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• W2073561387 abstract "Absent in melanoma 2 (AIM2) is a cytosolic double-stranded (dsDNA) sensor essential for innate immune responses against DNA viruses and bacteria such as Francisella and Listeria. Upon dsDNA engagement, the AIM2 amino-terminal pyrin domain (PYD) is responsible for downstream signaling to the adapter protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) through homotypic PYD-PYD interactions and the assembly of an inflammasome. Toward a better understanding of the AIM2 signaling mechanism, we determined the crystal structure of the human AIM2 PYD. The structure reveals a death domain fold with a short α3 helix that is buttressed by a highly conserved lysine residue at the α2 helix, which may stabilize the α3 helix for potential interaction with partner domains. The surface of the AIM2 PYD exhibits distinct charge distribution with highly acidic α1-α2 helices and highly basic α5-α6 helices. A prominent solvent-exposed hydrophobic patch formed by residues Phe-27 and Phe-28 at the α2 helix resembles a similar surface involved in the death effector domain homotypic interactions. Docking studies suggest that the AIM2 PYD may bind the AIM2 hematopoietic interferon-inducible nuclear (HIN) domain or ASC PYD using overlapping surface near the α2 helix. This may ensure that AIM2 interacts with the downstream adapter ASC only upon release of the autoinhibition by the dsDNA ligand. Our work thus unveils novel structural features of the AIM2 PYD and provides insights into the potential mechanisms of the PYD-HIN and PYD-PYD interactions important for AIM2 autoinhibition and inflammasome assembly. Absent in melanoma 2 (AIM2) is a cytosolic double-stranded (dsDNA) sensor essential for innate immune responses against DNA viruses and bacteria such as Francisella and Listeria. Upon dsDNA engagement, the AIM2 amino-terminal pyrin domain (PYD) is responsible for downstream signaling to the adapter protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) through homotypic PYD-PYD interactions and the assembly of an inflammasome. Toward a better understanding of the AIM2 signaling mechanism, we determined the crystal structure of the human AIM2 PYD. The structure reveals a death domain fold with a short α3 helix that is buttressed by a highly conserved lysine residue at the α2 helix, which may stabilize the α3 helix for potential interaction with partner domains. The surface of the AIM2 PYD exhibits distinct charge distribution with highly acidic α1-α2 helices and highly basic α5-α6 helices. A prominent solvent-exposed hydrophobic patch formed by residues Phe-27 and Phe-28 at the α2 helix resembles a similar surface involved in the death effector domain homotypic interactions. Docking studies suggest that the AIM2 PYD may bind the AIM2 hematopoietic interferon-inducible nuclear (HIN) domain or ASC PYD using overlapping surface near the α2 helix. This may ensure that AIM2 interacts with the downstream adapter ASC only upon release of the autoinhibition by the dsDNA ligand. Our work thus unveils novel structural features of the AIM2 PYD and provides insights into the potential mechanisms of the PYD-HIN and PYD-PYD interactions important for AIM2 autoinhibition and inflammasome assembly. AIM2 2The abbreviations used are: AIM2absent in melanoma 2PYDpyrin domainASCapoptosis-associated speck-like protein containing a CARDCARDcaspase recruitment domainHINhematopoietic interferon-inducible nuclearPYHINpyrin and hematopoietic interferon-inducible nuclearNLRNOD-like receptorNODnucleotide-binding oligomerization domainDDdeath domainDEDdeath effector domainMBPmaltose-binding proteinITCisothermal titration calorimetryPOPPYD-only proteinFADDFas-associated via death domain. is a major cytosolic dsDNA sensor that forms an inflammasome with the adapter ASC and procaspase-1 to activate proinflammatory cytokine processing (1Hornung V. Ablasser A. Charrel-Dennis M. Bauernfeind F. Horvath G. Caffrey D.R. Latz E. Fitzgerald K.A. AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC.Nature. 