Marchot P

General

Full name : Marchot Pascale

First name : Pascale

Mail : Centre National de la Recherche Scientifique \/ Aix-Marseille Universite UMR-7257\; Architecture et Fonction des Macromolecules Biologiques (AFMB)\; Facult des Sciences, Campus Luminy - Case 932\; 163 Avenue de Luminy\; Marseille cedex 09\; 13288

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City :

Country : France

Email : pascale.marchot@univ-amu.fr

Phone : +330491 825 579

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Website : \/\/www.afmb.univ-mrs.fr

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References (68)

Title : The ESTHER database on alpha\/beta hydrolase fold proteins - An overview of recent developments - Chatonnet_2023_Chem.Biol.Interact_14ChEPon_110671
Author(s) : Chatonnet A , Perochon M , Velluet E , Marchot P
Ref : Chemico-Biological Interactions , :110671 , 2023
Abstract : The ESTHER database, dedicated to ESTerases and alpha/beta-Hydrolase Enzymes and Relatives (https://bioweb.supagro.inra.fr/ESTHER/general?what=index), offers online access to a continuously updated, sequence-based classification of proteins harboring the alpha/beta hydrolase fold into families and subfamilies. In particular, the database proposes links to the sequences, structures, ligands and huge diversity of functions of these proteins, and to the related literature and other databases. Taking advantage of the promiscuity of enzymatic function, many engineered esterases, lipases, epoxide-hydrolases, haloalkane dehalogenases are used for biotechnological applications. Finding means for detoxifying those protein members that are targeted by insecticides, herbicides, antibiotics, or for reactivating human cholinesterases when inhibited by nerve gas, are still active areas of research. Using or improving the capacity of some enzymes to breakdown plastics with the aim to recycle valuable material and reduce waste is an emerging challenge. Most hydrolases in the superfamily are water-soluble and act on or are inhibited by small organic compounds, yet in a few subfamilies some members interact with other, unrelated proteins to modulate activity or trigger functional partnerships. Recent development in 3D structure prediction brought by AI-based programs now permits analysis of enzymatic mechanisms for a variety of hydrolases with no experimental 3D structure available. Finally, mutations in as many as 34 of the 120 human genes compiled in the database are now linked to genetic diseases, a feature fueling research on early detection, metabolic pathways, pharmacological treatment or enzyme replacement therapy. Here we review those developments in the database that took place over the latest decade and discuss potential new applications and recent and future expected research in the field.
ESTHER : Chatonnet_2023_Chem.Biol.Interact_14ChEPon_110671
PubMedSearch : Chatonnet_2023_Chem.Biol.Interact_14ChEPon_110671
PubMedID: 37582413

Title : Report from the 27th (Virtual) Meeting on Toxinology, Toxins: Mr Hyde or Dr Jekyll?, Organized by the French Society of Toxinology, 9-10 December 2021 - Ladant_2022_Toxins.(Basel)_14_
Author(s) : Ladant D , Marchot P , Diochot S , Prevost G , Popoff MR , Benoit E
Ref : Toxins (Basel) , 14 : , 2022
Abstract : The French Society of Toxinology (SFET) organized its 27th annual meeting on 9-10 December 2021 as a virtual meeting (e-RT27). The central theme of this meeting was "Toxins: Mr Hyde or Dr Jekyll?", emphasizing the latest findings on plant, fungal, algal, animal and bacterial toxins during 10 lectures, 15 oral communications (shorter lectures) and 20 posters shared by ca. 80 participants. The abstracts of lectures and posters, as well as the winners of the best oral communication and poster awards, are presented in this report.
ESTHER : Ladant_2022_Toxins.(Basel)_14_
PubMedSearch : Ladant_2022_Toxins.(Basel)_14_
PubMedID: 35202137

Title : The neuroligins and the synaptic pathway in Autism Spectrum Disorder - Trobiani_2020_Neurosci.Biobehav.Rev_119_37
Author(s) : Trobiani L , Meringolo M , Diamanti T , Bourne Y , Marchot P , Martella G , Dini L , Pisani A , De Jaco A , Bonsi P
Ref : Neurosci Biobehav Rev , 119 :37 , 2020
Abstract : The genetics underlying autism spectrum disorder (ASD) is complex and heterogeneous, and de novo variants are found in genes converging in functional biological processes. Neuronal communication, including trans-synaptic signaling involving two families of cell-adhesion proteins, the presynaptic neurexins and the postsynaptic neuroligins, is one of the most recurrently affected pathways in ASD. Given the role of these proteins in determining synaptic function, abnormal synaptic plasticity and failure to establish proper synaptic contacts might represent mechanisms underlying risk of ASD. More than 30 mutations have been found in the neuroligin genes. Most of the resulting residue substitutions map in the extracellular, cholinesterase-like domain of the protein, and impair protein folding and trafficking. Conversely, the stalk and intracellular domains are less affected. Accordingly, several genetic animal models of ASD have been generated, showing behavioral and synaptic alterations. The aim of this review is to discuss the current knowledge on ASD-linked mutations in the neuroligin proteins and their effect on synaptic function, in various brain areas and circuits.
ESTHER : Trobiani_2020_Neurosci.Biobehav.Rev_119_37
PubMedSearch : Trobiani_2020_Neurosci.Biobehav.Rev_119_37
PubMedID: 32991906

