Structural insights into serine protease inhibition by a marine invertebrate BPTI Kunitz-type inhibitor

Proteins isolated from marine invertebrates are frequently characterized by exceptional structural and functional properties. ShPI-1, a BPTI Kunitz-type inhibitor from the Caribbean Sea anemone Stichodactyla helianthus, displays activity not only against serine-, but also against cysteine-, and aspartate proteases. As an initial step to evaluate the molecular basis of its activities, we describe the crystallographic structure of ShPI-1 in complex with the serine protease bovine pancreatic trypsin at 1.7å resolution. The overall structure and the important enzyme-inhibitor interactions of this first invertebrate BPTI-like Kunitz-type inhibitor:trypsin complex remained largely conserved compared to mammalian BPTI-Kunitz inhibitor complexes. However, a prominent stabilizing role within the interface was attributed to arginine at position P3. Binding free-energy calculations indicated a 10-fold decrease for the inhibitor affinity against trypsin, if the P3 residue of ShPI-1 is mutated to alanine. Together with the increased role of Arg¹¹ at P3 position, slightly reduced interactions at the prime side (Pn') of the primary binding loop and at the secondary binding loop of ShPI-1 were detected. In addition, the structure provides important information for site directed mutagenesis to further optimize the activity of rShPI-1A for biotechnological applications.