Curariform antagonists bind in different orientations to the nicotinic receptor ligand binding domain

Curariform alkaloids competitively inhibit muscle acetylcholine receptors (AChR) by bridging the α and non-α subunits that form the ligand-binding site. Here we delineate bound orientations of d-tubocurarine (d-TC) and its methylated derivative metocurine using mutagenesis, ligand binding measurements, and computational methods. When tested against a series of lysine mutations in the ε subunit, the two antagonists show marked differences in the consequences of the mutations on binding affinity. The mutations εL117K, εY111K, and εL109K decrease affinity of metocurine by up to 3 orders of magnitude but only slightly alter affinity of d-TC. At the α subunit face of the binding site, the mutation αY198T decreases affinity of both antagonists, but αY198F preferentially enhances affinity of d-TC. Computation of antagonist docking orientations, based on our structural model of the α-ε site of the human AChR, indicates distinct orientations of each antagonist; the flatter metocurine fits into a pocket formed principally by the ε subunit, whereas the more compact d-TC spans the narrower crevasse between α and ε subunits. The side chains of εTyr-111 and εThr-117 juxtapose one of two quaternary nitrogens in metocurine but are remote from the equivalent quaternary nitrogen in d-TC, which instead closely approaches αTyr-198. The different docked orientations arise through tilt of the curariform scaffold by ∼60° normal to the nitrogen-nitrogen axis, together with a 20° rotation about the axis. The overall mutagenesis and computational results show that despite their similar structures, d-TC and metocurine bind in distinctly different orientations to the adult human AChR.