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Palmer Taylor - One of the best experts on this subject based on the ideXlab platform.
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Theoretical analysis of the structure of the peptide Fasciculin and its docking to acetylcholinesterase.
Protein science : a publication of the Protein Society, 2008Co-Authors: Harald K. L. Van Den Born, Palmer Taylor, Zoran Radić, Pascale Marchot, Igor F. TsigelnyAbstract:The Fasciculins are a family of closely related peptides that are isolated from the venom of mambas and exert their toxic action by inhibiting acetylcholinesterase (AChE). Fasciculins belong to the structural family of three-fingered toxins from Elapidae snake venoms, which include the alpha-neurotoxins that block the nicotinic acetylcholine receptor and the cardiotoxins that interact with cell membranes. The features unique to the known primary and tertiary structures of the Fasciculin molecule were analyzed. Loop I contains an arginine at position 11, which is found only in the Fasciculins and could form a pivotal anchoring point to AChE. Loop II contains five cationic residues near its tip, which are partly charge-compensated by anionic side chains in loop III. By contrast, the other three-fingered toxins show full charge compensation within loop II. The interaction of Fasciculin with the recognition site on acetylcholinesterase was investigated by estimating a precollision orientation followed by determination of the buried surface area of the most probable complexes formed, the electrostatic field contours, and the detailed topography of the interaction surface. This approach has led to testable models for the orientation and site of bound Fasciculin.
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Conformational Transitions in Protein-Protein Association: Binding of Fasciculin-2 to Acetylcholinesterase
Biophysical journal, 2006Co-Authors: Jennifer M. Bui, Palmer Taylor, Zoran Radić, J. Andrew MccammonAbstract:The neurotoxin Fasciculin-2 (FAS2) is a picomolar inhibitor of synaptic acetylcholinesterase (AChE). The dynamics of binding between FAS2 and AChE is influenced by conformational fluctuations both before and after protein encounter. Submicrosecond molecular dynamics trajectories of apo forms of Fasciculin, corresponding to different conformational substates, are reported here with reference to the conformational changes of loop I of this three-fingered toxin. This highly flexible loop exhibits an ensemble of conformations within each substate corresponding to its functions. The high energy barrier found between the two major substates leads to transitions that are slow on the timescale of the diffusional encounter of noninteracting FAS2 and AChE. The more stable of the two apo substates may not be the one observed in the complex with AChE. It seems likely that the more stable apo form binds rapidly to AChE and conformational readjustments then occur in the resulting encounter complex.
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Nanosecond Dynamics of Acetylcholinesterase Near the Active Center Gorge
The Journal of biological chemistry, 2004Co-Authors: Aileen E. Boyd, Palmer Taylor, Zoran Radić, Lilly Wong, Cristina S. Dunlop, David A. JohnsonAbstract:Abstract To delineate the role of peptide backbone flexibility and rapid molecular motion in acetylcholinesterase catalysis and inhibitor association, we investigated the decay of fluorescence anisotropy at three sites of fluorescein conjugation to cysteine-substitution mutants of the enzyme. One cysteine was placed in a loop at the peripheral site near the rim of the active center gorge (H287C); a second was in a helical region outside of the active center gorge (T249C); a third was at the tip of a small, flexible ω loop well separated from the gorge (A262C). Mutation and fluorophore conjugation did not appreciably alter catalytic or inhibitor binding parameters of the enzyme. The results show that each site examined was associated with a high degree of segmental motion; however, the A262C and H287C sites were significantly more flexible than the T249C site. Association of the active center inhibitor, tacrine, and the peripheral site peptide inhibitor, Fasciculin, had no effect on the anisotropy decay of fluorophores at positions 249 and 262. Fasciculin, but not tacrine, on the other hand, dramatically altered the decay profile of the fluorophore at the 287 position, in a manner consistent with Fasciculin reducing the segmental motion of the peptide chain in this local region. The results suggest that the motions of residues near the active center gorge and across from the Cys69-Cys96 ω loop are uncoupled and that ligand binding at the active center or the peripheral site does not influence acetylcholinesterase conformational dynamics globally, but induces primarily domain localized decreases in flexibility proximal to the bound ligand.
