Tsigelny I

General

Full name : Tsigelny Igor

First name : Igor

Mail : University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093

Zip Code :

City :

Country : USA

Email : itsigeln@ucsd.edu

Phone : 619-822-0953

Fax : 619-534-8248

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References (26)

Title : Acetylcholinesterase from the invertebrate Ciona intestinalis is capable of assembling into asymmetric forms when co-expressed with vertebrate collagenic tail peptide - Frederick_2008_FEBS.J_275_1309
Author(s) : Frederick A , Tsigelny I , Cohenour F , Spiker C , Krejci E , Chatonnet A , Bourgoin S , Richards G , Allen T , Whitlock MH , Pezzementi L
Ref : Febs J , 275 :1309 , 2008
Abstract : To learn more about the evolution of the cholinesterases (ChEs), acetylcholinesterase (AChE) and butyrylcholinesterase in the vertebrates, we investigated the AChE activity of a deuterostome invertebrate, the urochordate Ciona intestinalis, by expressing in vitro a synthetic recombinant cDNA for the enzyme in COS-7 cells. Evidence from kinetics, pharmacology, molecular biology, and molecular modeling confirms that the enzyme is AChE. Sequence analysis and molecular modeling also indicate that the cDNA codes for the AChE(T) subunit, which should be able to produce all three globular forms of AChE: monomers (G(1)), dimers (G(2)), and tetramers (G(4)), and assemble into asymmetric forms in association with the collagenic subunit collagen Q. Using velocity sedimentation on sucrose gradients, we found that all three of the globular forms are either expressed in cells or secreted into the medium. In cell extracts, amphiphilic monomers (G(1)(a)) and non-amphiphilic tetramers (G(4)(na)) are found. Amphiphilic dimers (G(2)(a)) and non-amphiphilic tetramers (G(4)(na)) are secreted into the medium. Co-expression of the catalytic subunit with Rattus norvegicus collagen Q produces the asymmetric A(12) form of the enzyme. Collagenase digestion of the A(12) AChE produces a lytic G(4) form. Notably, only globular forms are present in vivo. This is the first demonstration that an invertebrate AChE is capable of assembling into asymmetric forms. We also performed a phylogenetic analysis of the sequence. We discuss the relevance of our results with respect to the evolution of the ChEs in general, in deuterostome invertebrates, and in chordates including vertebrates.
ESTHER : Frederick_2008_FEBS.J_275_1309
PubMedSearch : Frederick_2008_FEBS.J_275_1309
PubMedID: 18279391
Gene_locus related to this paper: cioin-ACHE1 , cioin-ACHE2

Title : Inactivation of an invertebrate acetylcholinesterase by sulfhydryl reagents: a reconsideration of the implications for insecticide design - Rowland_2008_Chem.Biol.Interact_175_73
Author(s) : Rowland M , Tsigelny I , Wolfe M , Pezzementi L
Ref : Chemico-Biological Interactions , 175 :73 , 2008
Abstract : Previously we used site-directed mutagenesis, in vitro expression, and molecular modeling to investigate the inactivation of an invertebrate acetylcholinesterase, cholinesterase 2 from amphioxus, by the sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM). We created the mutants C310A, C466A, C310A/C466A and C310A/F312I to assess the roles of the two cysteines and a proposal that the increased rate of inactivation previously found in an F312I mutant was due to increased access of sulfhydryl reagents to Cys310. Our results indicated that both of the cysteines could be involved in inactivation by sulfhydryl reagents, but that the cysteine near the acyl pocket was more accessible. We speculated that the inactivation of aphid AChEs by sulfhydryl reagents was due to the presence of a cysteine homologous to Cys310 and proposed that this residue could be a target for a specific insecticide. Here we reconsider this proposal.
ESTHER : Rowland_2008_Chem.Biol.Interact_175_73
PubMedSearch : Rowland_2008_Chem.Biol.Interact_175_73
PubMedID: 18384763

Title : Thermal denaturation of wild type and mutant recombinant acetylcholinesterase from amphioxus: effects of the temperature of in vitro expression and of reversible inhibitors - Perrin_2008_Invert.Neurosci_8_147
Author(s) : Perrin B , Rowland M , Wolfe M , Tsigelny I , Pezzementi L
Ref : Invert Neurosci , 8 :147 , 2008
Abstract : We have studied the thermal inactivation at 37 degrees C of wild type and mutant ChE2 (C310A, F312I, C466A, C310A/F312I, and C310A/C466A) from amphioxus (Branchiostoma floridae) expressed in vitro in COS-7 monkey cells under three sets of conditions: 30 degrees C for 48 h, 30 degrees C for 24 h and 37 degrees C for 24 h, and 37 degrees C for 48 h. We found biphasic denaturation curves for all enzymes and conditions, except wild type and C310A ChE2 expressed at 30 degrees C for 48 h. Generally, single mutants are more unstable than wild type, and the double mutants are even more unstable. We propose a model involving stable and unstable conformations of the enzymes to explain these results, and we discuss the implications of the model. We also found a correlation between the melting temperature of the ChEs and the rates at which they denature at 37 degrees C, with the denaturation of the unstable conformation dominating the relationship. Reversible cholinergic inhibitors protect the ChEs from thermal denaturation, and in some cases produce monophasic denaturation curves; we also propose a model to explain this stabilization.
ESTHER : Perrin_2008_Invert.Neurosci_8_147
PubMedSearch : Perrin_2008_Invert.Neurosci_8_147
PubMedID: 18677525