Title : The Ig-like domain of Punctin\/MADD-4 is the primary determinant for interaction with the ectodomain of neuroligin NLG-1 - Platsaki_2020_J.Biol.Chem_295_16267
Author(s) : Platsaki S , Zhou X , Pinan-Lucarre B , Delauzun V , Tu H , Mansuelle P , Fourquet P , Bourne Y , Bessereau JL , Marchot P
Ref : Journal of Biological Chemistry , 295 :16267 , 2020
Abstract : Punctin/MADD-4, a member of the ADAMTSL extracellular matrix protein family, was identified as an anterograde synaptic organizer in the nematode Caenorhabditis elegans. At GABAergic neuromuscular junctions, the short isoform MADD-4B binds the ectodomain of neuroligin NLG-1, itself a postsynaptic organizer of inhibitory synapses. To identify the molecular bases of their partnership, we generated recombinant forms of the two proteins and carried out a comprehensive biochemical and biophysical study of their interaction, complemented by an in vivo localization study. We show that spontaneous proteolysis of MADD-4B first generates a shorter N-MADD-4B form, which comprises four thrombospondin (TSP) domains and one Ig-like domain and binds NLG-1. A second processing event eliminates the C-terminal Ig-like domain along with the ability of N-MADD-4B to bind NLG-1. These data identify the Ig-like domain as the primary determinant for N-MADD-4B interaction with NLG-1 in vitro We further demonstrate in vivo that this Ig-like domain is essential, albeit not sufficient per se, for efficient recruitment of GABA(A) receptors at GABAergic synapses in C. elegans The interaction of N-MADD-4B with NLG-1 is also disrupted by heparin, used as a surrogate for the extracellular matrix component, heparan sulfate. High-affinity binding of heparin/heparan sulfate to the Ig-like domain may proceed from surface charge complementarity, as suggested by homology three-dimensional modeling. These data point to N-MADD-4B processing and cell-surface proteoglycan binding as two possible mechanisms to regulate the interaction between MADD-4B and NLG-1 at GABAergic synapses.
ESTHER : Platsaki_2020_J.Biol.Chem_295_16267
PubMedSearch : Platsaki_2020_J.Biol.Chem_295_16267
PubMedID: 32928959

Title : Comparative mapping of selected structural determinants on the extracellular domains of cholinesterase-like cell-adhesion molecules - Comoletti_2020_Neuropharmacol__108381
Author(s) : Comoletti D , Trobiani L , Chatonnet A , Bourne Y , Marchot P
Ref : Neuropharmacology , :108381 , 2020
Abstract : Cell adhesion generally involve formation of homophilic or heterophilic protein complexes between two cells to form transcellular junctions. Neural cell-adhesion members of the alpha/beta-hydrolase fold superfamily of proteins use their extracellular or soluble cholinesterase-like domain to bind cognate partners across cell membranes, as illustrated by the neuroligins. These cell-adhesion molecules currently comprise the synaptic organizers neuroligins found in all phyla, along with three proteins found only in invertebrates: the guidance molecule neurotactin, the glia-specific gliotactin, and the basement membrane protein glutactin. Although these proteins share a cholinesterase-like fold, they lack one or more residues composing the catalytic triad responsible for the enzymatic activity of the cholinesterases. Conversely, they are found in various subcellular localisations and display specific disulfide bonding and N-glycosylation patterns, along with individual surface determinants possibly associated with recognition and binding of protein partners. Formation of non-covalent dimers typical of the cholinesterases is documented for mammalian neuroligins, yet whether invertebrate neuroligins and their neurotactin, gliotactin and glutactin relatives also form dimers in physiological conditions is unknown. Here we provide a brief overview of the localization, function, evolution, and conserved versus individual structural determinants of these cholinesterase-like cell-adhesion proteins.
ESTHER : Comoletti_2020_Neuropharmacol__108381
PubMedSearch : Comoletti_2020_Neuropharmacol__108381
PubMedID: 33166544

Title : An evolutionary perspective on the first disulfide bond in members of the cholinesterase-carboxylesterase (COesterase) family: Possible outcomes for cholinesterase expression in prokaryotes - Chatonnet_2019_Chem.Biol.Interact_13ChEPon_308_179
Author(s) : Chatonnet A , Brazzolotto X , Hotelier T , Lenfant N , Marchot P , Bourne Y
Ref : Chemico-Biological Interactions , 308 :179 , 2019
Abstract : Within the alpha/beta hydrolase fold superfamily of proteins, the COesterase group (carboxylesterase type B, block C, cholinesterases ...) diverged from the other groups through simultaneous integration of an N-terminal, first disulfide bond and a significant increase in the protein mean size. This first disulfide bond ties a large Cys loop, which in the cholinesterases is named the omega loop and forms the upper part of the active center gorge, essential for the high catalytic activity of these enzymes. In some non-catalytic members of the family, the loop may be necessary for heterologous partner recognition. Reshuffling of this protein portion occurred at the time of emergence of the fungi/metazoan lineage. Homologous proteins with this first disulfide bond are absent in plants but they are found in a limited number of bacterial genomes. In prokaryotes, the genes coding for such homologous proteins may have been acquired by horizontal transfer. However, the cysteines of the first disulfide bond are often lost in bacteria. Natural expression in bacteria of CO-esterases comprising this disulfide bond may have required compensatory mutations or expression of new chaperones. This disulfide bond may also challenge expression of the eukaryote-specific cholinesterases in prokaryotic cells. Yet recently, catalytically active human cholinesterase variants with enhanced thermostability were successfully expressed in E. coli. The key was the use of a peptidic sequence optimized through the Protein Repair One Stop Shop process, an automated structure- and sequence-based algorithm for expression of properly folded, soluble and stable eukaryotic proteins. Surprisingly however, crystal structures of the optimized cholinesterase variants expressed in bacteria revealed co-existing formed and unformed states of the first disulfide bond. Whether the bond never formed, or whether it properly formed then broke during the production/analysis process, cannot be inferred from the structural data. Yet, these features suggest that the recently acquired first disulfide bond is difficult to maintain in E. coli-expressed cholinesterases. To explore the fate of the first disulfide bond throughout the cholinesterase relatives, we reanalyzed the crystal structures of representative COesterases members from natural prokaryotic or eukaryotic sources or produced as recombinant proteins in E. coli. We found that in most cases this bond is absent.
ESTHER : Chatonnet_2019_Chem.Biol.Interact_13ChEPon_308_179
PubMedSearch : Chatonnet_2019_Chem.Biol.Interact_13ChEPon_308_179
PubMedID: 31100280

