Crystal structure of Aedes aegypti trypsin inhibitor in complex with μ‐plasmin reveals role for scaffold stability in Kazal‐type serine protease inhibitor

Kazal‐type protease inhibitor specificity is believed to be determined by sequence of the reactive‐site loop that make most, if not all, contacts with the serine protease. Here, we determined the complex crystal structure of Aedes aegypti trypsin inhibitor (AaTI) with μ‐plasmin, and compared its reactivities with other Kazal‐type inhibitors, infestin‐1 and infestin‐4. We show that the shortened 99‐loop of plasmin creates an S2 pocket, which is filled by phenylalanine at the P2 position of the reactive‐site loop of infestin‐4. In contrast, AaTI and infestin‐1 retain a proline at P2, rendering the S2 pocket unfilled, which leads to lower plasmin inhibitions. Furthermore, the protein scaffold of AaTI is unstable, due to an elongated Cys‐V to Cys‐VI region leading to a less compact hydrophobic core. Chimeric study shows that the stability of the scaffold can be modified by swapping of this Cys‐V to Cys‐VI region between AaTI and infestin‐4. The scaffold instability causes steric clashing of the bulky P2 residue, leading to significantly reduced inhibition of plasmin by AaTI or infestin‐4 chimera. Our findings suggest that surface loops of protease and scaffold stability of Kazal‐type inhibitor are both necessary for specific protease inhibition, in addition to reactive site loop sequence. PDB ID code: 7E50. PDB Code(s): 7E50.