Fungal β-trefoil trypsin inhibitors cnispin and cospin demonstrate the plasticity of the β-trefoil fold

The recently identified fungal protease inhibitors cnispin, from Clitocybe nebularis, and cospin, from Coprinopsis cinerea, are both β-trefoil proteins highly specific for trypsin. The reactive site residue of cospin, Arg27, is located on the β2-β3 loop. We show here, that the reactive site residue in cnispin is Lys127, located on the β11-β12 loop. Cnispin is a substrate-like inhibitor and the β11-β12 loop is yet another β-trefoil fold loop recruited for serine protease inhibition. By site-directed mutagenesis of the P1 residues in the β2-β3 and β11-β12 loops in cospin and cnispin, protease inhibitors with different specificities for trypsin and chymotrypsin inhibition have been engineered. Double headed inhibitors of trypsin or trypsin and chymotrypsin were prepared by introducing a second specific site residue into the β2-β3 loop in cnispin and into the β11-β12 loop in cospin. These results show that β-trefoil protease inhibitors from mushrooms exhibit broad plasticity of loop utilization in protease inhibition.