Title : Natural genomic amplification of cholinesterase genes in animals - Chatonnet_2017_J.Neurochem_142 Suppl 2_73
Author(s) : Chatonnet A , Lenfant N , Marchot P , Selkirk ME
Ref : Journal of Neurochemistry , 142 Suppl 2 :73 , 2017
Abstract : Tight control of the concentration of acetylcholine at cholinergic synapses requires precise regulation of the number and state of the acetylcholine receptors, and of the synthesis and degradation of the neurotransmitter. In particular, the cholinesterase activity has to be controlled exquisitely. In the genome of the first experimental models used (man, mouse, zebrafish and drosophila), there are only one or two genes coding for cholinesterases, whereas there are more genes for their closest relatives the carboxylesterases. Natural amplification of cholinesterase genes was first found to occur in some cancer cells and in insect species subjected to evolutionary pressure by insecticides. Analysis of the complete genome sequences of numerous representatives of the various metazoan phyla show that moderate amplification of cholinesterase genes is not uncommon in molluscs, echinoderms, hemichordates, prochordates or lepidosauria. Amplification of acetylcholinesterase genes is also a feature of parasitic nematodes or ticks. In these parasites, over-production of cholinesterase-like proteins in secreted products and the saliva are presumed to have effector roles related to host infection. These amplification events raise questions about the role of the amplified gene products, and the adaptation processes necessary to preserve efficient cholinergic transmission. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.
ESTHER : Chatonnet_2017_J.Neurochem_142 Suppl 2_73
PubMedSearch : Chatonnet_2017_J.Neurochem_142 Suppl 2_73
PubMedID: 28382676

Title : Hot Spots for Protein Partnerships at the Surface of Cholinesterases and Related alpha\/beta Hydrolase Fold Proteins or Domains-A Structural Perspective - Bourne_2017_Molecules_22_
Author(s) : Bourne Y , Marchot P
Ref : Molecules , 23 : , 2017
Abstract : The hydrolytic enzymes acetyl- and butyryl-cholinesterase, the cell adhesion molecules neuroligins, and the hormonogenic macromolecule thyroglobulin are a few of the many members of the alpha/beta hydrolase fold superfamily of proteins. Despite their distinctive functions, their canonical subunits, with a molecular surface area of ~20,000 A(2), they share binding patches and determinants for forming homodimers and for accommodating structural subunits or protein partners. Several of these surface regions of high functional relevance have been mapped through structural or mutational studies, while others have been proposed based on biochemical data or molecular docking studies. Here, we review these binding interfaces and emphasize their specificity versus potentially multifunctional character.
ESTHER : Bourne_2017_Molecules_22_
PubMedSearch : Bourne_2017_Molecules_22_
PubMedID: 29295471

Title : The three-finger toxin fold: a multifunctional structural scaffold able to modulate cholinergic functions - Kessler_2017_J.Neurochem_142 Suppl 2_7
Author(s) : Kessler P , Marchot P , Silva M , Servent D
Ref : Journal of Neurochemistry , 142 Suppl 2 :7 , 2017
Abstract : Three-finger fold toxins are miniproteins frequently found in Elapidae snake venoms. This fold is characterized by three distinct loops rich in beta-strands and emerging from a dense, globular core reticulated by four highly conserved disulfide bridges. The number and diversity of receptors, channels, and enzymes identified as targets of three-finger fold toxins is increasing continuously. Such manifold diversity highlights the specific adaptability of this fold for generating pleiotropic functions. Although this toxin superfamily disturbs many biological functions by interacting with a large diversity of molecular targets, the most significant target is the cholinergic system. By blocking the activity of the nicotinic and muscarinic acetylcholine receptors or by inhibiting the enzyme acetylcholinesterase, three-finger fold toxins interfere most drastically with neuromuscular junction functioning. Several of these toxins have become powerful pharmacological tools for studying the function and structure of their molecular targets. Most importantly, since dysfunction of these receptors/enzyme is involved in many diseases, exploiting the three-finger scaffold to create novel, highly specific therapeutic agents may represent a major future endeavor. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.
ESTHER : Kessler_2017_J.Neurochem_142 Suppl 2_7
PubMedSearch : Kessler_2017_J.Neurochem_142 Suppl 2_7
PubMedID: 28326549

Title : Cyclic imine toxins from dinoflagellates: a growing family of potent antagonists of the nicotinic acetylcholine receptors - Molgo_2017_J.Neurochem_142 Suppl 2_41
Author(s) : Molgo J , Marchot P , Araoz R , Benoit E , Iorga BI , Zakarian A , Taylor P , Bourne Y , Servent D
Ref : Journal of Neurochemistry , 142 Suppl 2 :41 , 2017
Abstract : We present an overview of the toxicological profile of the fast-acting, lipophilic macrocyclic imine toxins, an emerging family of organic compounds associated with algal blooms, shellfish contamination and neurotoxicity. Worldwide, shellfish contamination incidents are expanding; therefore, the significance of these toxins for the shellfish food industry deserves further study. Emphasis is directed to the dinoflagellate species involved in their production, their chemical structures, and their specific mode of interaction with their principal natural molecular targets, the nicotinic acetylcholine receptors, or with the soluble acetylcholine-binding protein, used as a surrogate receptor model. The dinoflagellates Karenia selliformis and Alexandrium ostenfeldii / A. peruvianum have been implicated in the biosynthesis of gymnodimines and spirolides, while Vulcanodinium rugosum is the producer of pinnatoxins and portimine. The cyclic imine toxins are characterized by a macrocyclic skeleton comprising 14-27 carbon atoms, flanked by two conserved moieties, the cyclic imine and the spiroketal ring system. These phycotoxins generally display high affinity and broad specificity for the muscle type and neuronal nicotinic acetylcholine receptors, a feature consistent with their binding site at the receptor subunit interfaces, composed of residues highly conserved among all nAChRs, and explaining the diverse toxicity among animal species. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.
ESTHER : Molgo_2017_J.Neurochem_142 Suppl 2_41
PubMedSearch : Molgo_2017_J.Neurochem_142 Suppl 2_41
PubMedID: 28326551

