Reversibly Bound and Covalently Attached Ligands Induce Conformational Changes in the Omega Loop, Cys69–Cys96, of Mouse Acetylcholinesterase

Abstract

We have used a combination of cysteine substitution mutagenesis and site-specific labeling to characterize the structural dynamics of mouse acetylcholinesterase (mAChE). Six cysteine-substituted sites of mAChE (Leu 76 , Glu 81 , Glu 84 , Tyr 124 , Ala 262 , and His 287 ) were labeled with the environmentally sensitive fluorophore, acrylodan, and the kinetics of substrate hydrolysis and inhibitor association were examined along with spectroscopic characteristics of the acrylodan-conjugated, cysteine-substituted enzymes. Residue 262, being well removed from the active center, appears unaffected by inhibitor binding. Following the binding of ligand, hypsochromic shifts in emission of acrylodan at residues 124 and 287, located near the perimeter of the gorge, reflect the exclusion of solvent and a hydrophobic environment created by the associated ligand. By contrast, the bathochromic shifts upon inhibitor binding seen for acrylodan conjugated to three omega loop (⍀ loop) residues 76, 81, and 84 reveal that the acrylodan side chains at these positions are displaced from a hydrophobic environment and become exposed to solvent. The magnitude of fluorescence emission shift is largest at position 84 and smallest at position 76, indicating that a concerted movement of residues on the ⍀ loop accompanies gorge closure upon ligand binding. Acrylodan modification of substituted cysteine at position 84 reduces ligand binding and steady-state kinetic parameters between 1 and 2 orders of magnitude, but a similar substitution at position 81 only minimally alters the kinetics. Thus, combined kinetic and spectroscopic analyses provide strong evidence that conformational changes of the ⍀ loop accompany ligand binding.Acetylcholinesterase (AChE), 1 a serine hydrolase in the ␣/␤-fold hydrolase protein superfamily (1), terminates nerve signals by catalyzing hydrolysis of the neurotransmitter acetylcholine at a diffusion limited rate (2, 3). The crystallographic structure of mouse AChE reveals a catalytic triad (Ser 203 , Glu 334 , and His 447 ) located at the bottom of a narrow active site gorge 20 Å in depth (4 -6). Because the cross-section of the physiological substrate acetylcholine is larger than the narrowest part of the gorge, the remarkably high turnover rate of AChE raises questions regarding substrate access to the catalytic site.Molecular dynamic simulations suggest that rapid fluctuations of gorge width combined with diffusion facilitated by electrostatic forces could enhance substrate accessibility (7-10). In addition, the high affinity and slowly dissociating complex of fasciculin and AChE retains slight residual catalytic activity (11, 12), despite the occlusion of the active site gorge by fasciculin as shown in the crystal structures (5, 13, 14). Rapid fluctuations in residues lining the gorge walls may leave transient gaps at the fasciculin-AChE interface and may account for residual activity.The large omega loop (⍀ loop), Cys 69 -Cys 96 , flanking the active site gorge in mouse AChE corresponds to the activation loop ...

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

“…The cysteine-substituted enzymes show kinetics of acetylcholine hydrolysis similar to wild type enzyme (11).…”

“…Interestingly, large hypsochromic shifts and enhancements of quantum yield are observed at positions 81 and 124. This pattern appears to be unusual, because the reversible active center ligands studied previously (11) and the dimethylphosphoryl conjugate formed from DDVP, which yields a phosphoryl serine with one methylene group shorter than echothiophate, confer little shift at position 124 and a large bathochromic shift at position 81. To confirm that the chromic shift is a result of conjugation of diethylphosphoryl group, not the retention of the thiocholine leaving group in the gorge, we examined the effect of paraoxon conjugation on chromic shift.…”

“…Excess dithiothreitol was removed by use of a G-50 Sephadex spin column (Roche Molecular Biochemicals) equilibrated in 10 mM Tris-HCl, 100 mM NaCl, 40 mM MgCl 2 , pH 8.0. Conditions for acrylodan labeling and stoichiometry estimates have been described previously (11). Stoichiometry of labeling of the various preparations, estimated from a comparison of enzyme concentration by protein (280 nm) to acrylodan (360 -380 nm) absorbance, ranged as follows: L76C, 0.7-1.0; E81C, 0.77-1.0; E84C, 0.77-1.0; Y124C, 0.78 -1.0; A262C, 0.69 -0.92; and H287C, 0.78 -1.0.…”

“…This residue is not anticipated to be influenced by ligand-induced changes in conformation. Our previous study showed a bathochromic emission shift of acrylodan conjugated at ⍀ loop residues 76, 81, and 84 upon binding of inhibitors, such as tacrine, edrophonium, huperzine A, and m-(N,N,N-trimethylammonio)trifluoromethyl acetophenone, that interact with Trp 86 in the choline binding site (11). Acrylodan fluorescence is exquisitely sensitive to dipole moment of the surrounding solvent or macromolecular milieu (12)(13)(14).…”

Contemporary paradigms for cholinergic ligand design guided by biological structure

“…10,11 By substitution of cysteines at designated positions in the molecule, it is possible to label selectively designated side chains and analyze both solvent exposure at equilibrium and dynamic fluctuations in molecular structure as a function of position of the label and occupation of the ligand. 10,12 From this approach, changes in the microscopic environment around the protein surface and dynamic structural fluctuations associated with ligand binding can be analyzed. Hence, part of the knowledge gap in the nsec time frame is satisfied with time-resolved fluorescence techniques.…”