Abstract
The nematode Ascaris suum primarily infects pigs, but also causes disease in humans. As part of its survival mechanism in the intestinal tract of the host, the worm produces a number of protease inhibitors, including pepsin inhibitor-3 (PI3), a 17 kDa protein. Recombinant PI3 expressed in E. coli has previously been shown to be a competitive inhibitor of a subgroup of aspartic proteinases: pepsin, gastricsin and cathepsin E. The previously determined crystal structure of the complex of PI3 with porcine pepsin (p. pepsin) showed that there are two regions of contact between PI3 and the enzyme. The first three N-terminal residues (QFL) bind into the prime side of the active site cleft and a polyproline helix (139-143) in the C-terminal domain of PI3 packs against residues 289-295 that form a loop in p. pepsin. Mutational analysis of both inhibitor regions was conducted to assess their contributions to the binding affinity for p. pepsin, human pepsin (h. pepsin) and several malarial aspartic proteases, the plasmepsins. Overall, the polyproline mutations have a limited influence on the Ki values for all the enzymes tested, with the values for p. pepsin remaining in the low-nanomolar range. The largest effect was seen with a Q1L mutant, with a 200-fold decrease in Ki for plasmepsin 2 from Plasmodium falciparum (PfPM2). Thermodynamic measurements of the binding of PI3 to p. pepsin and PfPM2 showed that inhibition of the enzymes is an entropy-driven reaction. Further analysis of the Q1L mutant showed that the increase in binding affinity to PfPM2 was due to improvements in both entropy and enthalpy.