Lines of communication within and between catalytic subunits of human acetylcholinesterase revealed by cryo- and room-temperature X-ray crystallography and by Small Angle X-ray Scattering (SAXS)

Abstract

Allosteric interactions have been discussed in the context of the acetylcholinesterase (EC 3.1.1.7; AChE) catalysis and inhibition for nearly sixty years, but even determination of more than 200 AChE X-ray structures has not helped to resolve a generally accepted atomic-level mechanism. Allosterism in inhibition of this key enzyme of cholinergic neurotransmission was first proposed by J.-P. Changeux for interaction of AChE with flaxedil and d-tubocurarine in early 1960s. It was followed by meticulous analyses of non-competitive fluorescent ligand binding by P. Taylor group and non-competitive components of reversible and covalent AChE inhibition by W.N. Aldridge and E. Reiner in early 1970s. The accumulated functional evidence of interactions between the active center and spatially remote sites of ligand binding appeared in apparent contradiction with small magnitudes of rarely-observed conformational changes of the 70 kDa catalytic AChE subunit in commonly used cryo- X-ray crystallographic analyses. We have turned to room-temperature X-ray crystallography to detect conformational diversity of active center human AChE (hAChE) residues reflected in distinct conformations of pyridinium aldoximes found to span active center and allosteric sites of the native and organophosphate (OP) inhibited hAChE. A sequence of conformational changes in the hAChE backbone, triggered by covalent OP inhibition can lead to dissociation of a hAChE homodimer, that we detected by SAXS, and identified structural elements of that allosteric effect. Furthermore, analysis of geometries of crystallographic hAChE homodimers, as well as homodimers of closely related alpha/beta hydrolase-fold proteins provided both evidence and suggestions for structural basis of allosteric interactions in those physiologically important proteins. 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].