Scaffold stability and P14′ residue steric hindrance in the differential inhibition of FXIIa by Aedes aegypti trypsin inhibitor versus Infestin-4

Kazal-type protease inhibitors strictly regulate Factor XIIa (FXIIa), a blood-clotting serine protease. However, when negatively charged surface of prosthetic device come into contact with FXII, it undergoes conformational change and auto-activation, leading to thrombus formation. Some research suggests that Kazal-type protease inhibitor specificity against FXIIa is governed solely by the reactive-site loop sequence, as this sequence makes most-if not all-of the direct contacts with FXIIa. Here, we sought to compare the inhibitory properties of two Kazal-type inhibitors, Infestin-4 (Inf4), a potent inhibitor of FXIIa, and Aedes aegypti trypsin inhibitor (AaTI), which does not inhibit FXIIa, to better understand Kazal-type protease specificity and determine the structural components responsible for inhibition. There are only three residue differences in the reactive-site loop between AaTI and Inf4. Through site-directed mutagenesis, we show that the reactive-site loop is only partially responsible for the inhibitory specificity of these proteases. The protein scaffold of AaTI is unstable due to an elongated C5C6 region. Through chimeric study, we show that swapping the protease-binding loop and the C5C6 region from Inf4 with that of AaTI can partially enhance the inhibitory activity of the AaTI_Inf4 chimera. Furthermore, the additional substitution of Asn at the P14′ position of AaTI with Gly (Gly27 in Inf4) absolves the steric clashing between AaTI and the surface 140-loop of FXIIa, and increases the inhibition of the chimeric AaTI to match that of wild-type Inf4. Our findings suggest that ancillary regions in addition to the reactive-site loop sequence are important factors driving Kazal-type inhibitor specificity.