2009; 458: 514-518Crossref PubMed Scopus (1773) Google Scholar, 2Fernandes-Alnemri T. Yu J.-W. Datta P. Wu J. Alnemri E.S. AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA.Nature. 2009; 458: 509-513Crossref PubMed Scopus (1334) Google Scholar, 3Roberts T.L. Idris A. Dunn J.A. Kelly G.M. Burnton C.M. Hodgson S. Hardy L.L. Garceau V. Sweet M.J. Ross I.L. Hume D.A. Stacey K.J. HIN-200 proteins regulate caspase activation in response to foreign cytoplasmic DNA.Science. 2009; 323: 1057-1060Crossref PubMed Scopus (667) Google Scholar, 4Bürckstümmer T. Baumann C. Blüml S. Dixit E. Dürnberger G. Jahn H. Planyavsky M. Bilban M. Colinge J. Bennett K.L. Superti-Furga G. An orthogonal proteomic-genomic screen identifies AIM2 as a cytoplasmic DNA sensor for the inflammasome.Nat. Immunol. 2009; 10: 266-272Crossref PubMed Scopus (809) Google Scholar). The AIM2 inflammasome is essential for immune responses against DNA viruses such as vaccinia virus and mouse cytomegalovirus and bacteria such as Francisella tularensis and Listeria monocytogenes (5Rathinam V.A. Jiang Z. Waggoner S.N. Sharma S. Cole L.E. Waggoner L. Vanaja S.K. Monks B.G. Ganesan S. Latz E. Hornung V. Vogel S.N. Szomolanyi-Tsuda E. Fitzgerald K.A. The AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses.Nat. Immunol. 2010; 11: 395-402Crossref PubMed Scopus (958) Google Scholar, 6Fernandes-Alnemri T. Yu J.-W. Juliana C. Solorzano L. Kang S. Wu J. Datta P. McCormick M. Huang L. McDermott E. Eisenlohr L. Landel C.P. Alnemri E.S. The AIM2 inflammasome is critical for innate immunity to Francisella tularensis.Nat. Immunol. 2010; 11: 385-393Crossref PubMed Scopus (558) Google Scholar, 7Sauer J.-D. Witte C.E. Zemansky J. Hanson B. Lauer P. Portnoy D.A. Listeria monocytogenes triggers AIM2-mediated pyroptosis upon infrequent bacteriolysis in the macrophage cytosol.Cell Host Microbe. 2010; 7: 412-419Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 8Kim S. Bauernfeind F. Ablasser A. Hartmann G. Fitzgerald K.A. Latz E. Hornung V. Listeria monocytogenes is sensed by the NLRP3 and AIM2 inflammasome.Eur. J. Immunol. 2010; 40: 1545-1551Crossref PubMed Scopus (206) Google Scholar, 9Ge J. Gong Y.-N. Xu Y. Shao F. Preventing bacterial DNA release and absent in melanoma 2 inflammasome activation by a Legionella effector functioning in membrane trafficking.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 6193-6198Crossref PubMed Scopus (92) Google Scholar). AIM2 was also reported to play a role in autoimmune disorders such as psoriasis through recognition of host DNA (10Dombrowski Y. Peric M. Koglin S. Kammerbauer C. Göss C. Anz D. Simanski M. Gläser R. Harder J. Hornung V. Gallo R.L. Ruzicka T. Besch R. Schauber J. Cytosolic DNA triggers inflammasome activation in keratinocytes in psoriatic lesions.Sci. Transl. Med. 2011; 3: 82ra38Crossref PubMed Scopus (312) Google Scholar). It belongs to the pyrin and HIN protein (PYHIN) family of proteins that includes interferon γ-inducible protein 16 (11Unterholzner L. Keating S.E. Baran M. Horan K.A. Jensen S.B. Sharma S. Sirois C.M. Jin T. Latz E. Xiao T.S. Fitzgerald K.A. Paludan S.R. Bowie A.G. IFI16 is an innate immune sensor for intracellular DNA.Nat. Immunol. 