Versatile Loops in Mycocypins Inhibit Three Protease Families

Abstract

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...

Entomotoxic and nematotoxic lectins and protease inhibitors from fungal fruiting bodies

“…Entomotoxic b (Cordara et al 2011;Grahn et al 2007;Grahn et al 2009;Rempel et al 2002;Winter et al 2002;Wohlschlager et al 2011) na Entomotoxic (Brzin et al 2000;Renko et al 2010;Sabotič et al 2007a;Sabotič et al 2006;Sabotič et al 2011;Šmid et al 2015 (91 % sequence similarity) and are approximately 32 % identical (43 % similar) to CGL2. Galectins CGL1 and CGL2 are highly abundant in young fruiting bodies but hardly produced in the vegetative mycelium of C. cinerea (Boulianne et al 2000;Plaza et al 2014).…”

“…Cathepsins B and H, that exhibit both endopeptidase and exopeptidase activity, are not or only very weakly inhibited by mycocypins. In addition to papain-like cysteine proteases (family C1), mycocypins also inhibit asparaginyl endopeptidase (AEP) also called legumain (family C13) while trypsin, but not AEP, is inhibited by macrocypin 4 (Renko et al 2010;Sabotič et al 2007a;Sabotič et al 2009). …”

“…In macrocypin 4, the asparagine is replaced by a lysine, enabling inhibition of trypsin. Consideration of the crystal structures leads to the conclusion that the binding loops of clitocypin and macrocypins exhibit substantial conformational flexibility during binding into the active site of their target enzymes (Renko et al 2010;.…”

“…Thus, dikaryotic fungi employ, in addition to a large repertoire of secondary metabolites, a plethora of proteins acting as deterrents or toxins in defense of their fruiting bodies (Spiteller 2015;Wang et al 2002). These proteins include lectins that bind to the glycans of glycoproteins or glycolipids in the digestive tract of fungivores , protease inhibitors that inhibit digestive proteases of fungivores (Renko et al 2010), biotinbinding proteins that sequester this essential cofactor (BleulerMartinez et al 2012), pore-forming proteins that cause cell lysis (Mancheno et al 2010;Ota et al 2014), RNA toxins (ribotoxins) that cleave or depurinate RNA molecules (Lacadena et al 2007), and other enzymes including proteases, oxidases, and phospholipases (Erjavec et al 2012). The first two groups of proteins: lectins and protease inhibitors, Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-7075-2) contains supplementary material, which is available to authorized users.…”

The Plasticity of the β-Trefoil Fold Constitutes an Evolutionary Platform for Protease Inhibition

“…It is commonly assumed that most members of this family have only a single reactive site loop, which for the archetypical soybean trypsin inhibitor (STI) is located between residues Ser-60 and Phe-66. However, several cases of inhibitors possessing two reactive sites, and thus binding two target molecules simultaneously, have been reported (7)(8)(9)(10). These have been dubbed "double-headed" or "Janus-type" inhibitors.…”