Title : Synaptic arrangement of the neuroligin\/beta-neurexin complex revealed by X-ray and neutron scattering - Comoletti_2007_Structure_15_693
Author(s) : Comoletti D , Grishaev A , Whitten AE , Tsigelny I , Taylor P , Trewhella J
Ref : Structure , 15 :693 , 2007
Abstract : Neuroligins are postsynaptic cell-adhesion proteins that associate with their presynaptic partners, the neurexins. Using small-angle X-ray scattering, we determined the shapes of the extracellular region of several neuroligin isoforms in solution. We conclude that the neuroligins dimerize via the characteristic four-helix bundle observed in cholinesterases, and that the connecting sequence between the globular lobes of the dimer and the cell membrane is elongated, projecting away from the dimer interface. X-ray scattering and neutron contrast variation data show that two neurexin monomers, separated by 107 A, bind at symmetric locations on opposite sides of the long axis of the neuroligin dimer. Using these data, we developed structural models that delineate the spatial arrangements of different neuroligin domains and their partnering molecules. As mutations of neurexin and neuroligin genes appear to be linked to autism, these models provide a structural framework for understanding altered recognition by these proteins in neurodevelopmental disorders.
ESTHER : Comoletti_2007_Structure_15_693
PubMedSearch : Comoletti_2007_Structure_15_693
PubMedID: 17562316
Gene_locus related to this paper: human-NLGN3 , human-NLGN4X

Title : A tryptophan in the bottleneck of the catalytic gorge of an invertebrate acetylcholinesterase confers relative resistance to carbamate and organophosphate inhibitors - Patel_2006_Cell.Biochem.Biophys_46_253
Author(s) : Patel R , Sanders R , Brown L , Baker S , Tsigelny I , Pezzementi L
Ref : Cell Biochem Biophys , 46 :253 , 2006
Abstract : Amphioxus, an invertebrate chordate, has two acetylcholinesterases (AChEs): cholinesterase 1 (ChE1) and cholinesterase 2 (ChE2). ChE1 is up to 329-fold more resistant to a variety of carbamate and organophosphate inhibitors, including a number of insecticides, when compared with ChE2. One difference between the two enzymes is at the position homologous to Phe331 in Torpedo AChE. In Torpedo AChE, this residue is a component of the hydrophobic subsite and defines one side of the bottleneck in the catalytic gorge of the enzyme. In ChE1, the homologous residue is Trp353; in ChE2, it is Phe353. We used site-directed mutagenesis to investigate the proposal that the resistance of ChE1 to inhibition by carbamates and organophosphates was due to this difference, creating a ChE1 W353F mutant to widen the bottleneck. The mutation virtually abolishes the difference in sensitivity to the inhibitors. The ChE1 W353F mutant is only 2- to 3-fold more resistant than ChE2 to carbamates and is actually 2.5- to 10-fold more sensitive to inhibition by organophosphates. The differences in resistance are due to different affinities of the enzymes for the inhibitors, not different reactivities. Molecular modeling supports the proposal that the difference in inhibition is due to the width of the bottleneck of the gorge. Our results have implications for insecticide resistance in insects, in particular mosquitoes and aphids.
ESTHER : Patel_2006_Cell.Biochem.Biophys_46_253
PubMedSearch : Patel_2006_Cell.Biochem.Biophys_46_253
PubMedID: 17272851

Title : Inactivation of an invertebrate acetylcholinesterase by sulfhydryl reagents: the roles of two cysteines in the catalytic gorge of the enzyme - Pezzementi_2006_Invert.Neurosci_6_47
Author(s) : Pezzementi L , Rowland M , Wolfe M , Tsigelny I
Ref : Invert Neurosci , 6 :47 , 2006
Abstract : We have used site-directed mutagenesis and molecular modeling to investigate the inactivation of an invertebrate acetylcholinesterase (AChE), ChE2 from amphioxus, by the sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM), creating various mutants, including C310A and C466A, and the double mutants C310A/C466A and C310A/F312I, to assess the relative roles of the two cysteines and a proposal that the increased rate of inactivation in the F312I mutant is due to increased access to Cys310. Our results suggest that both cysteines may be involved in inactivation by sulfhydryl reagents, but that the cysteine in the vicinity of the acyl pocket is more accessible. We speculate that the inactivation of aphid AChEs by sulfhydryl reagents is due to the presence of a cysteine homologous to Cys310. We also investigated the effects of various reversible cholinergic ligands, which bind to different subsites of the active site of the enzyme, on the rate of inactivation by DTNB of wild type ChE2 and ChE2 F312I. For the most part the inhibitors protect the enzymes from inactivation by DTNB. However, a notable exception is the peripheral site ligand propidium, which accelerates inactivation in the wild type ChE2, but retards inactivation in the F312I mutant. We propose that these opposing effects are the result of an altered allosteric signal transduction mechanism in the F312I mutant compared to the wild type ChE2.
ESTHER : Pezzementi_2006_Invert.Neurosci_6_47
PubMedSearch : Pezzementi_2006_Invert.Neurosci_6_47
PubMedID: 16586114