Title : Preface: Cholinergic Mechanisms - Prado_2017_J.Neurochem_142 Suppl 2_3
Author(s) : Prado MAM , Marchot P , Silman I
Ref : Journal of Neurochemistry , 142 Suppl 2 :3 , 2017
Abstract : This special issue is a companion to the meeting 'XVth International Symposium on Cholinergic Mechanisms', and is edited by Israel Silman, Marco Prado and Pascale Marchot. In the review articles, renowned researchers in the field capture key mechanisms of cholinergic neurotransmission, from genomic amplification of cholinesterase genes, splicing and post-translational modifications; features of the neuromuscular junction, implications of cholinergic circuitry that are relevant to addiction, anxiety and mood, to preclinical models, protein biomarkers, and clinical findings that are relevant to pathology, for example, developmental neurotoxicity. The broad variety of features reflects the impact of cholinergic mechanisms on many physiological events and emphasizes the importance of research in this area. This is the Preface for the special issue XVth International Symposium on Cholinergic Mechanisms.
ESTHER : Prado_2017_J.Neurochem_142 Suppl 2_3
PubMedSearch : Prado_2017_J.Neurochem_142 Suppl 2_3
PubMedID: 28791707

Title : A Triad of Crystals Sheds Light on MDGA Interference with Neuroligation - Thoumine_2017_Neuron_95_729
Author(s) : Thoumine O , Marchot P
Ref : Neuron , 95 :729 , 2017
Abstract : Neurexins and neuroligins form trans-synaptic complexes that promote synapse development. In this issue of Neuron, Aricescu and colleagues (Elegheert et al., 2017) complement and strengthen two recent reports by the Kim and Rudenko teams (Kim et al., 2017; Gangwar et al., 2017) to dissect the molecular determinants by which MDGAs challenge the neurexin-neuroligin partnership.
ESTHER : Thoumine_2017_Neuron_95_729
PubMedSearch : Thoumine_2017_Neuron_95_729
PubMedID: 28817794

Title : Relationships of human alpha\/beta hydrolase fold proteins and other organophosphate-interacting proteins - Lenfant_2016_Chem.Biol.Interact_259_343
Author(s) : Lenfant N , Bourne Y , Marchot P , Chatonnet A
Ref : Chemico-Biological Interactions , 259 :343 , 2016
Abstract : Organophosphates (OPs) are either found in nature or synthetized for use as pesticides, flame retardants, neurotoxic warfare agents or drugs (cholinergic enhancers in Alzheimer's disease and myasthenia gravis, or inhibitors of lipases in metabolic diseases). Because of the central role of acetylcholinesterase cholinergic neurotransmission in humans, one of the main purposes for using OPs is inactivation of the enzyme by phosphorylation of the nucleophilic serine residue in the active center. However, hundreds of serine hydrolases are expressed in the human proteome, and many of them are potential targets for OP adduction. In this review, we first situate the alpha/beta hydrolase fold proteins among the distinctively folded proteins known to interact with OPs, in particular the different lipases, peptidases, and enzymes hydrolyzing OPs. Second, we compile the human alpha/beta hydrolases and review those that have been experimentally shown to interact with OPs. Among the 120 human alpha/beta hydrolase fold proteins, 102 have a serine in the consensus GXSXG pentapeptide compatible with an active site, 6 have an aspartate or a cysteine as the active site nucleophile residue, and 12 evidently lack an active site. 76 of the 120 have been experimentally shown to bind an OP.
ESTHER : Lenfant_2016_Chem.Biol.Interact_259_343
PubMedSearch : Lenfant_2016_Chem.Biol.Interact_259_343
PubMedID: 27109753

Title : Steric and Dynamic Parameters Influencing In Situ Cycloadditions to Form Triazole Inhibitors with Crystalline Acetylcholinesterase - Bourne_2016_J.Am.Chem.Soc_138_1611
Author(s) : Bourne Y , Sharpless KB , Taylor P , Marchot P
Ref : Journal of the American Chemical Society , 138 :1611 , 2016
Abstract : Ligand binding sites on acetylcholinesterase (AChE) comprise an active center, at the base of a deep and narrow gorge lined by aromatic residues, and a peripheral site at the gorge entry. These features launched AChE as a reaction vessel for in situ click-chemistry synthesis of high-affinity TZ2PA6 and TZ2PA5 inhibitors, forming a syn-triazole upon cycloaddition within the gorge from alkyne and azide reactants bound at the two sites, respectively. Subsequent crystallographic analyses of AChE complexes with the TZ2PA6 regioisomers demonstrated that syn product association is accompanied by side chain reorganization within the gorge, freezing-in-frame a conformation distinct from an unbound state or anti complex. To correlate inhibitor dimensions with reactivity and explore whether in situ cycloaddition could be accelerated in a concentrated, crystalline template, we developed crystal-soaking procedures and solved structures of AChE complexes with the TZ2PA5 regioisomers and their TZ2/PA5 precursors (2.1-2.7 A resolution). The structures reveal motions of residue His447 in the active site and, unprecedentedly, residue Tyr341 at the gorge mouth, associated with TZ2 binding and coordinated with other side chain motions in the gorge that may guide AChE toward a transient state favoring syn-triazole formation. Despite precursor binding to crystalline AChE, coupling of rapid electric field fluctuations in the gorge with proper alignments of the azide and alkyne reactants to form the triazole remains a likely limiting step. These observations point to a prime requirement for AChE to interconvert dynamically between sequential conformations to promote favorable electrostatic factors enabling a productive apposition of the reactants for reactivity.
ESTHER : Bourne_2016_J.Am.Chem.Soc_138_1611
PubMedSearch : Bourne_2016_J.Am.Chem.Soc_138_1611
PubMedID: 26731630
Gene_locus related to this paper: mouse-ACHE

