Inhibitors of Different Structure Induce Distinguishing Conformations in the Omega Loop, Cys69–Cys96, of Mouse Acetylcholinesterase

Abstract

We have shown previously that association of reversible active site ligands induces a conformational change in an omega loop (⍀ loop), Cys 69 -Cys 96 , of acetylcholinesterase. The fluorophore acrylodan, site-specifically incorporated at positions 76, 81, and 84, on the external portion of the loop not lining the active site gorge, shows changes in its fluorescence spectrum that reflect the fluorescent side chain moving from a hydrophobic environment to become more solvent-exposed. This appears to result from a movement of the ⍀ loop accompanying ligand binding. We show here that the loop is indeed flexible and responds to conformational changes induced by both active center and peripheral site inhibitors (gallamine and fasciculin). Moreover, phosphorylation and carbamoylation of the active center serine shows distinctive changes in acrylodan fluorescence spectra at the ⍀ loop sites, depending on the chirality and steric dimensions of the covalently conjugated ligand. Capping of the gorge with fasciculin, although it does not displace the bound ligand, dominates in inducing a conformational change in the loop. Hence, the ligand-induced conformational changes are distinctive and suggest multiple loop conformations accompany conjugation at the active center serine. The fluorescence changes induced by the modified enzyme may prove useful in the detection of organophosphates or exposure to cholinesterase inhibitors. Acetylcholinesterase (AChE)1 plays a pivotal role in neurotransmission by terminating the action of neurotransmitter, acetylcholine, at neuromuscular junction and other cholinergic synapses (1-3). AChE is one of most efficient enzymes known with hydrolysis of its natural substrate reaching diffusion-controlled limits. Inhibitors of AChE target two sites in the active site gorge: an active center at the base of a narrow gorge 20 Å in depth and a peripheral site at the gorge rim (4). At the active center, a residue triad (Ser 203 -Glu 334 -His 447 ) promotes acyl transfer and hydrolysis of the substrate, whereas Trp 86 at the gorge base primarily stabilizes choline moiety of the substrate through a cationinteraction. Active site inhibitors block substrate binding either by associating with the tryptophan in the choline binding site (tacrine and edrophonium) or by reacting irreversibly with catalytic serine (carbamates and organophosphates). Peripheral site inhibitors, such as propidium and gallamine, inhibit catalytic activity through both steric blockade and allosterically altering catalytic efficiency of the active center residues (4 -8).To elucidate the conformational changes associated with mechanistically distinctive inhibitors, we developed a means for physically monitoring the conformation of purified mouse AChE by site-directed labeling with an environmentally sensitive fluorophore, acrylodan. Six single cysteine mutants were prepared for acrylodan conjugation (Fig. 1). Three were on the Cys 69 -Cys 96 omega loop (⍀ loop) flanking the active site gorge: L76C near the tip of the loop, and E81C and E...

Protein complex formation by acetylcholinesterase and the neurotoxin fasciculin-2 appears to involve an induced-fit mechanism

“…12, which is published as supporting information on the PNAS web site). Motions of the long omega loop of AChE, which are enhanced when FAS2 is bound to AChE, have also been shown in the previous MD and fluorescence anisotropy studies (17,24). Opening of the back door is highly correlated to the salt-bridge formation between FAS2:R11 and AChE:E91 (10).…”

Freeze-frame inhibitor captures acetylcholinesterase in a unique conformation

“…Hence, the flexibility of the AChE long ⍀ loop differs from the hinge-like motion of a homologous loop that, in the structurally related lipases, forms a rigid flap and opens only in the presence of the lipid substrate (43,44). Moreover, ligand binding to AChE may cause the gorge to collapse around the ligand, minimizing internal dimensions (41,42). This notion is supported by the observed repositioning of the Tyr-337 side chain and associated perturbation of Tyr-341, which not only alter the gorge shape but also enlarge its width at the position of constriction to accommodate the syn1 triazole (Fig.…”

“…Rapid fluctuations giving rise to transient enlargements of the gorge appear critical (40). Ligand binding evidently induces a closed gorge state, whereas the unliganded enzyme seems to fluctuate rapidly between multiple states with varying degrees of gorge openness (41,42).…”

Cholinesterase Structure: Identification of Mechanisms and Residues Involved in Organophosphate Inhibition and Enzyme Reactivation

“…Li the case of the omega loop, the fluorescence studies show that the inner portion excludes solvent water upon hgand binding, as would be predicted from the ligand occupation and the closure of the gorge around the hgand. More importantly, the outer portion of the omega loop exhibits increased curvature causing the side chains to move into the solvent, thereby showing a bathochromic emission shift of the acrylodan residue (Shi et al, 2001(Shi et al, ,2002. The residues showing this change in environment are a considerable distance from the binding site, so the conformational change is truly allosteric in character.…”

“…To this end, we have developed a physical approach to measure the effective dielectric constant of substituted side chains through fluorescence specfroscopy (Boyd et al, 2000;Shi et al, 2001Shi et al, , 2002. The studies are based on cysteine substitution mutagenesis of selected residues in the molecule.…”