Title : Dissection of synapse induction by neuroligins: effect of a neuroligin mutation associated with autism - Chubykin_2005_J.Biol.Chem_280_22365
Author(s) : Chubykin AA , Liu X , Comoletti D , Tsigelny I , Taylor P , Sudhof TC
Ref : Journal of Biological Chemistry , 280 :22365 , 2005
Abstract : To study synapse formation by neuroligins, we co-cultured hippocampal neurons with COS cells expressing wild type and mutant neuroligins. The large size of COS cells makes it possible to test the effect of neuroligins presented over an extended surface area. We found that a uniform lawn of wild type neuroligins displayed on the cell surface triggers the formation of hundreds of uniformly sized, individual synaptic contacts that are labeled with neurexin antibodies. Electron microscopy revealed that these artificial synapses contain a presynaptic active zone with docked vesicles and often feature a postsynaptic density. Neuroligins 1, 2, and 3 were active in this assay. Mutations in two surface loops of neuroligin 1 abolished neuroligin binding to neurexin 1beta, a presumptive presynaptic binding partner for postsynaptic neuroligins, and blocked synapse formation. An analysis of mutant neuroligins with an amino acid substitution that corresponds to a mutation described in patients with an autistic syndrome confirmed previous reports that these mutant neuroligins have a compromised capacity to be transported to the cell surface. Nevertheless, the small percentage of mutant neuroligins that reached the cell surface still induced synapse formation. Viewed together, our data suggest that neuroligins generally promote artificial synapse formation in a manner that is associated with beta-neurexin binding and results in morphologically well differentiated synapses and that a neuroligin mutation found in autism spectrum disorders impairs cell-surface transport but does not completely abolish synapse formation activity.
ESTHER : Chubykin_2005_J.Biol.Chem_280_22365
PubMedSearch : Chubykin_2005_J.Biol.Chem_280_22365
PubMedID: 15797875

Title : The Arg451Cys-neuroligin-3 mutation associated with autism reveals a defect in protein processing - Comoletti_2004_J.Neurosci_24_4889
Author(s) : Comoletti D , De Jaco A , Jennings LL , Flynn RE , Gaietta G , Tsigelny I , Ellisman MH , Taylor P
Ref : Journal of Neuroscience , 24 :4889 , 2004
Abstract : The neuroligins are a family of postsynaptic transmembrane proteins that associate with presynaptic partners, the beta-neurexins. Neurexins and neuroligins play a critical role in initiating formation and differentiation of synaptic junctions. A recent study reported that a mutation of neuroligin-3 (NL3), an X-linked gene, was found in siblings with autistic spectrum disorder in which two affected brothers had a point mutation that substituted a Cys for Arg451. To characterize the mutation at the biochemical level, we analyzed expression and activity of the mutated protein. Mass spectrometry comparison of the disulfide bonding pattern between the native and the mutated proteins indicates the absence of aberrant disulfide bonding, suggesting that the secondary structure of the mutated protein is conserved. However, the mutation separately affects protein expression and activity. The Cys mutation causes defective neuroligin trafficking, leading to retention of the protein in the endoplasmic reticulum. This, in turn, decreases the delivery of NL3 to the cell surface. Also, the small fraction of protein that reaches the cell membrane lacks or has markedly diminished beta-neurexin-1 (NX1beta) binding activity. Other substitutions for Arg451 allow for normal cellular expression but diminished affinity for NX1beta. Our findings reveal a cellular phenotype and loss of function for a congenital mutation associated with autistic spectrum disorders.
ESTHER : Comoletti_2004_J.Neurosci_24_4889
PubMedSearch : Comoletti_2004_J.Neurosci_24_4889
PubMedID: 15152050

