Conformational Remodeling of Femtomolar Inhibitor−Acetylcholinesterase Complexes in the Crystalline State

The active center of acetylcholinesterase (AChE), a target site for competitive inhibitors, resides centrosymmetric to the subunit at the base of a deep, narrow gorge lined by aromatic residues. At the gorge entry, a peripheral site encompasses overlapping binding loci for noncompetitive inhibitors, which alter substrate access to the gorge. The click-chemistry inhibitor TZ2PA6 links the active center ligand, tacrine, to the peripheral site ligand, propidium, through a biorthogonal reaction of an acetylene and an azide that forms either a <i>syn1</i> or an <i>anti1</i> triazole. Compared with wild-type mouse AChE, a Tyr337Ala mutant displays full catalytic activity, albeit with 2−3 orders of magnitude higher affinities for the TZ2PA6 <i>syn1</i> and <i>anti1</i> regioisomers, reflected in low femtomolar <i>K</i><sub>d</sub> values, diffusion-limited association, and dissociation half-times greater than 1 month and 1 week, respectively. Three structures of each of the co-crystallized <i>syn1</i> and <i>anti1</i> complexes of the Tyr337Ala mutant were solved at three distinct times of crystal maturation, consistent with or exceeding the half-lives of the complexes in solution, while crystalline complexes obtained from soaked Tyr337Ala crystals led to picturing “freshly formed” complexes. The structures, at 2.55−2.75Å resolution, reveal a range of unprecedented conformations of the bound regioisomers, not observed in the wild-type AChE complexes, associated with concerted positional rearrangements of side chains in the enzyme gorge. Moreover, time-dependent conformational remodeling of the crystalline complexes appears to correlate with the dissociation half-times of the solution complexes. Hence, for the tight-binding TZ2PA6 inhibitors, the initial complexes kinetically driven in solution slowly form more stable complexes governed by thermodynamic equilibrium and observable in mature crystals.