Distance distributions for the Tyr12Ce1 to Ser203N atoms.

<p>The soman-adducted structures show a significant increase in distances in comparison to the apo structures. This increase is due to the interactions of the phenol sidechain with the pinacolyl tail. A small peak at 14 Å in the apo distribution suggests a second conformation that allows ligand access to the catalytic triad.</p

A Wrench in the Works of Human Acetylcholinesterase: Soman Induced Conformational Changes Revealed by Molecular Dynamics Simulations

<div><p>Irreversible inactivation of human acetylcholinesterase (hAChE) by organophosphorous pesticides (OPs) and chemical weapon agents (CWA) has severe morbidity and mortality consequences. We present data from quantum mechanics/molecular mechanics (QM/MM) and 80 classical molecular dynamics (MD) simulations of the apo and soman-adducted forms of hAChE to investigate the effects on the dynamics and protein structure when the catalytic Serine 203 is phosphonylated. We find that the soman phosphonylation of the active site Ser203 follows a water assisted addition-elimination mechanism with the elimination of the fluoride ion being the highest energy barrier at 6.5 kcal/mole. We observe soman-dependent changes in backbone and sidechain motions compared to the apo form of the protein. These alterations restrict the soman-adducted hAChE to a structural state that is primed for the soman adduct to be cleaved and removed from the active site. The altered motions and resulting structures provide alternative pathways into and out of the hAChE active site. In the soman-adducted protein both side and back door pathways are viable for soman adduct access. Correlation analysis of the apo and soman adducted MD trajectories shows that the correlation of gorge entrance and back door motion is disrupted when hAChE is adducted. This supports the hypothesis that substrate and product can use two different pathways as entry and exit sites in the apo form of the protein. These alternative pathways have important implications for the rational design of medical countermeasures.</p></div

Comparison of motions of apo and soman-adducted hAChE.

<p>Cα rmsd values for the Omega loop are shown. In the soman-adducted protein the Omega loop motions are significantly lower than those in the apo protein. A second peak at 3 Å in the apo structures suggests an additional conformation is present.</p

Cα RMSD values and opening areas for the native and soman-adducted protein MD trajectories.^a

<p>^a The values exclude data points from the first 1 ns of each simulation and used every 10^th frame for a total of 4700 data points per trajectory. 40 trajectories for each protein state were performed for a total of 188000 data points. The root mean square deviation (rmsd) values are in units of Å and the standard deviations are shown in parentheses.</p><p>^b Rmsd values listed for the TcAChE proteins are calculated between the TcAChE crystal structures and the initial hAChE structure before MD is begun.</p><p>Triad contains Cα atoms from residues Trp86, Ser203, His447, and Glu334 (TcAChE:Trp84, Ser200, His440, and Glu327). Omega loop contains Cα atoms from residues 69 to 96 (Torpedo: 67 to 94).</p><p>286 loop contains residues 286 to 291</p><p>Gorge Entry contains Cα atoms from residues Tyr72, Leu76, His287, Glu292, and Tyr341 (TcAChE: Tyr70, Gln74, Asn280, Asp285, and Tyr334).</p><p>Side Door Entry contains backbone atoms from residues Asp74N, Leu76Cα, Met85Cα, and Asn87Cα (TcAChE: Asp72N, Gln74Cα, Met83Cα, and Asn85Cα)</p><p>Back Door contains Cα and sidechain atoms from residues Met85Cα, Val132Cg1, Tyr449Oh, and Glu452Cd (TcAChE: Val129Cg1, Tyr442Oh, and Glu445Cd).</p><p>Pinch Point contains backbone atoms from residues Trp86Hα, Tyr124Cα, and His447Cα (TcAChE: Trp84Hα, Tyr121Cα, and His440Cα).</p><p>Cα RMSD values and opening areas for the native and soman-adducted protein MD trajectories.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121092#t002fn001" target="_blank">^a</a></p

Normalized correlation matrices of the main chain Cα atoms in the apo and soman-adducted AChE.

<p>A backbone representation of the apo ache is shown (top) highlighting the circled regions (bottom); (A) residues 15–20 and 450–455, (B) residues 120–130 and 445–452, (C) residues 190–220 and 400–410, (D) residues 262–265 and 464–466.</p

Cross-sectional area distributions for three protein regions are shown for the apo (black) and soman-adducted (red) MD structures.

<p>Only the gorge entry and back door sidechain regions of the soman-adducted hAChE protein experience a shift to larger cross-sectional area values relative to the apo trajectories while the other areas are similar or diminished in area. These data suggest the soman adduct is shifting the protein into a more restrained set of conformations. The asterisk denotes the cross-sectional area of comparable atoms in the TcAChE apo and soman adducted structures.</p

The soman adduct in hAChE as viewed from the gorge entrance.

<p>Soman (yellow space-filled spheres) is mostly occluded by aromatic residues (blue space-filled spheres). The phosphoryl oxygen of the soman adduct is colored red. The aromatic sidechains sequester the soman adduct from the gorge thereby limiting access of traditional countermeasures. Figure created with VMD [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121092#pone.0121092.ref011" target="_blank">11</a>] and Tachyon [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121092#pone.0121092.ref012" target="_blank">12</a>].</p

The energy barriers for the adduction of soman to the active site Ser203 by QM calculations.

<p>Each step is a single DFT geometry optimization calculation. The structure (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121092#pone.0121092.s001" target="_blank">S1 Fig</a>) from the final geometry optimization was used as the starting point for the classical MD simulations of the soman-adducted human AChE. The 6.5 kcal/mole barrier value is similar to the experimentally determined value of 9.1 kcal/mole for that of sarin.</p

Normalized correlation matrices for all heavy atoms within 20 Å of the Ser203 hydroxyl oxygen atom for the apo and soman-adducted AChE.

<p>Residue numbers are shown for significant portions of the primary sequence. Positive correlations between near and distant residues is significantly decreased when soman is present. These areas of missing correlation are highlighted with ellipses. Additional negative correlation is observed between the 286 Loop and residues 443–455 in the soman adducted protein. The soman adduct appears to decouple positive correlative motions within a radius of 20 Å from the active site.</p

Acylation mechanism of hAChE by acetylcholine.

<p>The nucleophilic attack of the deprotonated Ser203 hydroxy group on the carbonyl carbon of acetylcholine and the reactivation of Ser203 by an activated water is shown.</p