Title : Crystal Structure of Snake Venom Acetylcholinesterase in Complex with Inhibitory Antibody Fragment Fab410 Bound at the Peripheral Site: EVIDENCE FOR OPEN AND CLOSED STATES OF A BACK DOOR CHANNEL - Bourne_2015_J.Biol.Chem_290_1522
Author(s) : Bourne Y , Renault L , Marchot P
Ref : Journal of Biological Chemistry , 290 :1522 , 2015
Abstract : The acetylcholinesterase found in the venom of Bungarus fasciatus (BfAChE) is produced as a soluble, non-amphiphilic monomer with a canonical catalytic domain but a distinct C terminus compared with the other vertebrate enzymes. Moreover, the peripheral anionic site of BfAChE, a surface site located at the active site gorge entrance, bears two substitutions altering sensitivity to cationic inhibitors. Antibody Elec410, generated against Electrophorus electricus acetylcholinesterase (EeAChE), inhibits EeAChE and BfAChE by binding to their peripheral sites. However, both complexes retain significant residual catalytic activity, suggesting incomplete gorge occlusion by bound antibody and/or high frequency back door opening. To explore a novel acetylcholinesterase species, ascertain the molecular bases of inhibition by Elec410, and document the determinants and mechanisms for back door opening, we solved a 2.7-A resolution crystal structure of natural BfAChE in complex with antibody fragment Fab410. Crystalline BfAChE forms the canonical dimer found in all acetylcholinesterase structures. Equally represented open and closed states of a back door channel, associated with alternate positions of a tyrosine phenol ring at the active site base, coexist in each subunit. At the BfAChE molecular surface, Fab410 is seated on the long Omega-loop between two N-glycan chains and partially occludes the gorge entrance, a position that fully reflects the available mutagenesis and biochemical data. Experimentally based flexible molecular docking supports a similar Fab410 binding mode onto the EeAChE antigen. These data document the molecular and dynamic peculiarities of BfAChE with high frequency back door opening, and the mode of action of Elec410 as one of the largest peptidic inhibitors targeting the acetylcholinesterase peripheral site.
ESTHER : Bourne_2015_J.Biol.Chem_290_1522
PubMedSearch : Bourne_2015_J.Biol.Chem_290_1522
PubMedID: 25411244
Gene_locus related to this paper: bunfa-ACHE

Title : Marine Macrocyclic Imines, Pinnatoxins A and G: Structural Determinants and Functional Properties to Distinguish Neuronal alpha7 from Muscle alpha1(2)betagammadelta nAChRs - Bourne_2015_Structure_23_1106
Author(s) : Bourne Y , Sulzenbacher G , Radic Z , Araoz R , Reynaud M , Benoit E , Zakarian A , Servent D , Molgo J , Taylor P , Marchot P
Ref : Structure , 23 :1106 , 2015
Abstract : Pinnatoxins are macrocyclic imine phycotoxins associated with algal blooms and shellfish toxicity. Functional analysis of pinnatoxin A and pinnatoxin G by binding and voltage-clamp electrophysiology on membrane-embedded neuronal alpha7, alpha4beta2, alpha3beta2, and muscle-type alpha12betagammadelta nicotinic acetylcholine receptors (nAChRs) reveals high-affinity binding and potent antagonism for the alpha7 and alpha12betagammadelta subtypes. The toxins also bind to the nAChR surrogate, acetylcholine-binding protein (AChBP), with low Kd values reflecting slow dissociation. Crystal structures of pinnatoxin-AChBP complexes (1.9-2.2 A resolution) show the multiple anchoring points of the hydrophobic portion, the cyclic imine, and the substituted bis-spiroketal and cyclohexene ring systems of the pinnatoxins that dictate tight binding between the opposing loops C and F at the receptor subunit interface, as observed for the 13-desmethyl-spirolide C and gymnodimine A congeners. Uniquely, however, the bulky bridged EF-ketal ring specific to the pinnatoxins extends radially from the interfacial-binding pocket to interact with the sequence-variable loop F and govern nAChR subtype selectivity and central neurotoxicity.
ESTHER : Bourne_2015_Structure_23_1106
PubMedSearch : Bourne_2015_Structure_23_1106
PubMedID: 26004441

Title : Tracking the origin and divergence of cholinesterases and neuroligins: the evolution of synaptic proteins - Lenfant_2014_J.Mol.Neurosci_53_362
Author(s) : Lenfant N , Hotelier T , Bourne Y , Marchot P , Chatonnet A
Ref : Journal of Molecular Neuroscience , 53 :362 , 2014
Abstract : A cholinesterase activity can be found in all kingdoms of living organism, yet cholinesterases involved in cholinergic transmission appeared only recently in the animal phylum. Among various proteins homologous to cholinesterases, one finds neuroligins. These proteins, with an altered catalytic triad and no known hydrolytic activity, display well-identified cell adhesion properties. The availability of complete genomes of a few metazoans provides opportunities to evaluate when these two protein families emerged during evolution. In bilaterian animals, acetylcholinesterase co-localizes with proteins of cholinergic synapses while neuroligins co-localize and may interact with proteins of excitatory glutamatergic or inhibitory GABAergic/glycinergic synapses. To compare evolution of the cholinesterases and neuroligins with other proteins involved in the architecture and functioning of synapses, we devised a method to search for orthologs of these partners in genomes of model organisms representing distinct stages of metazoan evolution. Our data point to a progressive recruitment of synaptic components during evolution. This finding may shed light on the common or divergent developmental regulation events involved into the setting and maintenance of the cholinergic versus glutamatergic and GABAergic/glycinergic synapses.
ESTHER : Lenfant_2014_J.Mol.Neurosci_53_362
PubMedSearch : Lenfant_2014_J.Mol.Neurosci_53_362
PubMedID: 24390353

