Canonical or noncanonical? Structural plasticity of serine protease‐binding loops in Kunitz‐STI protease inhibitors

The Kunitz‐Soybean Trypsin Inhibitor (Kunitz‐STI) family is a large family of proteins with most of its members being protease inhibitors. The versatility of the inhibitory profile and the structural plasticity of these proteins, make this family a promising scaffold for designing new multifunctional proteins. Historically, Kunitz‐STI inhibitors have been classified as canonical serine protease inhibitors, but new inhibitors with novel inhibition mechanisms have been described in recent years. Different inhibition mechanisms could be the result of different evolutionary pathways. In the present work, we performed a structural analysis of all the crystallographic structures available for Kunitz‐STI inhibitors to characterize serine protease‐binding loop structural features and locations. Our study suggests a relationship between the conformation of serine protease‐binding loops and the inhibition mechanism, their location in the β‐trefoil fold, and the plant source of the inhibitors. The classical canonical inhibitors of this family are restricted to plants from the Fabales order and bind their targets via the β4–β5 loop, whereas serine protease‐binding loops in inhibitors from other plants lie mainly in the β5–β6 and β9–β10 loops. In addition, we found that the β5–β6 loop is used to inhibit two different families of serine proteases through a steric blockade inhibition mechanism. This work will help to change the general perception that all Kunitz‐STI inhibitors are canonical inhibitors and proteins with protease‐binding loops adopting noncanonical conformations are exceptions. Additionally, our results will help in the identification of protease‐binding loops in uncharacterized or newly discovered inhibitors, and in the design of multifunctional proteins.