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inhibitors of different structure induce distinguishing conformations in the omega loop cys69 cys96 of mouse acetylcholinesterase
Journal of Biological Chemistry, 2002Co-Authors: Jianxin Shi, Zoran Radic, Palmer TaylorAbstract:Abstract We have shown previously that association of reversible active site ligands induces a conformational change in an omega loop (Ω loop), Cys69–Cys96, 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 Ω 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 Ω 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.
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Inhibitors of Different Structure Induce Distinguishing Conformations in the Omega Loop, Cys69–Cys96, of Mouse Acetylcholinesterase
Journal of Biological Chemistry, 2002Co-Authors: Jianxin Shi, Zoran Radić, Palmer TaylorAbstract:Abstract We have shown previously that association of reversible active site ligands induces a conformational change in an omega loop (Ω loop), Cys69–Cys96, 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 Ω 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 Ω 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.
Pascale Marchot - One of the best experts on this subject based on the ideXlab platform.
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Molecular Characterization of Monoclonal Antibodies that Inhibit Acetylcholinesterase by Targeting the Peripheral Site and Backdoor Region
2016Co-Authors: Yves Bourne, Jacques Grassi, Grégoire Mondielli, Patricia Lamourette, Didier Boquet, Pascale MarchotAbstract:The inhibition properties and target sites of monoclonal antibodies (mAbs) Elec403, Elec408 and Elec410, generated against Electrophorus electricus acetylcholinesterase (AChE), have been defined previously using biochemical and mutagenesis approaches. Elec403 and Elec410, which bind competitively with each other and with the peptidic toxin inhibitor Fasciculin, are directed toward distinctive albeit overlapping epitopes located at the AChE peripheral anionic site, which surrounds the entrance of the active site gorge. Elec408, which is not competitive with the other two mAbs nor Fasciculin, targets a second epitope located in the backdoor region, distant from the gorge entrance. To characterize the molecular determinants dictating their binding site specificity, we cloned and sequenced the mAbs; generated antigen-binding fragments (Fab) retaining the parental inhibition properties; and explored their structure-function relationships using complementary x-ray crystallography, homology modeling and flexible docking approaches. Hypermutation of one Elec403 complementarity-determining region suggests occurrence of antigen-driven selection towards recognition of the AChE peripheral site. Comparative analysis of the 1.9Å-resolution structure of Fab408 and of theoretical models of its Fab403 and Fab410 congeners evidences distinctive surface topographies and anisotropic repartitions of charges, consistent with their respective target sites and inhibitio
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Theoretical analysis of the structure of the peptide Fasciculin and its docking to acetylcholinesterase.
Protein science : a publication of the Protein Society, 2008Co-Authors: Harald K. L. Van Den Born, Palmer Taylor, Zoran Radić, Pascale Marchot, Igor F. TsigelnyAbstract:The Fasciculins are a family of closely related peptides that are isolated from the venom of mambas and exert their toxic action by inhibiting acetylcholinesterase (AChE). Fasciculins belong to the structural family of three-fingered toxins from Elapidae snake venoms, which include the alpha-neurotoxins that block the nicotinic acetylcholine receptor and the cardiotoxins that interact with cell membranes. The features unique to the known primary and tertiary structures of the Fasciculin molecule were analyzed. Loop I contains an arginine at position 11, which is found only in the Fasciculins and could form a pivotal anchoring point to AChE. Loop II contains five cationic residues near its tip, which are partly charge-compensated by anionic side chains in loop III. By contrast, the other three-fingered toxins show full charge compensation within loop II. The interaction of Fasciculin with the recognition site on acetylcholinesterase was investigated by estimating a precollision orientation followed by determination of the buried surface area of the most probable complexes formed, the electrostatic field contours, and the detailed topography of the interaction surface. This approach has led to testable models for the orientation and site of bound Fasciculin.
