Structural basis of trypsin inhibition and entomotoxicity of cospin, serine protease inhibitor involved in defense of Coprinopsis cinerea fruiting bodies |
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Authors: | Sabotič Jerica Bleuler-Martinez Silvia Renko Miha Avanzo Caglič Petra Kallert Sandra Štrukelj Borut Turk Dušan Aebi Markus Kos Janko Künzler Markus |
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Affiliation: | From the ‡Department of Biotechnology and ;the ¶Department of Biochemistry, Molecular, and Structural Biology, Jožef Stefan Institute and ;the ‖Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, Jamova 39, Ljubljana, Slovenia and ;the §Institute of Microbiology, Department of Biology, ETH Zürich, Zürich CH-8093, Switzerland |
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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. |
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Keywords: | Crystal Structure Fungi Host Defense Multifunctional Protein Protease Inhibitor Serine Protease Basidiomycete β-Trefoil Insect trypsin |
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