Conformational dynamics of plasmepsin X during inhibitor binding

The aspartic protease plasmepsin X (PMX) of the parasite Plasmodium is a promising drug target for novel malaria therapies. Two potent inhibitors of PMX are WM382 and WM4, which both include a guanidinium group that is in contact with the two catalytic aspartates of PMX in the bound complexes. In structural representations of the inhibitors, the guanidinium group is typically depicted as uncharged. However, pKa predictions with standard tools presented in this article indicate that the guanidinium groups of WM382 and WM4 are protonated and, thus, positively charged in the bound complexes. This positive charge is counterbalanced by a negatively charged catalytic aspartate D266 in PMX of Plasmodium falciparum, while the second catalytic aspartate D457 is uncharged. To investigate the interplay of the conformational dynamics of PMX and inhibitor (un)binding, we performed Hamiltonian replica exchange molecular dynamics (H-REMD) simulations starting from the predicted protonation state of the PMX-WM382 complex. On eight unbinding pathways enabled by weakened interactions of PMX and WM382 in the H-REMD simulations, we observed a dominant route of exit of the inhibitor from the binding pocket with a coupling to conformational changes in the "flap" of PMX, a {beta}-hairpin located above the binding pocket. In the bound complex, the flap adopts a closed conformation in which it tightly interacts with and covers the inhibitor. On the dominant route observed in our simulations, unbinding involves an open conformation of the flap that allows the inhibitor to exit the binding pocket. After unbinding, the flap adopts an occluded conformation in which the binding site is blocked by a bulky aromatic sidechain.