Inhibitor binding and conformational dynamics of plasmepsin X

The aspartic protease plasmepsin X (PMX) of the parasite Plasmodium is a promising drug target for novel malaria therapies. Two highly potent inhibitors of PMX are WM382 and WM4. In this thesis, we used molecular dynamics (MD) and Hamiltonian replica exchange molecular dynamics (H-REMD) simulations to investigate the binding of these two inhibitors to PMX. We first used empirical prediction methods to establish the protonation states of the inhibitors and of the two catalytic aspartates D266 and D457 of PMX. The guanidinium (CG) group of the inhibitors, which is in contact with the catalytic aspartates, is generally considered to be uncharged. However, our empirical predictions indicate that the CG group of the inhibitors is protonated and thus, positively charged, in the bound complexes, while the catalytic aspartate D266 of PMX is negatively charged. Binding free energy calculations based on MD simulations of the bound complexes in the four possible protonation states of the two aspartates D266 and D457 indicate strong binding in the protonation state in which only D266 is negatively charged, which confirms the empirical predictions. We then performed H-REMD simulations in the predicted protonation state of the complexes PMX-WM382 and PMX-WM4 with the aim to investigate the binding equilibrium of bound and unbound molecular conformations. Besides the expected high contact probabilities of PMX and the inhibitors in the binding pocket, the simulations indicate intermediate contact probabilities in the flap regio of PMX, which is located above the binding pocket. An investigation of conformational fluctuations in the flap region indicates a stabilization of the flap by the bound inhibitors. The torsional angle distributions of the inhibitors in the bound and unbound states of the simulations point out dominant bound inhibitor conformations and a loss of torsional freedom of the inhibitors during binding. Finally, we explored the unbinding pathways of WM382 observed in the HREMD simulations and the conformational changes of PMX during unbinding. We identified a dominant route of exit of the inhibitor from the binding pocket with a coupling to conformational changes in the flap region of PMX. In the bound state, the flap adopts a closed conformation in which it tightly interacts with the inhibitor. 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 with the binding site blocked by a bulky aromatic sidechain.