Crystal structure of the human NLRP9 pyrin domain suggests a distinct mode of inflammasome assembly Michael Marleaux1, Kanchan Anand1, Eicke Latz2 and Matthias Geyer1 1 Institute of Structural Biology, University of Bonn, Bonn, Germany 2 Institute of Innate Immunity, University of Bonn, Bonn, Germany Correspondence Inflammasomes are cytosolic multimeric signaling complexes of the innate M. Geyer, Institute of Structural Biology, immune system that induce activation of caspases. The NOD-like receptor Biomedical Center (BMZ), University of NLRP9 recruits the adaptor protein ASC to form an ASC-dependent inflam- Bonn, Venusberg Campus 1, 53127 Bonn, masome to limit rotaviral replication in intestinal epithelial cells, but only lit- Germany Tel: +49 228 287 51400 tle is known about the molecular mechanisms regulating and driving its E-mail: [email protected] assembly. Here, we present the crystal structure of the human NLRP9 pyrin domain (PYD). We show that NLRP9PYD is not able to self-polymerize nor (Received 19 April 2020, revised 17 May to nucleate ASC specks in HEK293T cells. A comparison with filament-form- 2020, accepted 1 June 2020, available ing PYDs revealed that NLRP9PYD adopts a conformation compatible with online 6 July 2020) filament formation, but several charge inversions of interfacing residues might cause repulsive effects that prohibit self-oligomerization. These results pro- doi:10.1002/1873-3468.13865 pose that inflammasome assembly of NLRP9 might differ largely from what Edited by Roosmarijn Vandenbroucke we know of other inflammasomes. Keywords: ASC specks; filament; inflammasomes; NLRP9; PYD; structure The innate immune system comprises germline-en- amplify signals from the PRR via an adaptor protein coded pattern recognition receptors (PRRs) that have to activate the downstream effector cysteine protease evolved to directly recognize a wide range of conserved caspase-1, which in turn processes the highly proin- pathogen-associated molecular patterns, danger-associ- flammatory cytokines pro-IL-1b and pro-IL-18 into ated molecular patterns, and homeostasis-altering their mature forms [4,5]. Active caspase-1 also cleaves molecular processes [1]. They fulfill diverse functions gasdermin D to induce pore formation in the host cell with importance not only in pathogen recognition but for the release of cytokines and pyroptotic cell death also in tissue homeostasis, apoptosis, graft-versus-host [5,6]. Notably, signal amplification is facilitated by uni- disease, early development, and even negative regula- fied nucleation-driven polymerization of the individual tion of inflammatory responses [2]. In the human inflammasome components [7–9]. As an example, immune system, PRRs group into five families, includ- NLRP3 is an inflammasomal sensor protein that ing Toll-like receptors, RIG-I-like receptors, C-type belongs to a subfamily of NLRs (termed NLRPs) con- lectin receptors, AIM2-like receptors (ALRs), and taining an N-terminal pyrin domain (PYD). After an NOD-like receptors (NLRs). Of these PRRs, ALRs initial priming step and activation by a wide range of and certain NLRs were found to oligomerize upon diverse stimuli, it recruits the bipartite protein ‘apopto- ligand stimulation and participate in the formation of sis-associated speck-like protein containing a caspase cytosolic multimeric signaling complexes, called inflam- recruitment domain’ (ASC) as an adaptor for caspase- masomes [3]. Canonical inflammasomes translate and 1 activation [8,10–12]. NLRP3 polymerizes with its Abbreviations GST, glutathione S-transferase; PYD, pyrin domain. FEBS Letters 594 (2020) 2383–2395 ª 2020 The Authors. FEBS Letters published by John Wiley & Sons Ltd 2383 on behalf of Federation of European Biochemical Societies This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. PYD structure of NLRP9 M. Marleaux et al. PYD to form the nucleation seed for ASC filament Materials and methods assembly using three major types of asymmetric inter- faces, which has also been described for NLRP6 and Cloning, expression, and purification of the AIM2 [7,13–15]. ASC filaments in turn nucleate the human NLRP9 pyrin domain formation of large caspase-1 filaments by caspase acti- vation and recruitment domain (CARD)/CARD inter- The coding sequence of a human NLRP9 cDNA clone (Gen- Bank accession NM_176820) corresponding to residues 1–97 actions, which finally leads to proximity-induced was amplified by PCR with specific oligonucleotides encom- autoproteolytic activation of caspase-1 [7]. Therefore, passing NcoI/EcoRI restriction sites at the 50 and 30 end, respec- inflammasome assembly significantly relies on homo- tively. The DNA product was cloned into a modified pGEX- typic interactions between PYD and CARD domains 4T-1 vector carrying an N-terminal glutathione S-transferase of the respective components. (GST) tag for affinity purification followed by a tobacco etch A less studied member of the NLRP subfamily is virus (TEV) protease