2010; 11: 997-1004Crossref PubMed Scopus (1184) Google Scholar), IFN-inducible protein X/PYHIN1, and myeloid cell nuclear differentiation antigen in the human genome (12Ludlow L.E. Johnstone R.W. Clarke C.J. The HIN-200 family: more than interferon-inducible genes?.Exp. Cell Res. 2005; 308: 1-17Crossref PubMed Scopus (136) Google Scholar, 13Kwon D. Yoon J.H. Shin S.Y. Jang T.H. Kim H.G. So I. Jeon J.H. Park H.H. A comprehensive manually curated protein-protein interaction database for the death domain superfamily.Nucleic Acids Res. 2012; 40: D331-D336Crossref PubMed Scopus (36) Google Scholar). AIM2 contains a carboxyl-terminal HIN domain that binds dsDNA and an amino-terminal PYD that is responsible for downstream signaling to the adapter protein ASC. Previous phylogenetic analysis of the PYD sequences revealed two separate clades among the mammalian PYHIN proteins, one containing the AIM2 orthologs and the other containing all other PYHIN proteins (14Cridland J.A. Curley E.Z. Wykes M.N. Schroder K. Sweet M.J. Roberts T.L. Ragan M.A. Kassahn K.S. Stacey K.J. The mammalian PYHIN gene family: phylogeny, evolution and expression.BMC Evol. Biol. 2012; 12: 140Crossref PubMed Scopus (135) Google Scholar). In agreement with its distinct sequence, the AIM2 PYD was shown to be the only PYHIN PYD that associates with ASC to form an inflammasome (1Hornung V. Ablasser A. Charrel-Dennis M. Bauernfeind F. Horvath G. Caffrey D.R. Latz E. Fitzgerald K.A. AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC.Nature. 2009; 458: 514-518Crossref PubMed Scopus (1773) Google Scholar, 2Fernandes-Alnemri T. Yu J.-W. Datta P. Wu J. Alnemri E.S. AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA.Nature. 2009; 458: 509-513Crossref PubMed Scopus (1334) Google Scholar). However, it remains unclear how the distinct sequence of the AIM2 PYD contributes to this unique function. absent in melanoma 2 pyrin domain apoptosis-associated speck-like protein containing a CARD caspase recruitment domain hematopoietic interferon-inducible nuclear pyrin and hematopoietic interferon-inducible nuclear NOD-like receptor nucleotide-binding oligomerization domain death domain death effector domain maltose-binding protein isothermal titration calorimetry PYD-only protein Fas-associated via death domain. The pyrin domains were found in both PYHIN proteins and Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) as signal transduction modules that adopt the six-helix bundle fold typical of the death domain superfamily (15Bertin J. DiStefano P.S. The PYRIN domain: a novel motif found in apoptosis and inflammation proteins.Cell Death Differ. 2000; 7: 1273-1274Crossref PubMed Scopus (159) Google Scholar, 16Martinon F. Hofmann K. Tschopp J. The pyrin domain: a possible member of the death domain-fold family implicated in apoptosis and inflammation.Curr. Biol. 2001; 11: R118-R120Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 17Staub E. Dahl E. Rosenthal A. The DAPIN family: a novel domain links apoptotic and interferon response proteins.Trends Biochem. Sci. 2001; 26: 83-85Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 18Pawłowski K. Pio F. Chu Z. Reed J.C. Godzik A. PAAD—a new protein domain associated with apoptosis, cancer and autoimmune diseases.Trends Biochem. Sci. 2001; 26: 85-87Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 19Fairbrother W.J. Gordon N.C. Humke E.W. O'Rourke K.M. Starovasnik M.A. Yin J.P. Dixit V.M. The PYRIN domain: a member of the death domain-fold superfamily.Protein Sci. 2001; 10: 1911-1918Crossref PubMed Scopus (125) Google Scholar). Besides PYD, the death domain superfamily includes the death domain (DD), death effector domain (DED), and caspase recruitment domain (CARD). Many of them are involved in the assembly of oligomeric multiprotein signaling complexes such as the PIDDosome (DD) (20Park H.H. Logette E. Raunser S. Cuenin S. Walz T. Tschopp J. Wu H. Death domain assembly mechanism revealed by crystal structure of the oligomeric PIDDosome core complex.Cell. 2007; 128: 533-546Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar), MyDDosome (DD) (21Lin S.