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Mechanism of acetylcholinesterase inhibition by Fasciculin: a 5-ns molecular dynamics simulation.
Journal of the American Chemical Society, 2002Co-Authors: Kaihsu Tai, Pascale Marchot, Yves Bourne, Tongye Shen, Richard H. Henchman, J. Andrew MccammonAbstract:Our previous molecular dynamics simulation (10 ns) of mouse acetylcholinesterase (EC 3.1.1.7) revealed complex fluctuation of the enzyme active site gorge. Now we report a 5-ns simulation of acetylcholinesterase complexed with Fasciculin 2. Fasciculin 2 binds to the gorge entrance of acetylcholinesterase with excellent complementarity and many polar and hydrophobic interactions. In this simulation of the protein-protein complex, where Fasciculin 2 appears to sterically block access of ligands to the gorge, again we observe a two-peaked probability distribution of the gorge width. When Fasciculin is present, the gorge width distribution is altered such that the gorge is more likely to be narrow. Moreover, there are large increases in the opening of alternative passages, namely, the side door (near Thr 75) and the back door (near Tyr 449). Finally, the catalytic triad arrangement in the acetylcholinesterase active site is disrupted with Fasciculin bound. These data support that, in addition to the steric obstruction seen in the crystal structure, Fasciculin may inhibit acetylcholinesterase by combined allosteric and dynamical means. Additional data from these simulations can be found at http://mccammon.ucsd.edu/.
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Electron paramagnetic resonance reveals altered topography of the active center gorge of acetylcholinesterase after binding of Fasciculin to the peripheral site
Biochimica et biophysica acta, 1999Co-Authors: Marjeta Šentjurc, Zoran Radić, Pascale Marchot, Slavko Pečar, Jure Stojan, Zoran GrubičAbstract:Abstract Fasciculin, a peptidic toxin from snake venom, inhibits mammalian and fish acetylcholinesterases (AChE) by binding to the peripheral site of the enzyme. This site is located at the rim of a narrow, deep gorge which leads to the active center triad, located at its base. The proposed mechanisms for AChE inhibition by Fasciculin include allosteric events resulting in altered conformation of the AChE active center gorge. However, a Fasciculin-induced altered topography of the active center gorge has not been directly demonstrated. Using electron paramagnetic resonance with the spin-labeled organophosphate 1-oxyl-2,2,6,6-tetramethyl-4-piperidinylethylphosphorofluoridate (EtOSL) specifically bound to the catalytic serine of mouse AChE (mAChE), we show that bound Fasciculin on mAChE slows down, but does not prevent phosphorylation of the active site serine by EtOSL and protects the gorge conformation against thermal denaturation. Most importantly, a restricted freedom of motion of the spin label bound to the Fasciculin-associated mAChE, compared to mAChE, is evidenced. Molecular models of mAChE and Fasciculin-associated mAChE with tethered EtOSL enantiomers indicate that this restricted motion is due to greater proximity of the S-EtOSL nitroxide radical to the W86 residue in the Fasciculin-associated enzyme. Our results demonstrate a topographical alteration indicative of a restricted conformation of the active center gorge of mAChE with bound Fasciculin at its rim.
