title: Structural basis of trypsin inhibition and entomotoxicity of cospin, serine protease inhibitor involved in defense of Coprinopsis cinerea fruiting bodies.
abstract: Cospin (PIC1) from Coprinopsis cinerea is a serine protease inhibitor with biochemical properties similar to those of the previously characterized fungal serine protease inhibitors, cnispin from Clitocybe nebularis and LeSPI from Lentinus edodes, classified in the family I66 of the MEROPS protease inhibitor classification. In particular, it exhibits a highly specific inhibitory profile as a very strong inhibitor of trypsin with K(i) in the picomolar range. Determination of the crystal structure revealed that the protein has a β-trefoil fold. Site-directed mutagenesis and mass spectrometry results have confirmed Arg-27 as the reactive binding site for trypsin inhibition. The loop containing Arg-27 is positioned between the β2 and β3 strands, distinguishing cospin from other β-trefoil-fold serine protease inhibitors in which β4-β5 or β5-β6 loops are involved in protease inhibition. Biotoxicity assays of cospin on various model organisms revealed a strong and specific entomotoxic activity against Drosophila melanogaster. The inhibitory inactive R27N mutant was not entomotoxic, associating toxicity with inhibitory activity. Along with the abundance of cospin in fruiting bodies of C. cinerea and the lack of trypsin-like proteases in the C. cinerea genome, these results suggest that cospin and its homologs are effectors of a fungal defense mechanism against fungivorous insects that function by specific inhibition of serine proteases in the insect gut.
authors.affiliation: Department of Biotechnology, Jožef Stefan Institute, Jamova 39, Ljubljana, Slovenia. Jerica.Sabotic@ijs.si
authors.forename: Jerica
authors.initials: J
authors.lastname: Sabotič
authors.forename: Silvia
authors.initials: S
authors.lastname: Bleuler-Martinez
authors.forename: Miha
authors.initials: M
authors.lastname: Renko
authors.forename: Petra
authors.initials: P
authors.lastname: Avanzo Caglič
authors.forename: Sandra
authors.initials: S
authors.lastname: Kallert
authors.forename: Borut
authors.initials: B
authors.lastname: Štrukelj
authors.forename: Dušan
authors.initials: D
authors.lastname: Turk
authors.forename: Markus
authors.initials: M
authors.lastname: Aebi
authors.forename: Janko
authors.initials: J
authors.lastname: Kos
authors.forename: Markus
authors.initials: M
authors.lastname: Künzler
mesh_terms: D000363:Agaricales / Q000737:chemistry*; D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D048690:Fruiting Bodies, Fungal / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014361:Trypsin Inhibitors / Q000737:chemistry*
chemical_list: D005656:Fungal Proteins; D014361:Trypsin Inhibitors