Structural determination of a new non-canonical inhibition complex between porcine trypsin and the potato Kunitz-STI M271.

The Kunitz-Soybean Trypsin Inhibitor (Kunitz-STI) family is a protein family found in different plants. Several of its members have been experimentally described as inhibitors of different classes of proteases from several organisms. Even though most of the previous crystallographic studies describing the protease inhibition interactions present by different Kunitz-STI family members have been focused on serine proteases, the protein family has been proposed as a promising scaffold for future multifunctional protease inhibitor with a probable wide range of applications. In this work, a new crystallographic complex formed between M271, a Kunitz-STI obtained from Solanum tuberosum, and porcine trypsin shows a new inhibitory interface in which M271 loops β1-β2 and β3-β4 play a central role. Additionally, the structural analysis demonstrates that the inhibition described here follows a non-canonical mechanism in which M271 loops β1-β2 and β3-β4 block the porcine trypsin active site. Our findings expand the molecular repertoire in which a Kunitz-STI can inhibit trypsin, stressing the role of the dynamical characteristics of the Kunitz-STI family members on the protein inhibition process. This work also gives a new example of the remarkable plasticity of the Kunitz-STI fold, in which an arrangement with eleven highly sequence-variable loops projected to the solvent demonstrates a simple answer for a multifunctional evolutionary tool to inhibit proteases.