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The Fasciculin-acetylcholinesterase interaction
Journal de la Societe de biologie, 1999Co-Authors: Pascale MarchotAbstract:L’acetylcholinesterase (AChE) est une enzyme-cle du mecanisme de transmission cholinergique. Les fas- ciculines, petites proteines « a trois doigts » isolees des venins de serpent mamba, sont des inhibiteurs puissants et selectifs des AChEs de mammiferes et poissons electriques. L’interaction Fasciculine-AChE constitue un exemple parfait d’interaction entre une toxine animale et son recepteur macromoleculaire. En effet, par leur selectivite, leur remarquable affinite, leur caractere d’inhibiteur allosterique, et leur nature proteique (donc accessible a la chimie des proteines et la biologie moleculaire), les Fasciculines constituent des outils de choix, « scalpels moleculaires » pour l’etude de l’AChE. Les travaux presentes dans ce chapitre ont ete menes selon une approche multidisciplinaire faisant intervenir des techniques distinctes, mais complementaires, ainsi que de nombreuses collaborations. Les resultats obtenus contribuent a la definition des bases structurales et dynamiques non seulement des mecanismes d'inhibition de l’AChE par les Fasciculines, mais egalement de la fonctionnalite du site peripherique de l’AChE, site distinct du site catalytique et doue de proprietes regulatrices.
Zoran Radić - One of the best experts on this subject based on the ideXlab platform.
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Theoretical analysis of the structure of the peptide Fasciculin and its docking to acetylcholinesterase.
Protein science : a publication of the Protein Society, 2008Co-Authors: Harald K. L. Van Den Born, Palmer Taylor, Zoran Radić, Pascale Marchot, Igor F. TsigelnyAbstract:The Fasciculins are a family of closely related peptides that are isolated from the venom of mambas and exert their toxic action by inhibiting acetylcholinesterase (AChE). Fasciculins belong to the structural family of three-fingered toxins from Elapidae snake venoms, which include the alpha-neurotoxins that block the nicotinic acetylcholine receptor and the cardiotoxins that interact with cell membranes. The features unique to the known primary and tertiary structures of the Fasciculin molecule were analyzed. Loop I contains an arginine at position 11, which is found only in the Fasciculins and could form a pivotal anchoring point to AChE. Loop II contains five cationic residues near its tip, which are partly charge-compensated by anionic side chains in loop III. By contrast, the other three-fingered toxins show full charge compensation within loop II. The interaction of Fasciculin with the recognition site on acetylcholinesterase was investigated by estimating a precollision orientation followed by determination of the buried surface area of the most probable complexes formed, the electrostatic field contours, and the detailed topography of the interaction surface. This approach has led to testable models for the orientation and site of bound Fasciculin.
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Conformational Transitions in Protein-Protein Association: Binding of Fasciculin-2 to Acetylcholinesterase
Biophysical journal, 2006Co-Authors: Jennifer M. Bui, Palmer Taylor, Zoran Radić, J. Andrew MccammonAbstract:The neurotoxin Fasciculin-2 (FAS2) is a picomolar inhibitor of synaptic acetylcholinesterase (AChE). The dynamics of binding between FAS2 and AChE is influenced by conformational fluctuations both before and after protein encounter. Submicrosecond molecular dynamics trajectories of apo forms of Fasciculin, corresponding to different conformational substates, are reported here with reference to the conformational changes of loop I of this three-fingered toxin. This highly flexible loop exhibits an ensemble of conformations within each substate corresponding to its functions. The high energy barrier found between the two major substates leads to transitions that are slow on the timescale of the diffusional encounter of noninteracting FAS2 and AChE. The more stable of the two apo substates may not be the one observed in the complex with AChE. It seems likely that the more stable apo form binds rapidly to AChE and conformational readjustments then occur in the resulting encounter complex.
