title: Structural basis for chitotetraose coordination by CGL3, a novel galectin-related protein from Coprinopsis cinerea.
abstract: Recent advances in genome sequencing efforts have revealed an abundance of novel putative lectins. Among these, many galectin-related proteins, characterized by many conserved residues but intriguingly lacking critical amino acids, have been found in all corners of the eukaryotic superkingdom. Here we present a structural and biochemical analysis of one representative, the galectin-related lectin CGL3 found in the inky cap mushroom Coprinopsis cinerea. This protein contains all but one conserved residues known to be involved in beta-galactoside binding in galectins. A Trp residue strictly conserved among galectins is changed to an Arg in CGL3 (R81). Accordingly, the galectin-related protein is not able to bind lactose. Screening of a glycan array revealed that CGL3 displays preference for oligomers of beta1-4-linked N-acetyl-glucosamines (chitooligosaccharides) and GalNAc beta 1-4GlcNAc (LacdiNAc). Carbohydrate-binding affinity of this novel lectin was quantified using isothermal titration calorimetry, and its mode of chitooligosaccharide coordination not involving any aromatic amino acid residues was studied by X-ray crystallography. Structural information was used to alter the carbohydrate-binding specificity and substrate affinity of CGL3. The importance of residue R81 in determining the carbohydrate-binding specificity was demonstrated by replacing this Arg with a Trp residue (R81W). This single-amino-acid change led to a lectin that failed to bind chitooligosaccharides but gained lactose binding. Our results demonstrate that, similar to the legume lectin fold, the galectin fold represents a conserved structural framework upon which dramatically altered specificities can be grafted by few alterations in the binding site and that, in consequence, many metazoan galectin-related proteins may represent lectins with novel carbohydrate-binding specificities.
authors.affiliation: Institute of Microbiology, ETH Zürich, Wolfgang-Pauli-Str. 10, CH-8093 Zürich, Switzerland.
authors.forename: Martin Andreas
authors.initials: MA
authors.lastname: Wälti
authors.forename: Piers Jamie
authors.initials: PJ
authors.lastname: Walser
authors.forename: Stéphane
authors.initials: S
authors.lastname: Thore
authors.forename: Anke
authors.initials: A
authors.lastname: Grünler
authors.forename: Michaela
authors.initials: M
authors.lastname: Bednar
authors.forename: Markus
authors.initials: M
authors.lastname: Künzler
authors.forename: Markus
authors.initials: M
authors.lastname: Aebi
mesh_terms: D000363:Agaricales / Q000201:enzymology / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017124:Conserved Sequence; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D037501:Galectin 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D037502:Galectin 3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D013816:Thermodynamics
chemical_list: D005656:Fungal Proteins; D037501:Galectin 2; D037502:Galectin 3; D009844:Oligosaccharides; C012238:chitotetrose