-C. Lo Y.-C. Wu H. Helical assembly in the MyD88-IRAK4-IRAK2 complex in TLR/IL-1R signalling.Nature. 2010; 465: 885-890Crossref PubMed Scopus (770) Google Scholar), apoptosomes (CARD) (22Acehan D. Jiang X. Morgan D.G. Heuser J.E. Wang X. Akey C.W. Three-dimensional structure of the apoptosome: implications for assembly, procaspase-9 binding, and activation.Mol. Cell. 2002; 9: 423-432Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar, 23Qi S. Pang Y. Hu Q. Liu Q. Li H. Zhou Y. He T. Liang Q. Liu Y. Yuan X. Luo G. Li H. Wang J. Yan N. Shi Y. Crystal structure of the Caenorhabditis elegans apoptosome reveals an octameric assembly of CED-4.Cell. 2010; 141: 446-457Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), and inflammasomes (PYD) (24Martinon F. Burns K. Tschopp J. The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-β.Mol. Cell. 2002; 10: 417-426Abstract Full Text Full Text PDF PubMed Scopus (4181) Google Scholar). Despite progress in the structural characterization of the DD, DED, and CARD signaling complexes, the molecular mechanisms of PYD-mediated signaling events remain poorly understood largely because of the lack of structural information on PYD-PYD complexes. To date, the structures of eight human PYDs and two mouse PYDs have been experimentally characterized, largely through nuclear magnetic resonance (NMR) spectroscopy. These include the PYDs from human ASC (Protein Data Bank codes 1UCP (25Liepinsh E. Barbals R. Dahl E. Sharipo A. Staub E. Otting G. The death-domain fold of the ASC PYRIN domain, presenting a basis for PYRIN/PYRIN recognition.J. Mol. Biol. 2003; 332: 1155-1163Crossref PubMed Scopus (120) Google Scholar) and 2KN6 (26de Alba E. Structure and interdomain dynamics of apoptosis-associated speck-like protein containing a CARD (ASC).J. Biol. Chem. 2009; 284: 32932-32941Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar)), NLRP1 (Protein Data Bank code 1PN5) (27Hiller S. Kohl A. Fiorito F. Herrmann T. Wider G. Tschopp J. Grütter M.G. Wüthrich K. NMR structure of the apoptosis- and inflammation-related NALP1 pyrin domain.Structure. 2003; 11: 1199-1205Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), NLRP3 (Protein Data Bank code 3QF2) (28Bae J.Y. Park H.H. Crystal structure of NALP3 protein pyrin domain (PYD) and its implications in inflammasome assembly.J. Biol. Chem. 2011; 286: 39528-39536Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), NLRP4 (Protein Data Bank code 4EWI) (29Eibl C. Grigoriu S. Hessenberger M. Wenger J. Puehringer S. Pinheiro A.S. Wagner R.N. Proell M. Reed J.C. Page R. Diederichs K. Peti W. Structural and functional analysis of the NLRP4 pyrin domain.Biochemistry. 2012; 51: 7330-7341Crossref PubMed Scopus (40) Google Scholar), NLRP7 (Protein Data Bank code 2KM6) (30Pinheiro A.S. Proell M. Eibl C. Page R. Schwarzenbacher R. Peti W. Three-dimensional structure of the NLRP7 pyrin domain: insight into pyrin-pyrin-mediated effector domain signaling in innate immunity.J. Biol. Chem. 2010; 285: 27402-27410Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), NLRP12 (Protein Data Bank code 2L6A) (31Pinheiro A.S. Eibl C. Ekman-Vural Z. Schwarzenbacher R. Peti W. The NLRP12 pyrin domain: structure, dynamics, and functional insights.J. Mol. Biol. 2011; 413: 790-803Crossref PubMed Scopus (56) Google Scholar), and