Crystal Structures of Fungal Tectonin in Complex with O-Methylated Glycans Suggest Key Role in Innate Immune Defense.

Innate immunity is the first line of defense against pathogens and predators. To initiate a response, it relies on the detection of invaders, where lectin-carbohydrate interactions play a major role. O-Methylated glycans were previously identified as non-self epitopes and conserved targets for defense effector proteins belonging to the tectonin superfamily. Here, we present two crystal structures of Tectonin 2 from the mushroom Laccaria bicolor in complex with methylated ligands, unraveling the molecular basis for this original specificity. Furthermore, they revealed the formation of a ball-shaped tetramer with 24 binding sites distributed at its surface, resembling a small virus capsid. Based on the crystal structures, a methylation recognition motif was identified and found in the sequence of many tectonins from bacteria to human. Our results support a key role of tectonins in innate defense based on a distinctive and conserved type of lectin-glycan interaction.

[1]  Richard A. Friesner,et al.  Flexible ligand docking with Glide. , 2007, Current protocols in bioinformatics.

[2]  S. Akira,et al.  Pathogen Recognition by the Innate Immune System , 2011, International reviews of immunology.

[3]  K. Henrick,et al.  Inference of macromolecular assemblies from crystalline state. , 2007, Journal of molecular biology.

[4]  D. Higgins,et al.  Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega , 2011, Molecular systems biology.

[5]  Michaela Wimmerová,et al.  The Fucose-binding Lectin from Ralstonia solanacearum , 2005, Journal of Biological Chemistry.

[6]  Christopher J. Knight,et al.  WAXSiS: a web server for the calculation of SAXS/WAXS curves based on explicit-solvent molecular dynamics , 2015, Nucleic Acids Res..

[7]  Maxim V. Petoukhov,et al.  New developments in the ATSAS program package for small-angle scattering data analysis , 2012, Journal of applied crystallography.

[8]  P. Gagneux,et al.  Glycan Evolution in Response to Collaboration, Conflict, and Constraint* , 2013, The Journal of Biological Chemistry.

[9]  Matthew P. Repasky,et al.  Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. , 2004, Journal of medicinal chemistry.

[10]  A. Imberty,et al.  Development of a competitive binding assay for the Burkholderia cenocepacia lectin BC2L-A and structure activity relationship of natural and synthetic inhibitors , 2016 .

[11]  Dirk Hauck,et al.  Methylated glycans as conserved targets of animal and fungal innate defense , 2014, Proceedings of the National Academy of Sciences.

[12]  Dmitri I. Svergun,et al.  Uniqueness of ab initio shape determination in small-angle scattering , 2003 .

[13]  I. Goldstein,et al.  Structure of mannose-specific snowdrop (Galanthus nivalis) lectin is representative of a new plant lectin family , 1995, Nature Structural Biology.

[14]  Ajit Varki,et al.  Discovery and Classification of Glycan-Binding Proteins , 2009 .

[15]  Xavier Robert,et al.  Deciphering key features in protein structures with the new ENDscript server , 2014, Nucleic Acids Res..

[16]  Vincent B. Chen,et al.  Correspondence e-mail: , 2000 .

[17]  E. Staudacher.  Methylation – an uncommon modification of glycans , 2012, Biological chemistry.

[18]  C. Huh,et al.  Cloning and characterization of Physarum polycephalum tectonins. Homologues of Limulus lectin L-6. , 1998, The Journal of biological chemistry.

[19]  Randy J. Read,et al.  Overview of the CCP4 suite and current developments , 2011, Acta crystallographica. Section D, Biological crystallography.

[20]  J. Claverie,et al.  Structure of the Escherichia coli TolB protein determined by MAD methods at 1.95 A resolution. , 1999, Structure.

[21]  J. Pierrat,et al.  The mycorrhiza helper Pseudomonas fluorescens BBc6R8 has a specific priming effect on the growth, morphology and gene expression of the ectomycorrhizal fungus Laccaria bicolor S238N. , 2007, The New phytologist.

