An enhanced molecular dynamics study of HPPK-ATP conformation space exploration and ATP binding to HPPK

HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase) catalyzes the transfer of pyrophosphate from ATP to HP (6-hydroxymethyl-7,8-dihydropterin). This first reaction in the folate biosynthetic pathway is an important target for potential antimicrobial agents. In this work, the mechanism by which HPPK traps and binds ATP is studied by molecular dynamics (MD)-based methods. Based on the ternary crystal structure of HPPK with an ATP mimic and HP, a complex of ATPMg(2) and HPPK is simulated and found to undergo small conformational changes with conventional MD, as does also conventional MD when started from the apo crystal structure. The introduction of restraints in the MD that serve to move HPPK-ATP from its ternary complex (closed) to apo-like (open) forms shows that throughout the restraint path ATP remains bound to HPPK. That ATP remains bound suggests that there is an ensemble of conformations with ATP bound to HPPK that span the apo to more ligand-bound-like conformations, consistent with the pre-existing equilibrium hypothesis of ligand binding, whereby a ligand can select from and bind to a broad range of protein conformations. In the apo-like conformations, ATPMg(2) remains bound to HPPK through a number of mainly salt-bridge-like interactions between several negatively charged residues and the two magnesium cations. The introduction of a reweight method that enhances the sampling of MD by targeting explicit terms in the force field helps define the interactions that bind ATP to HPPK. Using the reweight method, conformational and center of mass motions of ATP, driven by the breaking and making of hydrogen bonds and salt bridges, are identified that lead to ATP separating from HPPK. An elastic normal mode (ENM) approach to opening the ternary complex and closing the apo crystal structures was carried out. The ENM analysis of the apo structure analysis shows one mode that does have a closing motion of HPPK loops, but the direction does not correlate strongly with the loop motions that are required for forming the ternary complex.