Understanding the enzyme–ligand complex: insights from all-atom simulations of butyrylcholinesterase inhibition

Abstract

All-atom molecular dynamics simulations of butyrylcholinesterase (BChE) sans inhibitor and in complex with each of fifteen dialkyl phenyl phosphate derivatives were conducted to characterize inhibitor binding modes and strengths. Each system was sampled on the 250 ns timescale in explicit ionic solvent, for a total of over 4 μs of simulation time. A K-means algorithm was used to cluster the resulting structures into distinct binding modes, which were further characterized based on atomic-level contacts between inhibitor chemical groups and active site residues. Comparison of experimentally observed inhibition constants (K I) with the resulting contact tables provides structural explanations for relative binding coefficients and highlights several notable interaction motifs. These include ubiquitous contact between glycines in the oxyanion hole and the inhibitor phosphate group; a sterically-driven binding preference for positional isomers that extend aromaticity; a stereochemical binding preference for choline-containing inhibitors, which mimic natural BChE substrates; and the mechanically-induced opening of the omega loop region to fully expose the active site gorge in the presence of choline-containing inhibitors. Taken together, these observations can greatly inform future design of BChE inhibitors, and the approach reported herein is generalizable to other enzyme-inhibitor systems and similar complexes that depend on non-covalent molecular recognition.

A Comprehensive Review of Cholinesterase Modeling and Simulation

“…As expected, one of the most important sites for both cholinesterases is the catalytic active site (CAS), and the peripheral aromatic site (PAS) also plays an indispensable role in cholinesterase or ligand binding, while the Ω-loop (OML), acyl binding site (ABS), and oxyanion hole (OAH) sites are more essential in contributing to binding affinity and complex stability. Alvarado et al have provided a method of succinct graphical tabulation of BChE-ligand contacts and interactions, referred to as contact tables, that include these five sites and additional protein residues of interest [ 35 ], as discussed below. A detailed analysis of these binding sites is provided here in the same order that they are encountered by substrates and inhibitors upon entering and moving into the gorge.…”

“…In recent years, however, the aromatic properties of this binding site that are vital to cholinesterase function have driven the community to instead refer to this region as the “peripheral aromatic site”. Important amino acids in the PAS of AChE include serine, tyrosine, aspartic acid, and tryptophan [ 36 , 38 ], while notable PAS residues in BChE include asparagine, aspartic acid, glutamine, serine, and tyrosine [ 35 ], highlighting the polar, negatively charged, and electron-rich nature of residues in this site. As previously mentioned, one distinction between the cholinesterases is the aromatic nature of the residues surrounding the PAS in AChE, which is more aliphatic in BChE [ 10 ].…”

“…The catalytic active site (CAS, yellow in Figure 1 ) is located approximately 20 Å deep at the bottom of both the AChE and BChE gorges [ 12 , 24 ] and is made up of serine, glutamic acid, and histidine residues, prompting the name “catalytic triad” [ 24 , 35 ]. The CAS is surrounded by numerous aromatic and acidic residues [ 44 ] and is observed to engage in shorter hydrogen bonds in crystal and NMR structures than observed in simulation [ 45 , 46 ].…”

“…The oxyanion hole (OAH, orange in Figure 1 ), made of two glycines and one alanine [ 35 , 47 ], is generally a two-pronged site in many proteases and hydrolases; in the case of AChE, and subsequently BChE, the OAH is a three-pronged hole [ 61 ]. Early MD simulations of AChE phosphonylation suggested that the OAH exerts a pulling force on leaving groups during the alkylation step [ 45 ], and the OAH is known to lower the energy barrier for ACh hydrolysis in both cholinesterases [ 62 ].…”

“…More recently, this same laboratory studied an array of dialkyl phenyl phosphate inhibitors via MD simulation, including numerous phenyl substitutions that had been previously probed via docking calculations [ 146 ] and a small set of alkyl-to-cholinyl substitutions to mimic the chemistry of the natural substrates. It was noted therein that larger R-groups increase van der Waals contact area between the enzyme and the ligand, with S-enantiomers apparently binding more strongly than their R analogs [ 35 ]. In an effort to characterize the observed modes of binding in the flexible BChE-OP complexes, contact tables such as that shown in Figure 6 were used to highlight specific interactions between portions of the inhibitor and specific binding sites and amino acids in the BChE active site gorge.…”