[22]  Chang-jen Huang,et al.  Biochemical Properties and cDNa Cloning of Two New Lectins from the Plasma of Tachypleus tridentatus , 2001, The Journal of Biological Chemistry.

[23]  M. Künzler.  Hitting the Sweet Spot: Glycans as Targets of Fungal Defense Effector Proteins , 2015, Molecules.

[24]  Serge X. Cohen,et al.  Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7 , 2008, Nature Protocols.

[25]  Johannes Söding,et al.  Evolution of the β‐propeller fold , 2008, Proteins.

[26]  C. Sasakawa,et al.  A Tecpr1-dependent selective autophagy pathway targets bacterial pathogens. , 2011, Cell host & microbe.

[27]  C. Mueller-Dieckmann,et al.  Upgraded ESRF BM29 beamline for SAXS on macromolecules in solution , 2013, Journal of synchrotron radiation.

[28]  Philip R. Evans,et al.  An introduction to data reduction: space-group determination, scaling and intensity statistics , 2011, Acta crystallographica. Section D, Biological crystallography.

[29]  Adam Round,et al.  Determination of the GH3.12 protein conformation through HPLC-integrated SAXS measurements combined with X-ray crystallography. , 2013, Acta crystallographica. Section D, Biological crystallography.

[30]  Liisa Holm,et al.  Dali server: conservation mapping in 3D , 2010, Nucleic Acids Res..

[31]  M. Kiso,et al.  Six independent fucose-binding sites in the crystal structure of Aspergillus oryzae lectin. , 2016, Biochemical and biophysical research communications.

[32]  George M Sheldrick,et al.  Substructure solution with SHELXD. , 2002, Acta crystallographica. Section D, Biological crystallography.

[33]  Soichi Wakatsuki,et al.  Crystal Structure of the Human Cytosolic Sialidase Neu2 , 2005, Journal of Biological Chemistry.

[34]  C. Zipfel.  Plant pattern-recognition receptors. , 2014, Trends in immunology.

[35]  Martin Dragosits,et al.  Bisecting Galactose as a Feature of N-Glycans of Wild-type and Mutant Caenorhabditis elegans* , 2015, Molecular & Cellular Proteomics.

[36]  Christina Graf,et al.  Multivalency as a chemical organization and action principle. , 2012, Angewandte Chemie.

[37]  Narayanaswamy Srinivasan,et al.  Nucleic Acids Research Advance Access published June 21, 2007 PIC: Protein Interactions Calculator , 2007 .

[38]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[39]  H. Ushijima,et al.  Emergence and Disappearance of an Immune Molecule, an Antimicrobial Lectin, in Basal Metazoa , 2003, Journal of Biological Chemistry.

[40]  J. L. Ding,et al.  Molecular Interfaces of the Galactose-binding Protein Tectonin Domains in Host-Pathogen Interaction* , 2010, The Journal of Biological Chemistry.

[41]  M. Perduca,et al.  Three-dimensional structure and ligand-binding site of carp fishelectin (FEL). , 2015, Acta crystallographica. Section D, Biological crystallography.

[42]  Vladimir Brusic,et al.  BlockLogo: visualization of peptide and sequence motif conservation. , 2013, Journal of immunological methods.

[43]  M. Künzler,et al.  Dimerization of the fungal defense lectin CCL2 is essential for its toxicity against nematodes , 2016, Glycobiology.

[44]  Abraham Nudelman,et al.  NMR Chemical Shifts of Common Laboratory Solvents as Trace Impurities. , 1997, The Journal of organic chemistry.

[45]  David S. Goodsell,et al.  The RCSB protein data bank: integrative view of protein, gene and 3D structural information , 2016, Nucleic Acids Res..

[46]  W Bruce Turnbull,et al.  On the value of c: can low affinity systems be studied by isothermal titration calorimetry? , 2003, Journal of the American Chemical Society.

[47]  Sky W. Brubaker,et al.  Innate immune pattern recognition: a cell biological perspective. , 2015, Annual review of immunology.