POP1/ASC2 (Protein Data Bank code 2HM2) (32Natarajan A. Ghose R. Hill J.M. Structure and dynamics of ASC2, a pyrin domain-only protein that regulates inflammatory signaling.J. Biol. Chem. 2006; 281: 31863-31875Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) and mouse NLRP10 (Protein Data Bank code 2DO9) as well as the PYHIN family members myeloid cell nuclear differentiation antigen (Protein Data Bank code 2DBG; human) and p205b (Protein Data Bank code 2YU0; mouse). Interestingly, all known PYD structures have a uniquely short α3 helix compared with other DD superfamily members (33Kersse K. Verspurten J. Vanden Berghe T. Vandenabeele P. The death-fold superfamily of homotypic interaction motifs.Trends Biochem. Sci. 2011; 36: 541-552Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Previously, we reported the crystal structure of the AIM2 HIN domain in complex with dsDNA and found that the HIN domain binds dsDNA through electrostatic attraction, and the AIM2 PYD and HIN domain interaction maintains the receptor in an autoinhibited state in the absence of dsDNA (34Jin T. Perry A. Jiang J. Smith P. Curry J.A. Unterholzner L. Jiang Z. Horvath G. Rathinam V.A. Johnstone R.W. Hornung V. Latz E. Bowie A.G. Fitzgerald K.A. Xiao T.S. Structures of the HIN domain:DNA complexes reveal ligand binding and activation mechanisms of the AIM2 inflammasome and IFI16 receptor.Immunity. 2012; 36: 561-571Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). To examine the structure and function of the AIM2 PYD, we carried out crystallographic studies of the AIM2 PYD. The AIM2 PYD was crystallized as a fusion with maltose-binding protein (MBP). The structure reveals a typical death domain fold for the PYD with distinct surface charges and hydrophobic patches. We identify a highly conserved lysine residue at the α2 helix that stabilizes the short α3 helix, a common feature for all known PYD structures that has not been described previously. Our docking and binding studies suggest potential modes of the PYD-PYD and PYD-HIN associations through overlapping surface at the AIM2 PYD such that the AIM2 receptor signal transduction only occurs upon ligand engagement. The pyrin domain of human AIM2 (NCBI accession number NP_004824; residues 1–107) was cloned into a pET30a-derived vector with a non-cleavable amino-terminal MBP tag and a carboxyl-terminal hexahistidine tag. The MBP tag harbors mutations (D82A/K83A/E172A/N173A/K239A) designed to enhance its crystallization propensity (35Ullah H. Scappini E.L. Moon A.F. Williams L.V. Armstrong D.L. Pedersen L.C. Structure of a signal transduction regulator, RACK1, from Arabidopsis thaliana.Protein Science. 2008; 17: 1771-1780Crossref PubMed Scopus (93) Google Scholar, 36Moon A.F. Mueller G.A. Zhong X. Pedersen L.C. A synergistic approach to protein crystallization: combination of a fixed-arm carrier with surface entropy reduction.Protein Sci. 2010; 19: 901-913Crossref PubMed Scopus (115) Google Scholar). Transformed BL21(DE3) Codon Plus RIPL cells (Stratagene, Santa Clara, CA) were grown at 37 °C, induced with 0.3 mm isopropyl 1-thio-β-d-galactopyranoside at 18 °C for 4 h, harvested, and lysed in buffer A (20 mm Tris-HCl, pH 8.0, 100 mm NaCl) plus 5 mm imidazole, DNase (Biomatik, Wilmington, DE), and protease inhibitors (Roche Applied Science). Soluble protein from the cell lysate was purified by Hisprep IMAC column (GE Healthcare) and further purified using a XK26/60 Superdex 200 size exclusion column in buffer A supplemented with 5 mm maltose (Research Products International Corp., Mount Prospect, IL). The mutant AIM2 PYD constructs were produced using the Phusion site-directed mutagenesis kit (Thermo