Title : The neuroligins and their ligands: from structure to function at the synapse - Bourne_2014_J.Mol.Neurosci_53_387
Author(s) : Bourne Y , Marchot P
Ref : Journal of Molecular Neuroscience , 53 :387 , 2014
Abstract : The neuroligins are cell adhesion proteins whose extracellular domain belongs to the alpha/beta-hydrolase fold family of proteins, mainly containing enzymes and exemplified by acetylcholinesterase. The ectodomain of postsynaptic neuroligins interacts through a calcium ion with the ectodomain of presynaptic neurexins to form flexible trans-synaptic associations characterized by selectivity for neuroligin or neurexin subtypes. This heterophilic interaction, essential for synaptic differentiation, maturation, and maintenance, is regulated by gene selection, alternative mRNA splicing, and posttranslational modifications. Mutations leading to deficiencies in the expression, folding, maturation, and binding properties of either partner are associated with autism spectrum disorders. The currently available structural and functional data illustrate how these two families of cell adhesion molecules bridge the synaptic cleft to participate in synapse plasticity and support its dynamic nature. Neuroligin partners distinct from the neurexins, and which may undergo either trans or cis interaction, have also been described, and tridimensional structures of some of them are available. Our study emphasizes the partnership versatility of the neuroligin ectodomain associated with molecular flexibility and alternative binding sites, proposes homology models of the structurally non-characterized neuroligin partners, and exemplifies the large structural variability at the surface of the alpha/beta-hydrolase fold subunit. This study also provides new insights into possible surface binding sites associated with non-catalytic properties of the acetylcholinesterase subunit.
ESTHER : Bourne_2014_J.Mol.Neurosci_53_387
PubMedSearch : Bourne_2014_J.Mol.Neurosci_53_387
PubMedID: 24497299

Title : Proteins with an alpha\/beta hydrolase fold: Relationships between subfamilies in an ever-growing superfamily - Lenfant_2013_Chem.Biol.Interact_203_266
Author(s) : Lenfant N , Hotelier T , Bourne Y , Marchot P , Chatonnet A
Ref : Chemico-Biological Interactions , 203 :266 , 2013
Abstract : Alpha/beta hydrolases function as hydrolases, lyases, transferases, hormone precursors or transporters, chaperones or routers of other proteins. The amount of structural and functional available data related to this protein superfamily expands exponentially, as does the number of proteins classified as alpha/beta hydrolases despite poor sequence similarity and lack of experimental data. However the superfamily can be rationally divided according to sequence or structural homologies, leading to subfamilies of proteins with potentially similar functions. Since the discovery of proteins homologous to cholinesterases but devoid of enzymatic activity (e.g., the neuroligins), divergent functions have been ascribed to members of other subfamilies (e.g., lipases, dipeptidylaminopeptidase IV, etc.). To study the potentially moonlighting properties of alpha/beta hydrolases, the ESTHER database (for ESTerase and alpha/beta Hydrolase Enzymes and Relatives; http:\/\/bioweb.supagro.inra.fr/esther), which collects, organizes and disseminates structural and functional information related to alpha/beta hydrolases, has been updated with new tools and the web server interface has been upgraded. A new Overall Table along with a new Tree based on HMM models has been included to tentatively group subfamilies. These tools provide starting points for phylogenetic studies aimed at pinpointing the origin of duplications leading to paralogous genes (e.g., acetylcholinesterase versus butyrylcholinesterase, or neuroligin versus carboxylesterase). Another of our goals is to implement new tools to distinguish catalytically active enzymes from non-catalytic proteins in poorly studied or annotated subfamilies.
ESTHER : Lenfant_2013_Chem.Biol.Interact_203_266
PubMedSearch : Lenfant_2013_Chem.Biol.Interact_203_266
PubMedID: 23010363

Title : ESTHER, the database of the alpha\/beta-hydrolase fold superfamily of proteins: tools to explore diversity of functions - Lenfant_2013_Nucleic.Acids.Res_41_D423
Author(s) : Lenfant N , Hotelier T , Velluet E , Bourne Y , Marchot P , Chatonnet A
Ref : Nucleic Acids Research , 41 :D423 , 2013
Abstract : The ESTHER database, which is freely available via a web server (http:\/\/bioweb.supagro.inra.fr/esther) and is widely used, is dedicated to proteins with an alpha/beta-hydrolase fold, and it currently contains >30 000 manually curated proteins. Herein, we report those substantial changes towards improvement that we have made to improve ESTHER during the past 8 years since our 2004 update. In particular, we generated 87 new families and increased the coverage of the UniProt Knowledgebase (UniProtKB). We also renewed the ESTHER website and added new visualization tools, such as the Overall Table and the Family Tree. We also address two topics of particular interest to the ESTHER users. First, we explain how the different enzyme classifications (bacterial lipases, peptidases, carboxylesterases) used by different communities of users are combined in ESTHER. Second, we discuss how variations of core architecture or in predicted active site residues result in a more precise clustering of families, and whether this strategy provides trustable hints to identify enzyme-like proteins with no catalytic activity.
ESTHER : Lenfant_2013_Nucleic.Acids.Res_41_D423
PubMedSearch : Lenfant_2013_Nucleic.Acids.Res_41_D423
PubMedID: 23193256

Title : Molecular Characterization of Monoclonal Antibodies that Inhibit Acetylcholinesterase by Targeting the Peripheral Site and Backdoor Region - Bourne_2013_PLoS.One_8_e77226
Author(s) : Bourne Y , Renault L , Essono S , Mondielli G , Lamourette P , Boquet D , Grassi J , Marchot P
Ref : PLoS ONE , 8 :e77226 , 2013
Abstract : 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 1.9A-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 inhibition properties. Finally, a validated, data-driven docking model of the Fab403-AChE complex suggests a mode of binding at the PAS that fully correlates with the functional data. This comprehensive study documents the molecular peculiarities of Fab403 and Fab410, as the largest peptidic inhibitors directed towards the peripheral site, and those of Fab408, as the first inhibitor directed toward the backdoor region of an AChE and a unique template for the design of new, specific modulators of AChE catalysis.
ESTHER : Bourne_2013_PLoS.One_8_e77226
PubMedSearch : Bourne_2013_PLoS.One_8_e77226
PubMedID: 24146971

Title : Enzymatic activity and protein interactions in alpha\/beta hydrolase fold proteins: moonlighting versus promiscuity - Marchot_2012_Protein.Pept.Lett_19_132
Author(s) : Marchot P , Chatonnet A
Ref : Protein Pept Lett , 19 :132 , 2012
Abstract : Genes coding for members of the alpha/beta hydrolase fold superfamily of proteins are present in all known genomes. Although there is no common and essential function performed by these proteins shared in all living organisms, this fold has been used for a number of diverse functions. The ancestry of both enzymatic and protein-protein interaction capability of this structural scaffold made it an important tinkering tool kit for protein function evolution. Recently, enzymes known since a long time have been found to have a second function in acting promiscuously on alternative substrates, or to be true moonlighting proteins acting also as transporters, receptors, chaperones... The reverse situation has been encountered for adhesion proteins shown to be enzymes. This review, while not exhaustive, surveys some of the best-known examples of multiple functions in alpha/beta hydrolase fold proteins.
ESTHER : Marchot_2012_Protein.Pept.Lett_19_132
PubMedSearch : Marchot_2012_Protein.Pept.Lett_19_132
PubMedID: 21933125