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Nanosecond Dynamics of Acetylcholinesterase Near the Active Center Gorge
The Journal of biological chemistry, 2004Co-Authors: Aileen E. Boyd, Palmer Taylor, Zoran Radić, Lilly Wong, Cristina S. Dunlop, David A. JohnsonAbstract:Abstract To delineate the role of peptide backbone flexibility and rapid molecular motion in acetylcholinesterase catalysis and inhibitor association, we investigated the decay of fluorescence anisotropy at three sites of fluorescein conjugation to cysteine-substitution mutants of the enzyme. One cysteine was placed in a loop at the peripheral site near the rim of the active center gorge (H287C); a second was in a helical region outside of the active center gorge (T249C); a third was at the tip of a small, flexible ω loop well separated from the gorge (A262C). Mutation and fluorophore conjugation did not appreciably alter catalytic or inhibitor binding parameters of the enzyme. The results show that each site examined was associated with a high degree of segmental motion; however, the A262C and H287C sites were significantly more flexible than the T249C site. Association of the active center inhibitor, tacrine, and the peripheral site peptide inhibitor, Fasciculin, had no effect on the anisotropy decay of fluorophores at positions 249 and 262. Fasciculin, but not tacrine, on the other hand, dramatically altered the decay profile of the fluorophore at the 287 position, in a manner consistent with Fasciculin reducing the segmental motion of the peptide chain in this local region. The results suggest that the motions of residues near the active center gorge and across from the Cys69-Cys96 ω loop are uncoupled and that ligand binding at the active center or the peripheral site does not influence acetylcholinesterase conformational dynamics globally, but induces primarily domain localized decreases in flexibility proximal to the bound ligand.
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Inhibitors of Different Structure Induce Distinguishing Conformations in the Omega Loop, Cys69–Cys96, of Mouse Acetylcholinesterase
Journal of Biological Chemistry, 2002Co-Authors: Jianxin Shi, Zoran Radić, Palmer TaylorAbstract:Abstract We have shown previously that association of reversible active site ligands induces a conformational change in an omega loop (Ω loop), Cys69–Cys96, 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 Ω 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 Ω 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.
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Electron paramagnetic resonance reveals altered topography of the active center gorge of acetylcholinesterase after binding of Fasciculin to the peripheral site
Biochimica et biophysica acta, 1999Co-Authors: Marjeta Šentjurc, Zoran Radić, Pascale Marchot, Slavko Pečar, Jure Stojan, Zoran GrubičAbstract:Abstract Fasciculin, a peptidic toxin from snake venom, inhibits mammalian and fish acetylcholinesterases (AChE) by binding to the peripheral site of the enzyme. This site is located at the rim of a narrow, deep gorge which leads to the active center triad, located at its base. The proposed mechanisms for AChE inhibition by Fasciculin include allosteric events resulting in altered conformation of the AChE active center gorge. However, a Fasciculin-induced altered topography of the active center gorge has not been directly demonstrated. Using electron paramagnetic resonance with the spin-labeled organophosphate 1-oxyl-2,2,6,6-tetramethyl-4-piperidinylethylphosphorofluoridate (EtOSL) specifically bound to the catalytic serine of mouse AChE (mAChE), we show that bound Fasciculin on mAChE slows down, but does not prevent phosphorylation of the active site serine by EtOSL and protects the gorge conformation against thermal denaturation. Most importantly, a restricted freedom of motion of the spin label bound to the Fasciculin-associated mAChE, compared to mAChE, is evidenced. Molecular models of mAChE and Fasciculin-associated mAChE with tethered EtOSL enantiomers indicate that this restricted motion is due to greater proximity of the S-EtOSL nitroxide radical to the W86 residue in the Fasciculin-associated enzyme. Our results demonstrate a topographical alteration indicative of a restricted conformation of the active center gorge of mAChE with bound Fasciculin at its rim.
J C Fontecilla-camps - One of the best experts on this subject based on the ideXlab platform.
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Structure of Fasciculin 2 from green mamba snake venom: evidence for unusual loop flexibility.
Acta Crystallographica Section D Biological Crystallography, 1996Co-Authors: D. Housset, Pascale Marchot, Pierre E. Bougis, J. Navaza, J C Fontecilla-campsAbstract:The crystal structure of the snake toxin Fasciculin 2, a potent acetylcholinesterase inhibitor from the venom of the green mamba (Dendroaspis angusticeps), has been determined by the molecular-replacement method, using the Fasciculin 1 model and refined to 2.0 A resolution. The introduction of an overall anisotropic temperature factor improved significantly the quality of the electron-density map. It suggests, as it was also indicated by the packing, that the thermal motion along the unique axis direction is less pronounced than on the (ab) plane. The final crystallographic R factor is 0.188 for a model having r.m.s. deviations from ideality of 0.016 A for bond lengths and 2.01° for bond angles. As Fasciculin 1, Fasciculin 2 belongs to the three-finger class of Elapidae toxins, a structural group that also contains the α-neurotoxins and the cardiotoxins. Although the two Fasciculins have, overall, closely related structures, the conformation of loop I differs appreciably in the two molecules. The presence of detergent in crystallization medium in the case of Fasciculin 2 appears to be responsible for the displacement of the loop containing Thr9. This conformational change also results in the formation of a crystallographic dimer that displays extensive intermolecular interactions.