[48]  Gordon D. Brown,et al.  C-type lectins in immunity: recent developments , 2015, Current opinion in immunology.

[49]  David F. Smith,et al.  Recognition of Microbial Glycans by Human Intelectin , 2015, Nature Structural &Molecular Biology.

[50]  Anne Imberty,et al.  Fungal lectins: structure, function and potential applications. , 2013, Current opinion in structural biology.

[51]  S. Goda,et al.  Hemolytic Lectin CEL-III Heptamerizes via a Large Structural Transition from α-Helices to a β-Barrel during the Transmembrane Pore Formation Process* , 2014, The Journal of Biological Chemistry.

[52]  K. Shianna,et al.  Mutation in TECPR2 reveals a role for autophagy in hereditary spastic paraparesis. , 2012, American journal of human genetics.

[53]  C. Bewley,et al.  Griffithsin: An Antiviral Lectin with Outstanding Therapeutic Potential , 2016, Viruses.

[54]  P. Emsley,et al.  Features and development of Coot , 2010, Acta crystallographica. Section D, Biological crystallography.

[55]  Tom Alber,et al.  Sensor domain of the Mycobacterium tuberculosis receptor Ser/Thr protein kinase, PknD, forms a highly symmetric beta propeller. , 2004, Journal of molecular biology.

[56]  A. H. Wang,et al.  The many blades of the β-propeller proteins: conserved but versatile. , 2011, Trends in biochemical sciences.

[57]  N. Pannu,et al.  REFMAC5 for the refinement of macromolecular crystal structures , 2011, Acta crystallographica. Section D, Biological crystallography.

[58]  M. Künzler,et al.  O-Alkylated heavy atom carbohydrate probes for protein X-ray crystallography: Studies towards the synthesis of methyl 2-O-methyl-L-selenofucopyranoside , 2016, Beilstein journal of organic chemistry.

[59]  Airlie J. McCoy,et al.  Solving structures of protein complexes by molecular replacement with Phaser , 2006, Acta crystallographica. Section D, Biological crystallography.

[60]  Michaela Wimmerová,et al.  Fucose-binding Lectin from Opportunistic Pathogen Burkholderia ambifaria Binds to Both Plant and Human Oligosaccharidic Epitopes* , 2011, The Journal of Biological Chemistry.

[61]  S. Kawabata,et al.  A Novel Type of limulus Lectin-L6. PURIFICATION, PRIMARY STRUCTURE, AND ANTIBACTERIAL ACTIVITY (*) , 1995, The Journal of Biological Chemistry.

[62]  Anne Imberty,et al.  Membrane deformation by neolectins with engineered glycolipid binding sites. , 2014, Angewandte Chemie.

[63]  Vladimir Frecer,et al.  A Novel Human Tectonin Protein with Multivalent β-Propeller Folds Interacts with Ficolin and Binds Bacterial LPS , 2009, PloS one.

[64]  B. Nagar,et al.  X-ray crystal structure of a galactose-specific C-type lectin possessing a novel decameric quaternary structure. , 2004, Biochemistry.

[65]  Hiroki Noguchi,et al.  Computational design of a self-assembling symmetrical β-propeller protein , 2014, Proceedings of the National Academy of Sciences.

[66]  George M. Sheldrick,et al.  Experimental phasing with SHELXC/D/E: combining chain tracing with density modification , 2010, Acta crystallographica. Section D, Biological crystallography.

[67]  Anne Imberty,et al.  A Soluble Fucose-Specific Lectin from Aspergillus fumigatus Conidia - Structure, Specificity and Possible Role in Fungal Pathogenicity , 2013, PloS one.

[68]  Dmitri I. Svergun,et al.  Electronic Reprint Applied Crystallography Dammif, a Program for Rapid Ab-initio Shape Determination in Small-angle Scattering Applied Crystallography Dammif, a Program for Rapid Ab-initio Shape Determination in Small-angle Scattering , 2022 .

[69]  John A Tainer,et al.  Characterizing flexible and intrinsically unstructured biological macromolecules by SAS using the Porod-Debye law. , 2011, Biopolymers.