Title : Hydrolase versus other functions of members of the alpha\/beta-hydrolase fold superfamily of proteins -
Author(s) : Marchot P , Chatonnet A
Ref : Protein Pept Lett , 19 :130 , 2012
PubMedID: 21933117

Title : Structure-function relationships of the alpha\/beta-hydrolase fold domain of neuroligin: a comparison with acetylcholinesterase - Leone_2010_Chem.Biol.Interact_187_49
Author(s) : Leone P , Comoletti D , Taylor P , Bourne Y , Marchot P
Ref : Chemico-Biological Interactions , 187 :49 , 2010
Abstract : The neuroligins are postsynaptic cell adhesion proteins whose extracellular domain belongs to the alpha/beta-hydrolase fold family of proteins, a family characterized through the enzyme acetylcholinesterase (AChE) and other enzymes with various substrate specificities. Neuroligin associations with the pre-synaptic neurexins participate in synapse maturation and maintenance. Alternative splicing of the neuroligin and neurexin genes results in multiple isoforms and presumably regulation of activity, while mutations appear to be associated with autism spectrum disorders. The crystal structures of the extracellular, cell adhesion domain of three neuroligins (NL1, NL2 and NL4) revealed features that distinguish the neuroligins from their enzyme relatives and could not be predicted by homology modelling from an AChE template. The structures of NL1 and NL4 bound with a soluble beta-neurexin domain (Nrxbeta1) revealed the precise position and orientation of the bound Nrxbeta1 and the Ca(2+)-dependent interaction network at the complex interface. Herein we present an overview of the unbound and Nrxbeta1-bound neuroligin structures and compare them with structures of AChEs with and without a bound fasciculin partner. This study exemplifies how an alpha/beta-hydrolase fold domain tailored for catalysis varies to acquire adhesion properties, and defines three surface regions with distinctive locations and properties for homologous or heterologous partner association.
ESTHER : Leone_2010_Chem.Biol.Interact_187_49
PubMedSearch : Leone_2010_Chem.Biol.Interact_187_49
PubMedID: 20100470

Title : Structural determinants in phycotoxins and AChBP conferring high affinity binding and nicotinic AChR antagonism - Bourne_2010_Proc.Natl.Acad.Sci.U.S.A_107_6076
Author(s) : Bourne Y , Radic Z , Araoz R , Talley TT , Benoit E , Servent D , Taylor P , Molgo J , Marchot P
Ref : Proc Natl Acad Sci U S A , 107 :6076 , 2010
Abstract : Spirolide and gymnodimine macrocyclic imine phycotoxins belong to an emerging class of chemical agents associated with marine algal blooms and shellfish toxicity. Analysis of 13-desmethyl spirolide C and gymnodimine A by binding and voltage-clamp recordings on muscle-type alpha1(2)betagammadelta and neuronal alpha3beta2 and alpha4beta2 nicotinic acetylcholine receptors reveals subnanomolar affinities, potent antagonism, and limited subtype selectivity. Their binding to acetylcholine-binding proteins (AChBP), as soluble receptor surrogates, exhibits picomolar affinities governed by diffusion-limited association and slow dissociation, accounting for apparent irreversibility. Crystal structures of the phycotoxins bound to Aplysia-AChBP ( approximately 2.4A) show toxins neatly imbedded within the nest of ar-omatic side chains contributed by loops C and F on opposing faces of the subunit interface, and which in physiological conditions accommodates acetylcholine. The structures also point to three major features: (i) the sequence-conserved loop C envelops the bound toxins to maximize surface complementarity; (ii) hydrogen bonding of the protonated imine nitrogen in the toxins with the carbonyl oxygen of loop C Trp147 tethers the toxin core centered within the pocket; and (iii) the spirolide bis-spiroacetal or gymnodimine tetrahydrofuran and their common cyclohexene-butyrolactone further anchor the toxins in apical and membrane directions, along the subunit interface. In contrast, the se-quence-variable loop F only sparingly contributes contact points to preserve the broad receptor subtype recognition unique to phycotoxins compared with other nicotinic antagonists. These data offer unique means for detecting spiroimine toxins in shellfish and identify distinctive ligands, functional determinants and binding regions for the design of new drugs able to target several receptor subtypes with high affinity.
ESTHER : Bourne_2010_Proc.Natl.Acad.Sci.U.S.A_107_6076
PubMedSearch : Bourne_2010_Proc.Natl.Acad.Sci.U.S.A_107_6076
PubMedID: 20224036

Title : Structural insights into the exquisite selectivity of neurexin\/neuroligin synaptic interactions - Leone_2010_EMBO.J_29_2461
Author(s) : Leone P , Comoletti D , Ferracci G , Conrod S , Garcia SU , Taylor P , Bourne Y , Marchot P
Ref : EMBO Journal , 29 :2461 , 2010
Abstract : The extracellular domains of neuroligins and neurexins interact through Ca(2+) to form flexible trans-synaptic associations characterized by selectivity for neuroligin or neurexin subtypes. This heterophilic interaction, essential for synaptic maturation and differentiation, is regulated by gene selection, alternative mRNA splicing and post-translational modifications. A new, 2.6 A-resolution crystal structure of a soluble neurexin-1beta-neuroligin-4 (Nrx1beta-NL4) complex permits a detailed description of the Ca(2+)-coordinated interface and unveils concerted positional rearrangements of several residues of NL4, not observed in neuroligin-1, associated with Nrx1beta binding. Surface plasmon resonance analysis of the binding of structure-guided Nrx1beta mutants towards NL4 and neuroligin-1 shows that flexibility of the Nrx1beta-binding site in NL4 is reflected in a greater dissociation constant of the complex and higher sensitivity to ionic strength and pH variations. Analysis of neuroligin mutants points to critical functions for two respective residues in neuroligin-1 and neuroligin-2 in governing the affinity of the complexes. Although neuroligin-1 and neuroligin-2 have pre-determined conformations that respectively promote and prevent Nrx1beta association, unique conformational reshaping of the NL4 surface is required to permit Nrx1beta association.
ESTHER : Leone_2010_EMBO.J_29_2461
PubMedSearch : Leone_2010_EMBO.J_29_2461
PubMedID: 20543817
Gene_locus related to this paper: human-NLGN4X