Title : Structural characterization of recombinant soluble rat neuroligin 1: mapping of secondary structure and glycosylation by mass spectrometry - Hoffman_2004_Biochemistry_43_1496
Author(s) : Hoffman RC , Jennings LL , Tsigelny I , Comoletti D , Flynn RE , Sudhof TC , Taylor P
Ref : Biochemistry , 43 :1496 , 2004
Abstract : Neuroligins (NLs) are a family of transmembrane proteins that function in synapse formation and/or remodeling by interacting with beta-neurexins (beta-NXs) to form heterophilic cell adhesions. The large N-terminal extracellular domain of NLs, required for beta-NX interactions, has sequence homology to the alpha/beta hydrolase fold superfamily of proteins. By peptide mapping and mass spectrometric analysis of a soluble recombinant form of NL1, several structural features of the extracellular domain have been established. Of the nine cysteine residues in NL1, eight are shown to form intramolecular disulfide bonds. Disulfide pairings of Cys 117 to Cys 153 and Cys 342 to Cys 353 are consistent with disulfide linkages that are conserved among the family of alpha/beta hydrolase proteins. The disulfide bond between Cys 172 and Cys 181 occurs within a region of the protein encoded by an alternatively spliced exon. The disulfide pairing of Cys 512 and Cys 546 in NL1 yields a structural motif unique to the NLs, since these residues are highly conserved. The potential N-glycosylation sequons in NL1 at Asn 109, Asn 303, Asn 343, and Asn 547 are shown occupied by carbohydrate. An additional consensus sequence for N-glycosylation at Asn 662 is likely occupied. Analysis of N-linked oligosaccharide content by mass matching paradigms reveals significant microheterogeneous populations of complex glycosyl moieties. In addition, O-linked glycosylation is observed in the predicted stalk region of NL1, prior to the transmembrane spanning domain. From predictions based on sequence homology of NL1 to acetylcholinesterase and the molecular features of NL1 established from mass spectrometric analysis, a novel topology model for NL three-dimensional structure has been constructed.
ESTHER : Hoffman_2004_Biochemistry_43_1496
PubMedSearch : Hoffman_2004_Biochemistry_43_1496
PubMedID: 14769026
Gene_locus related to this paper: ratno-1neur

Title : Amino acids defining the acyl pocket of an invertebrate cholinesterase - Pezzementi_2003_Comp.Biochem.Physiol.B.Biochem.Mol.Biol_136_813
Author(s) : Pezzementi L , Johnson K , Tsigelny I , Cotney J , Manning E , Barker A , Merritt S
Ref : Comparative Biochemistry & Physiology B Biochem Mol Biol , 136 :813 , 2003
Abstract : Amphioxus (Branchiostoma floridae) cholinesterase 2 (ChE2) hydrolyzes acetylthiocholine (AsCh) almost exclusively. We constructed a homology model of ChE2 on the basis of Torpedo californica acetylcholinesterase (AChE) and found that the acyl pocket of the enzyme resembles that of Drosophila melanogaster AChE, which is proposed to be comprised of Phe330 (Phe290 in T. californica AChE) and Phe440 (Val400), rather than Leu328 (Phe288) and Phe330 (Phe290), as in vertebrate AChE. In ChE2, the homologous amino acids are Phe312 (Phe290) and Phe422 (Val400). To determine if these amino acids define the acyl pocket of ChE2 and its substrate specificity, and to obtain information about the hydrophobic subsite, partially comprised of Tyr352 (Phe330) and Phe353 (Phe331), we performed site-directed mutagenesis and in vitro expression. The aliphatic substitution mutant F312I ChE2 hydrolyzes AsCh preferentially but also butyrylthiocholine (BsCh), and the change in substrate specificity is due primarily to an increase in k(cat) for BsCh; K(m) and K(ss) are also altered. F422L and F422V produce enzymes that hydrolyze BsCh and AsCh equally due to an increase in k(cat) for BsCh and a decrease in k(cat) for AsCh. Our data suggest that Phe312 and Phe422 define the acyl pocket. We also screened mutants for changes in sensitivity to various inhibitors. Y352A increases the sensitivity of ChE2 to the bulky inhibitor ethopropazine. Y352A decreases inhibition by BW284c51, consistent with its role as part of the choline-binding site. Aliphatic replacement mutations produce enzymes that are more sensitive to inhibition by iso-OMPA, presumably by increasing access to the active site serine. Y352A, F353A and F353V make ChE2 considerably more resistant to inhibition by eserine and neostigmine, suggesting that binding of these aromatic inhibitors is mediated by pi-pi or cation-pi interactions at the hydrophobic site. Our results also provide information about the aromatic trapping of the active site histidine and the inactivation of ChE2 by sulfhydryl reagents.
ESTHER : Pezzementi_2003_Comp.Biochem.Physiol.B.Biochem.Mol.Biol_136_813
PubMedSearch : Pezzementi_2003_Comp.Biochem.Physiol.B.Biochem.Mol.Biol_136_813
PubMedID: 14662305