Scientific, Waltham, MA), and the expression and purification were carried essentially the same as for the wild type protein. The human ASC PYD (residues 1–91) coding sequence was cloned into a pET30a-derived vector with a tobacco etch virus protease cleavable amino-terminal GB1 tag. Expression and purification were carried out similarly to the AIM2 PYD except that the GB1 tag was removed by tobacco etch virus cleavage. Purified MBP-PYD protein was concentrated to 20 mg/ml before setting up hanging drops for vapor diffusion crystallization. Multiple commercial crystallization screens were tested using the Mosquito crystallization robot (TTP Labtech, UK). Rod-shaped crystals grew at room temperature in 1 week using a well solution containing 20% polyethylene glycol (PEG) 3350 and 0.1 m potassium acetate at pH 4.0. 20% ethylene glycol (v/v) was added to the reservoir solution as the cryoprotectant to freeze the crystals in liquid nitrogen for x-ray diffraction data collection. X-ray diffraction data were collected at the General Medical Sciences and Cancer Institutes Collaborative Access Team (GM/CA-CAT) of the Advanced Photon Source, Argonne National Laboratory. Data were processed with the HKL2000 program suite (37Otwinowski Z. Minor W. Processing of x-ray diffraction data.Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38527) Google Scholar). The solvent content was 53.2% for one molecule of the fusion protein per asymmetric unit. The structure was determined by molecular replacement with Phaser (38McCoy A.J. Grosse-Kunstleve R.W. Adams P.D. Winn M.D. Storoni L.C. Read R.J. Phaser crystallographic software.J. Appl. Crystallogr. 2007; 40: 658-674Crossref PubMed Scopus (14444) Google Scholar) from the CCP4 program suite (39Potterton E. Briggs P. Turkenburg M. Dodson E. A graphical user interface to the CCP4 program suite.Acta Crystallogr. D Biol. Crystallogr. 2003; 59: 1131-1137Crossref PubMed Scopus (1067) Google Scholar). An MBP structure from the Protein Data Bank (code 3DM0) was used as the search model (35Ullah H. Scappini E.L. Moon A.F. Williams L.V. Armstrong D.L. Pedersen L.C. Structure of a signal transduction regulator, RACK1, from Arabidopsis thaliana.Protein Science. 2008; 17: 1771-1780Crossref PubMed Scopus (93) Google Scholar). Electron density maps calculated with phases from the MBP search model clearly showed positive densities for the AIM2 PYD. The PYD model was manually built with Coot (40Emsley P. Cowtan K. Coot: model-building tools for molecular graphics.Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23229) Google Scholar) and refined with Phenix.refine (41Adams P.D. Afonine P.V. Bunkóczi G. Chen V.B. Davis I.W. Echols N. Headd J.J. Hung L.-W. Kapral G.J. Grosse-Kunstleve R.W. McCoy A.J. Moriarty N.W. Oeffner R. Read R.J. Richardson D.C. Richardson J.S. Terwilliger T.C. Zwart P.H. PHENIX: a comprehensive Python-based system for macromolecular structure solution.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 213-221Crossref PubMed Scopus (16439) Google Scholar). The final structure contains 467 residues of which residues Met-371 to Lys-467 correspond to the first 97 residues of the AIM2 receptor (NCBI accession number NP_004824). Validation by the MolProbity server (42Chen V.B. Arendall 3rd, W.B. Headd J.J. Keedy D.A. Immormino R.M. Kapral G.J. Murray L.W. Richardson J.S. Richardson D.C. MolProbity: all-atom structure validation for macromolecular crystallography.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 12-21Crossref PubMed Scopus (9818) Google Scholar) and Research Collaboratory for Structural Bioinformatics ADIT validation server (43Yang H. Guranovic V. Dutta S. Feng Z. Berman H.M. Westbrook J.D. Automated and accurate deposition of structures solved by x-ray diffraction to the Protein Data Bank.Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 1833-1839Crossref PubMed Scopus (221) Google Scholar) showed that 97.5% of all protein residues were in the favored regions of the Ramachandran plot with no outliers. Electrostatic surfaces were calculated with program Delphi (v4) (44Honig B. Nicholls A. Classical electrostatics in biology and chemistry.Science. 1995; 268: 1144-1149Crossref PubMed Scopus (2528) Google Scholar) and displayed with PyMOL (Schrödinger, LLC). Calculation of the solvent-accessible area was performed with the program Areaimol from the CCP4 program suite (39Potterton E. Briggs P. Turkenburg M. Dodson E. A graphical user interface to the CCP4 program suite.Acta Crystallogr. D Biol. Crystallogr. 2003; 59: 1131-1137Crossref PubMed Scopus (1067) Google Scholar, 45Lee B. Richards F.M. The interpretation of protein structures: estimation of static accessibility.J. Mol. Biol. 1971; 55: 379-400Crossref PubMed Scopus (5336) Google Scholar). Sequence alignment of the PYDs was performed with the program MEGA5 (46Tamura K. Peterson D. Peterson N. Stecher G. Nei M. Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods.Mol. Biol. Evol. 2011; 28: 2731-2739Crossref PubMed Scopus (34846) Google Scholar) with minor adjustments. The phylogenic tree was calculated using the maximum likelihood method in MEGA5, and the reliability of the tree was tested with bootstrapping for 1000 replications. The sequence conservation scores were calculated by the ConSurf server (47Ashkenazy H. Erez E. Martz E. Pupko T. Ben-Tal N. ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids.Nucleic Acids Res. 2010; 38: W529-W533Crossref PubMed Scopus (1338) Google Scholar) using the sequence alignment in Fig. 1. Docking of the AIM2 PYD (Protein Data Bank code 3VD8) with the ASC PYD (Protein Data Bank code 1UCP) or the AIM2 HIN domain (Protein Data Bank code 3RN2) was performed with the web server ClusPro (48Kozakov D. Hall D.R. Beglov D. Brenke R. Comeau S.R. Shen Y. Li K. Zheng J. Vakili P. Paschalidis I.Ch. Vajda S. Achieving reliability and high accuracy in automated protein docking: Cluspro, PIPER, SDU, and stability analysis in CAPRI rounds 13–19.Proteins. 2010; 78: 3124-3130Crossref PubMed Scopus (194) Google Scholar), taking into account both shape complementarity and electrostatic charge interactions. An interactive docking program, Hex (v6.3) (49Ritchie D.W. Venkatraman V. Ultra-fast FFT protein docking on graphics processors.Bioinformatics. 2010; 26: 2398-2405Crossref PubMed Scopus (274) Google Scholar), was also used for comparison. No preidentification of residues within or outside the contact surface was specified. The choice of either domain as the stationary receptor resulted in a similar mode of interactions for the AIM2 PYD (see FIGURE 5, FIGURE 6): the top docking models consistently placed the α2 helix of the AIM2 PYD at the PYD-PYD and PYD-HIN interfaces. The top ranked docking model for the PYD-HIN complex was subjected to energy minimization using the relax mode of the Rosetta (v3.4) program (50Kuhlman B. Dantas G. Ireton G.C. Varani G. Stoddard B.L. Baker D. Design of a novel globular protein fold with atomic-level accuracy.Science. 