Protease inhibitors from the Kunitz family (I3 in MEROPS) are 20-25 kDa proteins widely distributed in plant kingdom. They share a conserved b-trefoil fold in which variable loops are involved in interactions with proteases. Kunitz inhibitors target serine proteases using the canonical (Laskowski) mechanism based on a single binding loop with conserved structure.
Here, we present a set of high-resolution crystal structures of two potato Kunitz inhibitors in complex with trypsin and chymotrypsin. We identified a new, non-canonical type of reactive site that binds both serine proteases. It is formed by two separate loops interacting with the S1 and S1´ pockets. Through this structural mechanism, the non-canonical reactive site is stabilized against proteolysis by the target proteases, providing a functional advantage over the canonical design.