cleavage site. The expression construct NLRP9, which was first described as a maternal was confirmed by DNA sequencing and transformed into – effect gene in bovine [16 19]. Upon recognition of Escherichia coli BL21(DE3) cells. Expression cultures were ini- low molecular weight dsRNA by the endogenous tially grown at 37 °C in LB medium, supplemented with À1 RNA helicase DHX9, its mouse analogue NLRP9b 100 mgÁL ampicillin. When OD600 reached 0.8, cells were was described to form an inflammasome with ASC cooled to 16 °C before protein expression was induced by addi- and caspase-1 [20,21]. This inflammasomal complex tion of 0.55 mM IPTG. Bacteria were further grown overnight mediates pyroptosis of intestinal epithelial cells at 16 °C, harvested by centrifugation, and stored at À80 °C. (IECs), thereby limiting rotaviral replication in mice For purification, cells were resuspended in buffer A [20,21]. Additionally, NLRP9b deficiency resulted in (50 mM HEPES pH 7.5, 150 mM NaCl, and 10 mM bME) less pulmonary histopathological alterations in a containing 1 mM phenylmethylsulfonyl fluoride and À mouse model of acute lung injury and markedly ele- 1mgÁL 1 DNAse I, and lysed by the addition of lysozyme vated proinflammatory cytokine and chemokine levels, and further sonication. The lysate was cleared from cell deb- as well as ROS production, in a LPS-activated mouse ris by centrifugation at 70 000 g for 30 min. The protein epithelial cell line [22]. Also, expression of NLRP9 in containing an N-terminal GST tag was captured using a human IECs and DHX9-dependent association with GSTrap column (GE Healthcare, Freiburg, Germany), and ASC upon stimulation with rotavirus could be shown the unbound fraction was washed off with buffer B (50 mM b [21]. Moreover, upregulation of expression was found HEPES pH 7.5, 300 mM NaCl, 10 mM ME), followed by upon inflammatory stimulus ROS and interferon-c in buffer A. After elution with buffer A containing 10 mM reduced glutathione, the GST tag was cleaved off overnight brain pericytes and cerebral endothelial cells, respec- at 4 °C by the addition of 1 : 100 w/w TEV protease. Protein tively [23,24]. However, the role of human NLRP9 in was further purified using a Superdex 75 gel filtration col- innate immunity is not yet clarified and elucidation of umn 16/600 (GE Healthcare) that was connected to a its inflammasome function would be valuable. This GSTrap column for prolonged retention of the cleaved GST holds particularly true, as alterations in NLRP9 tag and pre-equilibrated with buffer C (50 mM HEPES pH expression were linked to several inflammatory dis- 7.5, 150 mM NaCl, 1 mM TCEP). Peak fractions containing eases, including urothelial carcinoma, multiple sclero- NLRP9PYD were pooled, concentrated to 31.7 mgÁmLÀ1, sis, juvenile idiopathic arthritis, familial late-onset snap-frozen in liquid nitrogen, and stored at À80 °C. Alzheimer disease, and infection with Helicobacter pylori [25–31]. To gain first insights into NLRP9 inflammasome Crystallization and diffraction data collection assembly, we expressed and purified recombinant Protein solution was crystallized by the hanging-drop vapor- PYD human NLRP9 and studied its capability to self- diffusion method at 15 °C. Initial crystals were obtained by polymerize into filaments and nucleate ASC speck for- JCSG + screen [32] (0.2 M potassium formate, 20% PYD mation. We found that NLRP9 exists as a mono- PEG3350). The optimal hexagonal-shaped crystals were mer under near-physiological conditions in vitro and in grown with 1 : 1 ratio of protein and precipitant solution by PYD cells. In contrast to NLRP3 , overexpression of using 0.1 M Bicine pH 9.0, 0.2 M sodium thiocyanate, 0.1 M murine or human NLRP9PYD did not nucleate ASC sodium formate, and 25–27% v/w PEG3350. Crystals were speck formation in HEK293T cells. We determined the cryoprotected with 15% ethylene glycol in the mother liquor crystal structure of human NLRP9PYD to study the and flash-cooled in liquid nitrogen. molecular differences from filament-forming PYDs Data were collected at the synchrotron beamlines of and discuss its implications for the assembly of the Swiss Light Source (SLS) in Villigen, Switzerland, and NLRP9 inflammasome. Deutsches Elektronen-Synchrotron (DESY) in Hamburg, 2384 FEBS Letters 594 (2020) 2383–2395 ª 2020 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies M. Marleaux et al. PYD structure of NLRP9 Germany. Presented diffraction data up to 1.95 A resolu- Table 1. Crystallographic data collection and refinement statistics. tion were collected from native crystals at 100K on the X- Data collection ray diffraction PX1 beamline using Eiger detector at SLS. Space group P6522 a, b, c (A) 33.33, 33.33, 311.09 Resolution (A)a 28.74–1.95 (2.02–1.95) Data processing, structure determination, and Rmerge (%) 8.25 (98.49) refinement I/r (I) 18.58 (1.84) Completeness (%) 99.52 (99.23) Data were processed and scaled using the XDS program CC1/2 0.998 (0.916) package [33].