Shifts in Cα backbone conformation in X-ray structures of human acetylcholinesterase covalently inhibited by organophosphorates and organophosphoramidates revealed by PACCT3 comparative analysis

Protein backbone conformation of human acetylcholinesterase (hAChE; EC 3.1.1.7) the key enzyme in cholinergic neurotransmission, observed in over 40 PDB deposited X-ray structures appears largely identical. An exception was found in hAChE covalently inhibited by the organophosphate (OP) paraoxon where ethoxy substituent of the diethylphosphorylated active serine (Ser203), unable to fit into limited size of the acyl pocket, distorts the acyl pocket loop by shifting its Cα backbone. This obstructs access into the active center gorge and restricts ability of nucleophilic antidotes to reactivate inhibited hAChE.

We have analyzed six recently released X-ray structures of hAChE covalently inhibited by a series of phosphoramidate OPs known as Novichoks, A-230, A-232 and A-234, with or without reactivating antidote HI6, bound reversibly. A large phosphoramido substituent, identical in all three Novichoks fills the choline binding site of inhibited hAChE tightly, while remaining methyl (A-230), methoxy (A-232) or ethoxy (A-234) substituents are directed to the acyl pocket. We have explored, using PACCT3 (Pairwise Alpha Carbon Comparison Tool, available from www.ZENODO.org) whether conformations of the acyl pocket loop or of other backbone domains shifted in three conjugated hAChEs. Furthermore, we explored whether reversible binding of HI6 influenced backbone conformations of three conjugates. Our findings indicate that unlike diethylphosphorylated conjugate, in spite of their larger size, conjugated Novichok OPs do not alter the acyl pocket and cause minimal shifts in protein conformation. Nevertheless, the whole Ser203 bound OP conjugate appears shifted by ~ 1.5 Å towards the choline binding site and away from the acyl pocket. Several smaller α-helix shifts will be discussed.

This research was supported by the CounterACT Program, National Institutes of Health Office of the Director (NIH OD), and the National Institute of Neurological Disorders and Stroke (NINDS), [Grant Numbers U01 NS083451 and R21 NS098998].