Title : Residues in the epsilon subunit of the nicotinic acetylcholine receptor interact to confer selectivity of waglerin-1 for the alpha-epsilon subunit interface site - Molles_2002_Biochemistry_41_7895
Author(s) : Molles BE , Tsigelny I , Nguyen PD , Gao SX , Sine SM , Taylor P
Ref : Biochemistry , 41 :7895 , 2002
Abstract : Waglerin-1 (Wtx-1) is a 22-amino acid peptide that competitively antagonizes muscle nicotinic acetylcholine receptors (nAChRs). Previous work demonstrated that Wtx-1 binds to mouse nAChRs with higher affinity than receptors from rats or humans, and distinguished residues in alpha and epsilon subunits that govern the species selectivity. These studies also showed that Wtx-1 binds selectively to the alpha-epsilon binding site with significantly higher affinity than to the alpha-delta binding site. Here we identify residues at equivalent positions in the epsilon, gamma, and delta subunits that govern Wtx-1 selectivity for one of the two binding sites on the nAChR pentamer. Using a series of chimeric and point mutant subunits, we show that residues Gly-57, Asp-59, Tyr-111, Tyr-115, and Asp-173 of the epsilon subunit account predominantly for the 3700-fold higher affinity of the alpha-epsilon site relative to that of the alpha-gamma site. Similarly, we find that residues Lys-34, Gly-57, Asp-59, and Asp-173 account predominantly for the high affinity of the alpha-epsilon site relative to that of the alpha-delta site. Analysis of combinations of point mutations reveals that Asp-173 in the epsilon subunit is required together with the remaining determinants in the epsilon subunit to achieve Wtx-1 selectivity. In particular, Lys-34 interacts with Asp-173 to confer high affinity, resulting in a DeltaDeltaG(INT) of -2.3 kcal/mol in the epsilon subunit and a DeltaDeltaG(INT) of -1.3 kcal/mol in the delta subunit. Asp-173 is part of a nonhomologous insertion not found in the acetylcholine binding protein structure. The key role of this insertion in Wtx-1 selectivity indicates that it is proximal to the ligand binding site. We use the binding and interaction energies for Wtx-1 to generate structural models of the alpha-epsilon, alpha-gamma, and alpha-delta binding sites containing the nonhomologous insertion.
ESTHER : Molles_2002_Biochemistry_41_7895
PubMedSearch : Molles_2002_Biochemistry_41_7895
PubMedID: 12069578

Title : Tryptophan fluorescence reveals conformational changes in the acetylcholine binding protein - Hansen_2002_J.Biol.Chem_277_41299
Author(s) : Hansen SB , Radic Z , Talley TT , Molles BE , Deerinck TJ , Tsigelny I , Taylor P
Ref : Journal of Biological Chemistry , 277 :41299 , 2002
Abstract : The recent characterization of an acetylcholine binding protein (AChBP) from the fresh water snail, Lymnaea stagnalis, shows it to be a structural homolog of the extracellular domain of the nicotinic acetylcholine receptor (nAChR). To ascertain whether the AChBP exhibits the recognition properties and functional states of the nAChR, we have expressed the protein in milligram quantities from a synthetic cDNA transfected into human embryonic kidney (HEK) cells. The protein secreted into the medium shows a pentameric rosette structure with ligand stoichiometry approximating five sites per pentamer. Surprisingly, binding of acetylcholine, selective agonists, and antagonists ranging from small alkaloids to larger peptides results in substantial quenching of the intrinsic tryptophan fluorescence. Using stopped-flow techniques, we demonstrate rapid rates of association and dissociation of agonists and slow rates for the alpha-neurotoxins. Since agonist binding occurs in millisecond time frames, and the alpha-neurotoxins may induce a distinct conformational state for the AChBP-toxin complex, the snail protein shows many of the properties expected for receptor recognition of interacting ligands. Thus, the marked tryptophan quenching not only documents the importance of aromatic residues in ligand recognition, but establishes that the AChBP will be a useful functional as well as structural surrogate of the nicotinic receptor.
ESTHER : Hansen_2002_J.Biol.Chem_277_41299
PubMedSearch : Hansen_2002_J.Biol.Chem_277_41299
PubMedID: 12235129

Title : Common EF-hand motifs in cholinesterases and neuroligins suggest a role for Ca2+ binding in cell surface associations - Tsigelny_2000_Protein.Sci_9_180
Author(s) : Tsigelny I , Shindyalov IN , Bourne PE , Sudhof TC , Taylor P
Ref : Protein Science , 9 :180 , 2000
Abstract : Comparisons of protein sequence via cyclic training of Hidden Markov Models (HMMs) in conjunction with alignments of three-dimensional structure, using the Combinatorial Extension (CE) algorithm, reveal two putative EF-hand metal binding domains in acetylcholinesterase. Based on sequence similarity, putative EF-hands are also predicted for the neuroligin family of cell surface proteins. These predictions are supported by experimental evidence. In the acetylcholinesterase crystal structure from Torpedo californica, the first putative EF-hand region binds the Zn2+ found in the heavy metal replacement structure. Further, the interaction of neuroligin 1 with its cognate receptor neurexin depends on Ca2+. Thus, members of the alpha,beta hydrolase fold family of proteins contain potential Ca2+ binding sites, which in some family members may be critical for heterologous cell associations.
ESTHER : Tsigelny_2000_Protein.Sci_9_180
PubMedSearch : Tsigelny_2000_Protein.Sci_9_180
PubMedID: 10739260