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Structure of Fasciculin 2 from green mamba snake venom: evidence for unusual loop flexibility.
Acta crystallographica. Section D Biological crystallography, 1996Co-Authors: D. Housset, J. Navaza, P Marchot, P E Bougis, J C Fontecilla-campsAbstract:The crystal structure of the snake toxin Fasciculin 2, a potent acetylcholinesterase inhibitor from the venom of the green mamba (Dendroaspis angusticeps), has been determined by the molecular-replacement method, using the Fasciculin 1 model and refined to 2.0 A resolution. The introduction of an overall anisotropic temperature factor improved significantly the quality of the electron-density map. It suggests, as it was also indicated by the packing, that the thermal motion along the unique axis direction is less pronounced than on the (ab) plane. The final crystallographic R factor is 0.188 for a model having r.m.s. deviations from ideality of 0.016 A for bond lengths and 2.01 degrees for bond angles. As Fasciculin 1, Fasciculin 2 belongs to the three-finger class of Elapidae toxins, a structural group that also contains the alpha-neurotoxins and the cardiotoxins. Although the two Fasciculins have, overall, closely related structures, the conformation of loop I differs appreciably in the two molecules. The presence of detergent in crystallization medium in the case of Fasciculin 2 appears to be responsible for the displacement of the loop containing Thr9. This conformational change also results in the formation of a crystallographic dimer that displays extensive intermolecular interactions.
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1.9-A resolution structure of Fasciculin 1, an anti-acetylcholinesterase toxin from green mamba snake venom.
The Journal of biological chemistry, 1992Co-Authors: P Marchot, P E Bougis, J C Fontecilla-campsAbstract:Abstract The crystal structure of Fasciculin 1, a potent acetylcholinesterase inhibitor from green mamba snake venom, has been solved by the multiple isomorphous replacement method complemented with anomalous scattering and subsequently refined at 1.9-A resolution. The overall structure of Fasciculin is similar to those of the short alpha-neurotoxins and cardiotoxins, with a dense core rich in disulfide bridges and three long loops disposed as the central fingers of a hand. A comparison of these three prototypic toxin types shows that Fasciculin 1 has structural features that are intermediate between those of the other two molecules. Its core region, which can be defined as a continuous stretch of conserved residues, is very similar to that of erabutoxin b, whereas the orientation of its long loops resembles that of cardiotoxin VII4. This result introduces a new element in the study of phylogenetic relationships of snake toxins and suggests that, after divergency from an ancestral gene, convergent evolution may have played an important factor in the evolution of these proteins. In Fasciculin 1, several arginine and lysine residues are well ordered and relatively exposed to the solvent medium and may play a role in the binding to the peripheral site of acetylcholinesterases.
Pierre E. Bougis - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of mouse acetylcholinesterase by Fasciculin: crystal structure of the complex and mutagenesis of Fasciculin.
