Structural Basis of Trypsin Inhibition and Entomotoxicity of Cospin, Serine Protease Inhibitor Involved in Defense of Coprinopsis cinerea Fruiting Bodies

Jerica Sabotič,Silvia Bleuler-Martinez,Miha Renko,Petra Avanzo Caglič,Sandra Kallert,Borut Štrukelj,Dušan Turk,Markus Aebi,Janko Kos,Markus Künzler

doi:10.1074/jbc.m111.285304

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.

Highlights

Mushrooms are a rich source of novel proteins with unique features
The loop containing Arg-27 is positioned between the ␤2 and ␤3 strands, distinguishing cospin from other ␤-trefoil-fold serine protease inhibitors in which ␤4-␤5 or ␤5-␤6 loops are involved in protease inhibition
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

Summary

Background

Mushrooms are a rich source of novel proteins with unique features. Results: Cospin, a trypsin-specific protease inhibitor, has a ␤-trefoil fold and is toxic against the fruit fly. 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 It exhibits a highly specific inhibitory profile as a very strong inhibitor of trypsin with Ki in the picomolar range. A few serine protease inhibitors from basidiomycete fruiting bodies have been isolated and characterized, serine proteases constitute the predominant proteolytic activity in these structures [1] These inhibitors include two isomeric inhibitors of serine proteases, IA-1 and IA-2, from Pleurotus ostreatus, POIA1 and POIA2 [7], belonging to family I9 in the MEROPS classification, a serine proteinase inhibitor from Lentinus edodes [8], and cnispin, a trypsin-specific inhibitor from Clitocybe nebularis [4], the latter two belonging to family I66 [9]. We present evidence for its biological role in defense against fungivorous insects in C. cinerea fruiting bodies

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DISCUSSION