Title : Subunit interface selective toxins as probes of nicotinic acetylcholine receptor structure - Taylor_2000_Pflugers.Arch_440_R115
Author(s) : Taylor P , Malanz S , Molles BE , Osaka H , Tsigelny I
Ref : Pflugers Arch , 440 :R115 , 2000
Abstract : The pentametric assembly of the nicotinic acetylcholine receptor with two of the five subunit interfaces serving as a ligand binding sites offers an opportunity to distinguish features on the surfaces of the subunits, and their ligand specificity characteristics. The receptor from mammalian muscle, with its circular order of homologous subunits (alphagamma alphadelta beta), assembles in a unique arrangement. The residues governing assembly can be ascertained through mutagenesis. Selectivity of certain natural toxins is sufficient to distinguish between sites at the alphagamma and alphadelta subunit interfaces. By interchanging residues on the gamma and delta subunits through mutagenesis, and ascertaining how they interact with the alpha subunit, determinants forming the binding sites can be delineated. The alpha-conotoxins show a 10,000-fold preference for the alphadelta over alphagamma subunit interface with alphaepsilon falling in between. The waglerins show a 2,000-fold preference for alphaepsilon over the alphagamma and alphadelta interfaces. Finally, the alpha-neurotoxin from N. mossambica mossambica shows a 10,000-fold preference for the alphagamma and alphadelta interfaces over alphaepsilon. Identification of interactive residues through mutagenesis, when coupled with homology modeling of domains and site-directed residue modification, has revealed important elements of receptor structure.
ESTHER : Taylor_2000_Pflugers.Arch_440_R115
PubMedSearch : Taylor_2000_Pflugers.Arch_440_R115
PubMedID: 11005635

Title : Analysis of cholinesterase inactivation and reactivation by systematic structural modification and enantiomeric selectivity - Taylor_1999_Chem.Biol.Interact_119-120_3
Author(s) : Taylor P , Wong L , Radic Z , Tsigelny I , Bruggemann R , Hosea NA , Berman HA
Ref : Chemico-Biological Interactions , 119-120 :3 , 1999
Abstract : We show here with a congeneric series of Rp- and Sp-alkoxymethyl phosphonothiolates of known absolute stereochemistry that chiral selectivity in their reaction with acetylcholinesterase can be described in terms of discrete orientational and steric requirements. Stereoselectivity depends on acyl pocket dimensions, which govern leaving group orientation and a productive association of the phosphonyl oxygen in the oxyanion hole. Overall geometry is consistent with a pentavalent intermediate where the attacking serine and leaving group are at apical positions. Oxime reactivation of the phosphonylated enzyme occurs through a similar associative intermediate presumably forming an oxime phosphonate. The oximes of differing structure show distinct angles of attacking the phosphate where the attack angles and access to the phosphorus are constrained in the sterically impacted gorge. Hence, efficacy of oxime reactivation is dependent on both oxime and conjugated phosphonate structures.
ESTHER : Taylor_1999_Chem.Biol.Interact_119-120_3
PubMedSearch : Taylor_1999_Chem.Biol.Interact_119-120_3
PubMedID: 10421434

Title : Binding orientation of thealpha-neurotoxins with the nicotinic acetylcholine receptor -
Author(s) : Osaka H , Ackermann EJ , Kanter J , Tsigelny I , Sine SM , Taylor P
Ref : Journal de Physiologie (Paris) , 92 :476 , 1998
PubMedID:

Title : Fast Molecular Docking Methods -
Author(s) : Ten Eyck LF , Mandell J , Kotlovyi V , Tsigelny I
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :357 , 1998
PubMedID:

Title : Oxime Reactivation of Acetylcholinesterase Inhibited by Enantiomeric Organophosphates -
Author(s) : Bruggemann RJ , Radic Z , Tsigelny I , Taylor P
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :377 , 1998
PubMedID:

Title : Metal Binding Motifs in Cholinesterases and Neuroligins -
Author(s) : Tsigelny I , Matsumura T , Sudhof TC , Taylor P
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :407 , 1998
PubMedID:

Title : Toxins selective for subunit interfaces as probes of nicotinic acetylcholine receptor structure - Taylor_1998_J.Physiol.Paris_92_79
Author(s) : Taylor P , Osaka H , Molles BE , Sugiyama N , Marchot P , Ackermann EJ , Malany S , McArdle JJ , Sine SM , Tsigelny I
Ref : Journal de Physiologie (Paris) , 92 :79 , 1998
Abstract : The pentameric structure of the nicotinic acetylcholine receptor with two of the five subunit interfaces serving as ligand binding sites offers an opportunity to distinguish features on the surfaces of the subunits and their ligand specificity characteristics. This problem has been approached through the study of assembly of subunits and binding characteristics of selective peptide toxins. The receptor, with its circular order of homologous subunits (alpha gamma alpha delta beta), assembles in only one arrangement, and through mutagenesis, the residues governing assembly can be ascertained. Selectivity of certain toxins is sufficient to readily distinguish between sites at the alpha gamma and alpha delta interfaces. By interchanging residues on the gamma and delta subunits, and ascertaining how they interact with the alpha-subunit, determinants forming the binding sites can be delineated. The alpha-conotoxins, which contain two disulfide loops and 12-14 amino acids, show a 10,000-fold preference for the alpha delta over the alpha gamma subunit interface with alpha epsilon falling between the two. The waglerins, as 22-24 amino acid peptides with a single core disulfide loop, show a 2000-fold preference for alpha epsilon over the alpha gamma and alpha delta interfaces. Finally, the 6700 Da short alpha-neurotoxin from N. mossambica mossambica shows a 10,000-fold preference for the alpha gamma and alpha delta interfaces over alpha epsilon. Selective mutagenesis enables one to also distinguish alpha-neurotoxin binding at the alpha gamma and alpha delta subunits. This information, when coupled with homology modeling of domains and site-directed residue modification, reveals important elements of receptor structure and conformation.
ESTHER : Taylor_1998_J.Physiol.Paris_92_79
PubMedSearch : Taylor_1998_J.Physiol.Paris_92_79
PubMedID: 9782448