2003; 302: 1364-1368Crossref PubMed Scopus (1240) Google Scholar).FIGURE 6Docking of the AIM2 PYD and HIN domains. A, superposition of the top 10 docking models of the AIM2 PYD-HIN domain complex with the HIN domain as the stationary receptor by the ClusPro server. The AIM2 PYD and HIN domain are colored orange and cyan, respectively. B, the structure of the AIM2 HIN-DNA complex (Protein Data Bank code 3RN2) is shown for comparison. C, superposition of the top 10 docking models of the AIM2 PYD-HIN complex with the PYD as the stationary receptor by the ClusPro server. The 10 models were superimposed on their HIN domains. D, the original 10 docking models for C are superimposed on their PYDs with the AIM2 residues Asp-19, Glu-20, Asp-23, Phe-27, and Phe-28 shown as yellow spheres. E, the top scoring model from A was energy-minimized with Rosetta and is shown in ribbons. The PYD and HIN domain are colored orange and cyan, respectively. The PYD helices and termini are labeled. The open book view of the PYD-HIN interface is shown as electrostatic surface in F (HIN) and G (PYD). The basic residues in HIN are labeled in yellow, and acidic residues in PYD are labeled in black.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Purified MBP or MBP-tagged wild type or mutant AIM2 PYD samples (200 μg) were immobilized with 50 μl of amylose beads (New England Biolabs, Ipswich, MA) in buffer B (20 mm HEPES-Na, pH 7.4, 100 mm NaCl). After three washing steps using buffer B, 200 μg of AIM2 HIN protein was incubated with the beads followed by three washing steps with buffer B. The bound proteins were eluted with 50 μl of buffer B plus 10 mm maltose and analyzed by SDS-PAGE. The GAL4-based Matchmaker yeast two-hybrid system from Clontech was used to examine the interaction between the AIM2 PYD and the ASC PYD. The PYD coding sequences were cloned into the pGBKT7 plasmid encoding DNA-binding domain and the pGADT7 plasmid encoding activation domain. Combinations of DNA-binding domain and activation domain plasmid pairs were transformed into yeast strain AH109. The cells were plated on agar plates of minimum synthetic dropout medium without leucine and tryptophan (−Leu/−Trp) to select for transformants. Single colonies were then picked and replicated onto −His/−Leu/−Trp plates. Growth of the colonies at 30 °C was recorded 72 h later. The association between the AIM2 PYD and HIN domain was measured using an ITC200 calorimeter (Microcal, Piscataway, NJ) at 288 K. All protein samples were dialyzed extensively in buffer B. The wild type or mutant MBP-AIM2 PYD at 0.3 mm was titrated into ∼200 μl of purified AIM2 HIN domain at 30 μm. The binding isotherm data were analyzed with the program Origin using a single site binding model after subtracting the buffer dilution background. A 5′,6-fluorescein-labeled 20-mer DNA oligo with a sequence of 5′-ccatcaaagagagaaagagc-3′ (Integrated DNA Technologies, Coralville, IA) was dissolved in buffer B and annealed with its reverse complement DNA oligo. A 4 nm concentration of the probe was mixed with 100 nm purified AIM2 HIN domain, and increasing concentrations of the wild type or mutant MBP-AIM2 PYD were added to the above DNA-HIN complex. The mixtures were then aliquoted in triplets into black 96-well plates, and the fluorescence polarization was measured with a Paradigm spectrometer (Molecular Devices, Sunnyvale, CA). Data were analyzed using the program Prism (GraphPad, San Diego, CA). There are a total of 22 PYD-containing proteins in the human genome (13Kwon D. Yoon J.H. Shin S.Y. Jang T.H. Kim H.G. So I. Jeon J.H. Park H.H. A comprehensive manually curated protein-protein interaction database for the death domain superfamily.Nucleic Acids Res. 2012; 40: D331-D336Crossref PubMed Scopus (36" @default.
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• W2073561387 title "Structure of the Absent in Melanoma 2 (AIM2) Pyrin Domain Provides Insights into the Mechanisms of AIM2 Autoinhibition and Inflammasome Assembly" @default.
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