Toxicon : official journal of the International Society on Toxinology, 1998Co-Authors: Pascale Marchot, Pierre E. Bougis, Yves Bourne, Claudine N. Prowse, Palmer TaylorAbstract:Fasciculins are members of the superfamily of three-fingered peptidic toxins from Elapidae venoms. They selectively inhibit mammalian and electric fish acetylcholinesterases (AChE) with Ki values in the pico- to nanomolar range. Kinetic studies performed in solution indicate that Fasciculin does not totally occlude ligand access to the active site of AChE, but rather binds to a peripheral site of the enzyme to inhibit catalysis, perhaps allosterically. The crystal structure of the Fas2-mouse AChE complex delineated a large contact area consistent with the low dissociation constant of the complex; the Fas2 and AChE residues participating in the binding interface were unambiguously established, and major hydrophobic interactions were identified. The structure however suggests that Fasciculin totally occludes substrate entry into the catalytic site of AChE, and does not reveal to what extent each contact between Fas2 and AChE contributes to the overall binding energy. New probes, designed to delineate the individual contributions of the Fasciculin residues to the complex formation and conformation, were generated by site-directed mutagenesis of a synthetic Fas2 gene. A fully processed recombinant Fasciculin, rFas2, that is undistinguishable from the natural, venom-derived Fas2, was expressed in a mammalian system; fourteen mutants, encompassing 16 amino acid residues distributed among the three loops (fingers) of Fas2, were developed from both the kinetic and structural data and analyzed for inhibition of mouse AChE. The determinants identified by the structural and the functional approaches do coincide. However, only a few of the many residues which make up the overall interactive site of the Fas2 molecule provide the strong interactions required for high affinity binding and enzyme inhibition. Potential drug design from the Fasciculin molecule is discussed.
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Fasciculin Inhibition of Mouse Acetylcholinesterase
Structure and Function of Cholinesterases and Related Proteins, 1998Co-Authors: Pascale Marchot, Pierre E. Bougis, Yves Bourne, Claudine N. Prowse, Joan R. Kanter, Jonathan Eads, Palmer TaylorAbstract:Fasciculins are 7 kDa proteins that contain four disulfide bridges and are isolated from mamba venoms. They are members of the superfamily of three-fingered Elapidae snake toxins that include the selective nicotinic acetylcholine receptor blockers, α- and κ-neurotoxins, the subtype-specific muscarinic acetylcholine receptor agonists, termed “muscarinic toxins”, the L-type calcium channel blockers, calciceptine and FS2, the GPIIa-IIIa antagonist and platelet aggregtion inhibitor, RGD-containing dendroaspin (or mambin), and the cell membrane lytic cardiotoxins (or cytotoxins) [1, and references therein]. Despite a highly conserved structural motif, the toxins in this family are directed to diverse targets, yet their individual modes of action are highly selective: Fasciculins inhibit mammalian, electric fish, and some snake venom acetylcholinesterases (AChE) with Ki values in the pico- to nanomolar range, but micromolar concentrations are required to inhibit avian and insect AChEs, and butyrylcholinesterase [cf. 2 for review].
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Expression and Activity of Mutants of Fasciculin, a Peptidic Acetylcholinesterase Inhibitor from Mamba Venom
The Journal of biological chemistry, 1997Co-Authors: Pascale Marchot, Zoran Radić, Shelley Camp, Pierre E. Bougis, Claudine N. Prowse, Joan R. Kanter, Elizabeth J. Ackermann, Palmer TaylorAbstract:Abstract Fasciculin, a selective peptidic inhibitor of acetylcholinesterase, is a member of the three-fingered peptide toxin superfamily isolated from snake venoms. The availability of a crystal structure of a Fasciculin 2 (Fas2)-acetylcholinesterase complex affords an opportunity to examine in detail the interaction of this toxin with its target site. To this end, we constructed a synthetic Fasciculin gene with an appropriate leader peptide for expression and secretion from mammalian cells. Recombinant wild-type Fas2, expressed and amplified in Chinese hamster ovary cells, was purified to homogeneity and found to be identical in composition and biological activities to the