Title : A model of the nicotinic receptor extracellular domain based on sequence identity and residue location - Tsigelny_1997_Biophys.J_73_52
Author(s) : Tsigelny I , Sugiyama N , Sine SM , Taylor P
Ref : Biophysical Journal , 73 :52 , 1997
Abstract : We have modeled the extracellular domains of individual subunits (amino acids 31-200) in the nicotinic acetylcholine receptor using sequence homology with copper binding proteins of known crystal structure, plastocyanin and pseudoazurin, and data from recent site-specific mutagenesis, antibody mapping, and site-directed labelling studies. These data formed an initial model that was refined using molecular dynamics and mechanics as well as electrostatic and solvation energy calculations. The sequences between residues 31 and 164 in the alpha 1-subunit and corresponding residues in homologous receptor subunits show similarity with the core sequence of the cation binding site in plastocyanin and pseudoazurin, a region in the template proteins characterized by multiple hairpin loops. In addition to defining the subunit interfaces that comprise the site for agonist and competitive antagonist binding in more detail, the findings show that negatively charged residues cluster in domains arranged to diminish electrostatic free energy of the complex. Electrostatic factors also appear to distinguish the ligand binding interfaces, alpha gamma and alpha delta, from the other three interfaces on the pentameric receptor.
ESTHER : Tsigelny_1997_Biophys.J_73_52
PubMedSearch : Tsigelny_1997_Biophys.J_73_52
PubMedID: 9199771

Title : Determining ligand orientation and transphosphonylation mechanisms on acetylcholinesterase by Rp, Sp enantiomer selectivity and site-specific mutagenesis - Taylor_1997_Enantiomer_2_249
Author(s) : Taylor P , Hosea NA , Tsigelny I , Radic Z , Berman HA
Ref : Enantiomer , 2 :249 , 1997
Abstract : Acetylcholinesterase, an enzyme of the serine hydrolase family, catalyzes the rapid hydrolysis of certain carboxyl esters. Other acyl esters efficiently transacylate the enzyme with a subsequent, slow deacylation step. Of these, the phosphoryl and phosphonyl esters are perhaps of greatest mechanistic interest since individual enantiomers of known absolute stereochemistry can be isolated and their interactions with the dissymmetric enzyme active site examined. We describe here studies of a series of enantiomeric Rp- and Sp-alkylphosphonates interacting with mouse acetylcholinesterase. Since the acetylcholinesterase is generated by recombinant DNA methods, mutant enzymes can be made with specific replacements of individual amino acid side chains. Individual amino acid replacements in the acyl pocket, the choline subsite and at the active center gorge entry have been generated, and the reaction kinetics of the mutant enzymes analyzed. These studies have shown that substitution of aliphatic amino acids for phenylalanines 295 and 297 in the acyl pocket diminishes, and in some cases, actually inverts chiral preferences. The combined structure-activity approach, where both ligand and enzyme are modified systematically, has enabled us to show that the restricted dimensions of the acyl pocket in the active center dictate enantiomeric selectivity. Moreover, the reactions of compounds of known absolute stereochemistry show three requirements for efficient transphosphonylation: (a) apposition of the phosphate with the gamma-oxygen on Ser 203 to form a pentavalent, presumed trigonal bipyramidal intermediate, (b) polarization of the phosphonyl oxygen bond by its positioning in the oxyanion hole, and (c) positioning the leaving group towards the gorge exit.
ESTHER : Taylor_1997_Enantiomer_2_249
PubMedSearch : Taylor_1997_Enantiomer_2_249
PubMedID: 9676269

