Human butyrylcholinesterase-cocaine binding pathway and free energy profiles by molecular dynamics and potential of mean force simulations

Abstract

In the present study, we have performed combined molecular dynamics and potential of mean force (PMF) simulations to determine the enzyme-substrate (ES) binding pathway and the corresponding free energy profiles for wild-type butyrylcholinesterase (BChE) binding with (-)/(+)-cocaine and for the A328W/Y332G mutant binding with (-)-cocaine. According to the PMF simulations, for each ES binding system, the substrate first binds with the enzyme at a peripheral anionic site around the entrance of the active-site gorge to form the first ES complex (ES1-like) during the binding process. Further evolution from the ES1-like complex to the nonprereactive ES complex is nearly barrierless, with a free energy barrier lower than 1.0 kcal/mol. So, the nonprereactive ES binding process should be very fast. The rate-determining step of the entire ES binding process is the subsequent evolution from the nonprereactive ES complex to the prereactive ES complex. Further accounting for the entire ES binding process, the PMF-based simulations qualitatively reproduced the relative order of the experimentally derived binding free energies (ΔG(bind)), although the simulations systematically overestimated the magnitude of the binding affinity and systematically underestimated the differences between the ΔG(bind) values. The obtained structural and energetic insights into the entire ES binding process provide a valuable base for future rational design of high-activity mutants of BChE as candidates for an enzyme therapy for cocaine overdose and abuse.

Figure 1

Plots of important distances tracked through MD simulation on the nonprereactive complex for wild-type BChE-(+)-cocaine binding. W82---N20 represents the distance from the center of aromatic side chain of W82 to the nitrogen atom at the cationic head of (+)-cocaine; Y332---COCbenzoyl means the distance from the center of aromatic side chain of Y332 to the center of benzoyl group of (+)-cocaine; F329---COCmethyl represents the distance from the center of aromatic side chain of F329 to the center of methyl ester group of (+)-cocaine; G116H---O33 represents the distance between the backbone hydrogen of residue G116 to the carbonyl oxygen at the benzoyl ester of (+)-cocaine; S198HG-H438NE and H438HD-E325OE represent the hydrogen bonding distances within the catalytic triad residues S198-H438-E325 of the enzyme; S198OG---C32 represents the distance from the OG atom of the hydroxyl group at the side chain of residue S198 to the carbonyl carbon atom at the benzoyl ester of (+)-cocaine, and the H438O---NH⁺ for the distance from the backbone oxygen atom of H438 to the proton at the cationic head of (+)-cocaine.

Figure 2

MD-simulated nonprereactive complex structure for (+)-cocaine binding with wild-type BChE. BChE is shown as gold ribbon. (+)-cocaine molecule and residues of BChE within 5 Å around (+)-cocaine are shown in the stick style and colored by the atom type. The distances from the proton at the cationic head of (+)-cocaine to the backbone oxygen atom of residue H438 and from the carbonyl oxygen at the benzoyl group of (+)-cocaine to the OG atom at the hydroxyl group of residue S198 are represented as dashed lines and labeled. Labeled also are the hydrogen bonds formed within the catalytic triad S198-H438-E325 of BChE.

Figure 3

Free energy profiles determined for (−)-cocaine binding with wild-type BChE (black curve) and the A328W/Y332G mutant (blue curve) and for (+)-cocaine binding with wild-type BChE (red curve). The reaction coordinate was defined as the distance between the mass center of all non-hydrogen atoms of either (−)-cocaine or (+)-cocaine and the mass center of all non-hydrogen atoms belonging to the E197 and I442 side chains of BChE.

Figure 4

Free energy profiles for the formation of prereactive complex for wild-type BChE binding with (−)-cocaine (left), wild-type BChE binding with (+)-cocaine (middle), and the A328W/Y332G mutant binding with (−)-cocaine (right).

Scheme 1