Mycocypins, clitocypins and macrocypins, are cysteine protease inhibitors isolated from the mushrooms Clitocybe nebularis and Macrolepiota procera. Lack of sequence homology to other families of protease inhibitors suggested that mycocypins inhibit their target cysteine protease by a unique mechanism and that a novel fold may be found. The crystal structures of the complex of clitocypin with the papain-like cysteine protease cathepsin V and of macrocypin and clitocypin alone have revealed yet another motif of binding to papain like-cysteine proteases, which in a yet unrevealed way occludes the catalytic residue. The binding is associated with a peptide-bond flip of glycine that occurs before or concurrently with the inhibitor docking. Mycocypins possess a -trefoil fold, the hallmark of Kunitz-type inhibitors. It is a tree-like structure with two loops in the root region, a stem comprising a six-stranded -barrel, and two layers of loops (6 ؉ 3) in the crown region. The two loops that bind to cysteine cathepsins belong to the lower layer of the crown loops, whereas a single loop from the crown region can inhibit trypsin or asparaginyl endopeptidase, as demonstrated by site-directed mutagenesis. These loops present a versatile surface with the potential to bind to additional classes of proteases. When appropriately engineered, they could provide the basis for possible exploitation in crop protection.Inhibition of foreign protease activity is a widespread defense mechanism in plants against their pests, pathogens, and parasites (1). Protein inhibitors of proteases are present in a variety of plant tissues. They can be deployed alone or together with a variety of small molecules (2). It has been known for a long time that the expression of protease inhibitors is increased in injured plant leaves (3) and that their expression can be induced as a response to attack by insects or pathogens (2).Given the negative environmental effects of chemical pesticides used in crop protection, it is important to explore alternative approaches, such as the incorporation of genes encoding protease inhibitors into plants. Transgenic plants expressing various protease inhibitors have shown enhanced levels of insect resistance; however, the adaptive capacity of insect digestive proteases limits the use of single protease inhibitors (4, 5). The use of hybrid protease inhibitors with multiple inhibitory activity could, however, affect the functional properties of the fused inhibitors (6). Incorporation of genes encoding a range of protease inhibitors is to run the risk of deleterious modification of plants, but the use of a single protease inhibitor with versatile functionality could be the way forward.With these in mind, we have undertaken structural and mechanistic studies of the cysteine protease inhibitors clitocypin (Clt) 3 from mushroom Basidiomycetes Clitocybe nebularis and macrocypins (Mcp) from Macrolepiota procera. Based on the lack of sequence similarity to other protease inhibitors, they form separate protease inhibitor famil...