Fasciculin 2 Binds to the Peripheral Site on Acetylcholinesterase and Inhibits Substrate Hydrolysis by Slowing a Step Involving Proton Transfer during Enzyme Acylation

Eastman, Jean; Wilson, Erica J.; Červeñanský, Carlos; Rosenberry, Terrone L.

doi:10.1074/jbc.270.34.19694

Cited by 89 publications

(96 citation statements)

References 26 publications

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“…5). Residual activity for the FAS2-bound enzyme has been observed in experimental studies (14,26,27). It seems possible that when the main gorge entrance is sealed off the charge distribution of the back door creates an electric field, guiding substrates to the active site via this opening.…”

Section: Resultsmentioning

confidence: 98%

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

Bui

McCammon

2006

Proc. Natl. Acad. Sci. U.S.A.

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Specific, rapid association of protein complexes is essential for all forms of cellular existence. The initial association of two molecules in diffusion-controlled reactions is often influenced by the electrostatic potential. Yet, the detailed binding mechanisms of proteins highly depend on the particular system. A complete protein complex formation pathway has been delineated by using structural information sampled over the course of the transformation reaction. The pathway begins at an encounter complex that is formed by one of the apo forms of neurotoxin fasciculin-2 (FAS2) and its high-affinity binding protein, acetylcholinesterase (AChE), followed by rapid conformational rearrangements into an intermediate complex that subsequently converts to the final complex as observed in crystal structures. Formation of the intermediate complex has also been independently captured in a separate 20-ns molecular dynamics simulation of the encounter complex. Conformational transitions between the apo and liganded states of FAS2 in the presence and absence of AChE are described in terms of their relative free energy profiles that link these two states. The transitions of FAS2 after binding to AChE are significantly faster than in the absence of AChE; the energy barrier between the two conformational states is reduced by half. Conformational rearrangements of FAS2 to the final liganded form not only bring the FAS2͞AChE complex to lower energy states, but by controlling transient motions that lead to opening or closing one of the alternative passages to the active site of the enzyme also maximize the ligand's inhibition of the enzyme.conformational transitions ͉ protein-protein binding

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Section: Resultsmentioning

confidence: 98%

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

Bui

McCammon

2006

Proc. Natl. Acad. Sci. U.S.A.

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“…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.…”

mentioning

confidence: 99%

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

Shi

Boyd²,

Radić

et al. 2001

Journal of Biological Chemistry

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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 ...

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“…This seemingly less accessible position of the serine raises questions regarding ease of substrate entry and product dissociation, which have been addressed, but incompletely resolved, through molecular dynamics simulations and site-directed mutagenesis studies (9 -12). Furthermore, co-crystallization of the tight binding snake toxin fasciculin with mouse and Torpedo californica AChE shows complete occlusion of the active site gorge (7,13) despite small inhibitors remaining accessible to react with the active site serine of the complex, albeit at reduced rates (14,15). These residual rates suggest either the presence of alternative entry points for these inhibitors or breathing motions that open a gap between the fasciculin and AChE interfaces.…”

mentioning

confidence: 99%

Probing the Active Center Gorge of Acetylcholinesterase by Fluorophores Linked to Substituted Cysteines

Boyd

Marnett²,

Wong

et al. 2000

Journal of Biological Chemistry

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To examine the influence of individual side chains in governing rates of ligand entry into the active center gorge of acetylcholinesterase and to characterize the dynamics and immediate environment of these residues, we have conjugated reactive groups with selected charge and fluorescence characteristics to cysteines substituted by mutagenesis at specific positions on the enzyme. Insertion of side chains larger than in the native tyrosine at position 124 near the constriction point of the active site gorge confers steric hindrance to affect maximum catalytic throughput (k cat /K m ) and rates of diffusional entry of trifluoroketones to the active center. Smaller groups appear not to present steric constraints to entry; however, cationic side chains selectively and markedly reduce cation ligand entry through electrostatic repulsion in the gorge. Acetylcholinesterase (AChE), 1 a serine hydrolase in the ␣/␤ fold protein superfamily (1), functions at cholinergic synapses to terminate nerve signals by catalyzing ester hydrolysis of the neurotransmitter acetylcholine (2, 3). To enhance synaptic efficiency, AChE has evolved to function rapidly and can catalyze acetylcholine hydrolysis at near diffusion-limited rates (4, 5). Several crystal structures of AChE have been solved, revealing notable features of the tertiary structure (6 -8). The active site serine resides at the bottom of a deep and contorted gorge lined primarily with aromatic amino acid side chains. This seemingly less accessible position of the serine raises questions regarding ease of substrate entry and product dissociation, which have been addressed, but incompletely resolved, through molecular dynamics simulations and site-directed mutagenesis studies (9 -12). Furthermore, co-crystallization of the tight binding snake toxin fasciculin with mouse and Torpedo californica AChE shows complete occlusion of the active site gorge (7, 13) despite small inhibitors remaining accessible to react with the active site serine of the complex, albeit at reduced rates (14, 15). These residual rates suggest either the presence of alternative entry points for these inhibitors or breathing motions that open a gap between the fasciculin and AChE interfaces. Fluorescent ligands can be used to probe structural characteristics of proteins in solution. The prototypic AChE peripheral site ligand propidium, for instance, elucidated the nature of a peripheral binding site for inhibitors, remote from the active site serine residue (16). Furthermore, fluorescent phosphonates, which conjugate with the active site serine, were utilized to measure the hydrophobicity of the active site gorge, the contribution of charge to active center accessibility, and the distance between the reactive serine and the peripheral site years before a crystal structure was solved (17,18). Potentially, site-directed mutagenesis on recombinant DNA-derived AChE should render a broader range of discrete positions available for selective fluorescence labeling on the enzyme surface.Cysteine substitution mutag...

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Fasciculin 2 Binds to the Peripheral Site on Acetylcholinesterase and Inhibits Substrate Hydrolysis by Slowing a Step Involving Proton Transfer during Enzyme Acylation

Cited by 89 publications

References 26 publications

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

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

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

Probing the Active Center Gorge of Acetylcholinesterase by Fluorophores Linked to Substituted Cysteines

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