venom-derived toxin. Sixteen mutations at positions where the crystal structure of the complex indicates a significant interfacial contact point or determinant of conformation were generated. Two mutants of loop I, T8A/T9A and R11Q, ten mutants of the longest loop II, R24T, K25L, R27W, R28D, H29D, ΔPro30, P31R, K32G, M33A, and V34A/L35A, and two mutants of loop III, D45K and K51S, were expressed transiently in human embryonic kidney cells. Inhibitory potencies of the Fas2 mutants toward mouse AChE were established, based on titration of the mutants with a polyclonal anti-Fas2 serum. The Arg27, Pro30, and Pro31 mutants each lost two or more orders of magnitude in Fas2 activity, suggesting that this subset of three residues, at the tip of loop II, dominates the loop conformation and interaction of Fas2 with the enzyme. The Arg24, Lys32, and Met33 mutants lost about one order of magnitude, suggesting that these residues make moderate contributions to the strength of the complex, whereas the Lys25, Arg28, Val34-Leu35, Asp45, and Lys51 mutants appeared as active as Fas2. The Thr8-Thr9, Arg11, and His29 mutants showed greater ratios of inhibitory activity to immunochemical titer than Fas2. This may reflect immunodominant determinants in these regions or intramolecular rearrangements in conformation that enhance the interaction. Of the many Fas2 residues that lie at the interface with acetylcholinesterase, only a few appear to provide substantial energetic contributions to the high affinity of the complex.
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Structure of Fasciculin 2 from green mamba snake venom: evidence for unusual loop flexibility.
Acta Crystallographica Section D Biological Crystallography, 1996Co-Authors: D. Housset, Pascale Marchot, Pierre E. Bougis, J. Navaza, J C Fontecilla-campsAbstract:The crystal structure of the snake toxin Fasciculin 2, a potent acetylcholinesterase inhibitor from the venom of the green mamba (Dendroaspis angusticeps), has been determined by the molecular-replacement method, using the Fasciculin 1 model and refined to 2.0 A resolution. The introduction of an overall anisotropic temperature factor improved significantly the quality of the electron-density map. It suggests, as it was also indicated by the packing, that the thermal motion along the unique axis direction is less pronounced than on the (ab) plane. The final crystallographic R factor is 0.188 for a model having r.m.s. deviations from ideality of 0.016 A for bond lengths and 2.01° for bond angles. As Fasciculin 1, Fasciculin 2 belongs to the three-finger class of Elapidae toxins, a structural group that also contains the α-neurotoxins and the cardiotoxins. Although the two Fasciculins have, overall, closely related structures, the conformation of loop I differs appreciably in the two molecules. The presence of detergent in crystallization medium in the case of Fasciculin 2 appears to be responsible for the displacement of the loop containing Thr9. This conformational change also results in the formation of a crystallographic dimer that displays extensive intermolecular interactions.
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Structural Determinants of Fasciculin Specificity for Acetylcholinesterase
Enzymes of the Cholinesterase Family, 1995Co-Authors: Pascale Marchot, Zoran Radić, Shelley Camp, Pierre E. Bougis, Palmer TaylorAbstract:Fasciculins are the only known peptide inhibitors of acetylcholinesterase (AChE) with a high degree of selectivity. They are found in mamba snake venoms and have been shown to display powerful inhibitory activity toward mammalian and fish AChE. To date, four iso-Fasciculins have been characterized: Fasciculin 1 (FASI) and Fasciculin 2 (FAS2) from the venom of Dendroaspis angusticeps (Rodriguez-Ithurralde et al., 1983), toxin C from the venom of D. polylepis (Joubert and Taljaard, 1978), and Fasciculin 3 (FAS3) from a venom of D. viridis (Marchot et al., 1993). The early pharmacological and biochemical studies of FAS2, carried out both in vivo and in vitro on various AChE-containing tissues (Karlsson et al., 1984; Lin et al., 1987), showed that i) FAS2 inhibits several (but not all) AChEs from different sources, ii) inhibition is of pseudo-irreversible type with Ki values of about 10−11 M., iii) FAS2 is able to displace propidium, known as a specific probe for a peripheral anionic site of AChE (See Harvey et al., 1984 for a review).
