereas XIAPF495A failed to detectably conjugate Ub chains (Fig 4D). Two XLP2-derived RING mutations (G466X and P482R) were also examined and, in line with our previous report (Damgaard et al, 2012), both mutations abrogate XIAP's Ub ligase activity and impaired its ability to ubiquitylate RIPK2 [Fig 4D; (Damgaard et al, 2012)]. Thus, although XLP2-derived BIR2 mutations affected RIPK2 ubiquitylation similar to XLP2-derived RING mutations, they do not affect XIAP's intrinsic Ub ligase activity. An important function of the Ub chains conjugated by XIAP at the NOD2 signalling complex is to enhance the association of the LUBAC subunits HOIP, HOIL-1 and SHARPIN with the complex (Damgaard et al, 2012). To address if LUBAC recruitment to NOD2 is affected by XLP2-BIR2 mutations, we ectopically expressed HA-NOD2 in wild-type cells and in XIAP-deficient cells reconstituted with XIAPWT or XIAPXLP2-BIR2 variants. Immunoprecipitation of HA-NOD2 showed that XIAPXLP2-BIR2 variants failed to mediate recruitment of LUBAC subunits to the NOD2 complex, whereas XIAPWT mediated the recruitment of LUBAC subunits to similar levels as observed in wild type cells (Fig 4E, compare lanes 10–12, and lane 11 with lanes 13–16). Thus, XLP2-BIR2 mutations specifically abolish RIPK2 binding leading to impaired RIPK2 ubiquitylation and recruitment of LUBAC to the NOD2 signalling complex. RIPK2 binding is mediated by residues in XIAP's BIR2 IBM-binding pocket and can be antagonized by Smac Prompted by these findings, we investigated how the individual XLP2 mutations impact on BIR2 structure and/or function. The NMR structure of the BIR2 domain (Sun et al, 1999) indicates that five of the six XLP2-derived mutations are likely to disturb the overall structure/folding of the BIR2 domain (Fig 5A, left panel): residue C203 is one of four conserved residues that coordinate the Zn2+ ion required for folding of the domain (Fig 1B and 5A, left panel; open circles above the BIR2 sequence in Fig 1B denote the Zn2+-coordinating residues). W173 and V198 are positioned in the core of the domain and contribute to stabilization of the domain through hydrophobic interactions. G188 is positioned at a conserved tight loop linking α-helix 2 with the three-stranded antiparallel β-sheet that forms the central part of the domain. R166 is located in α-helix 1 with its side chain facing towards α-helix 2 in the core of the domain and is likely to also contribute to the structural stability of the domain. In contrast, L207 is surface-exposed and together with residues D214 and E219 form the cleft of the IBM-bindi