Structures of paraoxon-inhibited human acetylcholinesterase reveal perturbations of the acyl loop and the dimer interface

Abstract

Irreversible inhibition of the essential nervous system enzyme acetylcholinesterase by organophosphate nerve agents and pesticides may quickly lead to death. Oxime reactivators currently used as antidotes are generally less effective against pesticide exposure than nerve agent exposure, and pesticide exposure constitutes the majority of cases of organophosphate poisoning in the world. The current lack of published structural data specific to human acetylcholinesterase organophosphate-inhibited and oxime-bound states hinders development of effective medical treatments. We have solved structures of human acetylcholinesterase in different states in complex with the organophosphate insecticide, paraoxon, and oximes. Reaction with paraoxon results in a highly perturbed acyl loop that causes a narrowing of the gorge in the peripheral site that may impede entry of reactivators. This appears characteristic of acetylcholinesterase inhibition by organophosphate insecticides but not nerve agents. Additional changes seen at the dimer interface are novel and provide further examples of the disruptive effect of paraoxon. Ternary structures of paraoxon-inhibited human acetylcholinesterase in complex with the oximes HI6 and 2-PAM reveals relatively poor positioning for reactivation. This study provides a structural foundation for improved reactivator design for the treatment of organophosphate intoxication. Proteins 2016; 84:1246-1256. © 2016 Wiley Periodicals, Inc.

The structural and biochemical impacts of monomerizing human acetylcholinesterase

“…Recently, the plasticity within the active site of hAChE has been implicated in the ability of the enzyme to be inhibited by nerve agents and rescued by reactivating agents . To explore whether the flexibility of the active site was overly impacted by the monomerization of hAChE by the mutations L380R/F535K, the kinetic constants of (P R/S ) – VX inhibition and subsequent rescue by HI‐6 were obtained.…”

“…The efficacy of reactivators was shown to vary based on the OP, the stereochemistry of the OP, and the species of AChE . The flexibility of the active site, in particular the acyl loop, has recently been shown to play a role in the ability of nerve agents to inhibit hAChE and subsequent reactivation . Although known to play a role in the inhibition and reactivation of hAChE, probing the exact influence that the plasticity of hAChE's active site has on these events has been problematic.…”

“…Final crystal conditions were obtained by screening pH and sodium citrate concentrations. Using crystals generated under these conditions, a 2.09 Å data set was obtained in the space group P6 5 22, which differs from previous hAChE space groups . Using molecular replacement with one subunit of hAChE (PDB http://firstglance.jmol.org/fg.htm?mol=4EY4), a solution was readily found (Table ).…”

“…Outside the dimer interface, the presence of a loop at residues 491–499 is the most noticeable structural difference between the structures of the dimers and monomers. This loop in structures of the dimer is rarely complete and usually lacks electron density, implicating structural flexibility . As a result, modeling of this region has been sparse.…”

Overlay-independent comparisons of X-ray structures reveal small, systematic conformational changes in liganded acetylcholinesterase

“…The clear and significant difference between two covalent conjugates is in the acyl pocket where sharp, blue 1.2 Å positive "Δ distance" peak is observed at the position 296 of POX-hAChE, combined with smaller, 0.26 Å expansion of the peripheral site Trp286 and opposed by 0.8-0.6 Å contractions at positions 291 and 292 of the acyl pocket. This severe distortion of the acyl pocket loop specific for the POX-hAChE was well described before (9) and is a consequence of a lack of space in the hAChE acyl pocket for stabilization of phosphorus bound ethoxy substituent in the POX conjugate as opposed to much smaller methyl substituent in the sarin conjugate that fits available acyl pocket space well. In addition, or as a consequence several C-terminal alpha helices move closer to the active serine.…”