Title : Aspartate 74 as a primary determinant in acetylcholinesterase governing specificity to cationic organophosphonates - Hosea_1996_Biochemistry_35_10995
Author(s) : Hosea NA , Radic Z , Tsigelny I , Berman HA , Quinn DM , Taylor P
Ref : Biochemistry , 35 :10995 , 1996
Abstract : Through site-specific mutagenesis, we examined the determinants on acetylcholinesterase which govern the specificity and reactivity of three classes of substrates: enantiomeric alkyl phosphonates, trifluoromethyl acetophenones, and carboxyl esters. By employing cationic and uncharged pairs of enantiomeric alkyl methylphosphonyl thioates of known absolute stereochemistry, we find that an aspartate residue near the gorge entrance (D74) is responsible for the enhanced reactivity of the cationic organophosphonates. Removal of the charge with the mutation D74N causes a near equal reduction in the reaction rate constants for the Rp and Sp enantiomers and exerts a greater influence on the cationic organophosphonates than on the charged trimethylammonio trifluoromethyl acetophenone and acetylthiocholine. This pattern of reactivity suggests that the orientation of the leaving group for both enantiomers is directed toward the gorge exit and in apposition to Asp 74. Replacement of tryptophan 86 with alanine in the choline subsite also diminishes the reaction rates for cationic organophosphonates, although to a lesser extent than with the D74N mutation, while not affecting the reactions with the uncharged compounds. Hence, reaction with cationic OPs depends to a lesser degree on Trp 86 than on Asp 74. Docking of Sp and Rp cycloheptyl methylphosphonyl thiocholines and thioethylates in AChE as models of the reversible complex and transition state using molecular dynamics affords structural insight into the spatial arrangement of the substituents surrounding phosphorus prior to and during reaction. The leaving group of the Rp and Sp enantiomers, regardless of charge, is directed to the gorge exit and toward Asp 74, an orientation unique to tetrahedral ligands.
ESTHER : Hosea_1996_Biochemistry_35_10995
PubMedSearch : Hosea_1996_Biochemistry_35_10995
PubMedID: 8718893

Title : Amino acid residues controlling reactivation of organophosphonyl conjugates of acetylcholinesterase by mono- and bisquaternary oximes - Ashani_1995_J.Biol.Chem_270_6370
Author(s) : Ashani Y , Radic Z , Tsigelny I , Vellom DC , Pickering NA , Quinn DM , Doctor BP , Taylor P
Ref : Journal of Biological Chemistry , 270 :6370 , 1995
Abstract : Single and multiple site mutants of recombinant mouse acetylcholinesterase (rMoAChE) were inhibited with racemic 7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide (MEPQ) and the resulting mixture of two enantiomers, CH3PR,S(O)(OC2H5)-AChE(EMPR,S-AChE), were subjected to reactivation with 2-(hydroxyiminomethyl)-1-methylpyridinium methanesulfonate (P2S) and 1-(2'-hydroxyiminomethyl-1'-pyridinium)-3-(4"-carbamoyl-1"- pyridinium)-2-oxapropane dichloride (HI-6). Kinetic analysis of the reactivation profiles revealed biphasic behavior with an approximate 1:1 ratio of two presumed reactivatable enantiomeric components. Equilibrium dissociation and kinetic rate constants for reactivation of site-specific mutant enzymes were compared with those obtained for wild-type rMoAChE, tissue-derived Torpedo AChE and human plasma butyrylcholinesterase. Substitution of key amino acid residues at the entrance to the active-site gorge (Trp-286, Tyr-124, Tyr-72, and Asp-74) had a greater influence on the reactivation kinetics of the bisquaternary reactivator HI-6 compared with the monoquaternary reactivator P2S. Replacement of Phe-295 by Leu enhanced reactivation by HI-6 but not by P2S. Of residues forming the choline-binding subsite, the E202Q mutation had a dominant influence where reactivation by both oximes was decreased 16- to 33-fold. Residues Trp-86 and Tyr-337 in this subsite showed little involvement. These kinetic findings, together with energy minimization of the oxime complex with the phosphonylated enzyme, provide a model for differences in the reactivation potencies of P2S and HI-6. The two kinetic components of oxime reactivation of MEPQ-inhibited AChEs arise from the chirality of O-ethyl methylphosphonyl moieties conjugated with Ser-203 and may be attributable to the relative stability of the phosphonyl oxygen of the two enantiomers in the oxyanion hole.
ESTHER : Ashani_1995_J.Biol.Chem_270_6370
PubMedSearch : Ashani_1995_J.Biol.Chem_270_6370
PubMedID: 7890775

Title : Amino Acid Residues that Control Mono- and Bisquaternary Oxime-Induced Reactivation of O-Ethyl Methylphosphonylated Cholinesterases -
Author(s) : Ashani Y , Radic Z , Tsigelny I , Vellom DC , Pickering NA , Quinn DM , Doctor BP , Taylor P
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :133 , 1995
PubMedID:

Title : Theoretical analysis of the structure of the peptide fasciculin and its docking to acetylcholinesterase - van den Born_1995_Prot.Sci_4_703
Author(s) : van den Born HK , Radic Z , Marchot P , Taylor P , Tsigelny I
Ref : Protein Science , 4 :703 , 1995
Abstract : 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.
ESTHER : van den Born_1995_Prot.Sci_4_703
PubMedSearch : van den Born_1995_Prot.Sci_4_703
PubMedID: 7613468