Title : Conformational remodeling of femtomolar inhibitor-acetylcholinesterase complexes in the crystalline state - Bourne_2010_J.Am.Chem.Soc_132_18292
Author(s) : Bourne Y , Radic Z , Taylor P , Marchot P
Ref : Journal of the American Chemical Society , 132 :18292 , 2010
Abstract : The active center of acetylcholinesterase (AChE), a target site for competitive inhibitors, resides centrosymmetric to the subunit at the base of a deep, narrow gorge lined by aromatic residues. At the gorge entry, a peripheral site encompasses overlapping binding loci for noncompetitive inhibitors, which alter substrate access to the gorge. The click-chemistry inhibitor TZ2PA6 links the active center ligand, tacrine, to the peripheral site ligand, propidium, through a biorthogonal reaction of an acetylene and an azide that forms either a syn1 or an anti1 triazole. Compared with wild-type mouse AChE, a Tyr337Ala mutant displays full catalytic activity, albeit with 2-3 orders of magnitude higher affinities for the TZ2PA6 syn1 and anti1 regioisomers, reflected in low femtomolar K(d) values, diffusion-limited association, and dissociation half-times greater than 1 month and 1 week, respectively. Three structures of each of the co-crystallized syn1 and anti1 complexes of the Tyr337Ala mutant were solved at three distinct times of crystal maturation, consistent with or exceeding the half-lives of the complexes in solution, while crystalline complexes obtained from soaked Tyr337Ala crystals led to picturing "freshly formed" complexes. The structures, at 2.55-2.75 A resolution, reveal a range of unprecedented conformations of the bound regioisomers, not observed in the wild-type AChE complexes, associated with concerted positional rearrangements of side chains in the enzyme gorge. Moreover, time-dependent conformational remodeling of the crystalline complexes appears to correlate with the dissociation half-times of the solution complexes. Hence, for the tight-binding TZ2PA6 inhibitors, the initial complexes kinetically driven in solution slowly form more stable complexes governed by thermodynamic equilibrium and observable in mature crystals.
ESTHER : Bourne_2010_J.Am.Chem.Soc_132_18292
PubMedSearch : Bourne_2010_J.Am.Chem.Soc_132_18292
PubMedID: 21090615
Gene_locus related to this paper: mouse-ACHE

Title : Insecticide resistance through mutations in cholinesterases or carboxylesterases: data mining in the ESTHER database - Hotelier_2010_J.Pestic.Sci_35_315
Author(s) : Hotelier T , Negre V , Marchot P , Chatonnet A
Ref : Journal of Pesticide Science , 35 :315 , 2010
Abstract : Resistance of arthropods to organophosphates and carbamates used as insecticides is mainly due to mutations in genes encoding carboxylesterase or acetylcholinesterase members of the alpha/beta-hydrolase fold superfamily of proteins. Mutations that have been described at the molecular level concern 24 species, 31 genes and 32 identical positions in the aligned aminoacid sequences. Seven of these positions are found in more than four species and can be considered as hot spots for mutations. Mutations in one single gene also result in cross resistance to pyrethroids. These figures along with all pieces of information related to these mutations can be recovered from the ESTHER database, dedicated to the alpha/beta-hydrolase fold superfamily (http:\/\/bioweb.supagro.inra.fr/esther), through built-in or custom made queries. A sequence alignment of enzymes involved in resistance with highlighted mutated amino acid residues is provided. Selecting one amino acid residue leads to all information about mutations analyzed at this position. Links to the related literature are also available.
ESTHER : Hotelier_2010_J.Pestic.Sci_35_315
PubMedSearch : Hotelier_2010_J.Pestic.Sci_35_315
PubMedID:

Title : Structural determinants for interaction of partial agonists with acetylcholine binding protein and neuronal alpha7 nicotinic acetylcholine receptor - Hibbs_2009_EMBO.J_28_3040
Author(s) : Hibbs RE , Sulzenbacher G , Shi J , Talley TT , Conrod S , Kem WR , Taylor P , Marchot P , Bourne Y
Ref : EMBO Journal , 28 :3040 , 2009
Abstract : The pentameric acetylcholine-binding protein (AChBP) is a soluble surrogate of the ligand binding domain of nicotinic acetylcholine receptors. Agonists bind within a nest of aromatic side chains contributed by loops C and F on opposing faces of each subunit interface. Crystal structures of Aplysia AChBP bound with the agonist anabaseine, two partial agonists selectively activating the alpha7 receptor, 3-(2,4-dimethoxybenzylidene)-anabaseine and its 4-hydroxy metabolite, and an indole-containing partial agonist, tropisetron, were solved at 2.7-1.75 A resolution. All structures identify the Trp 147 carbonyl oxygen as the hydrogen bond acceptor for the agonist-protonated nitrogen. In the partial agonist complexes, the benzylidene and indole substituent positions, dictated by tight interactions with loop F, preclude loop C from adopting the closed conformation seen for full agonists. Fluctuation in loop C position and duality in ligand binding orientations suggest molecular bases for partial agonism at full-length receptors. This study, while pointing to loop F as a major determinant of receptor subtype selectivity, also identifies a new template region for designing alpha7-selective partial agonists to treat cognitive deficits in mental and neurodegenerative disorders.
ESTHER : Hibbs_2009_EMBO.J_28_3040
PubMedSearch : Hibbs_2009_EMBO.J_28_3040
PubMedID: 19696737

Title : Structural analysis of the synaptic protein neuroligin and its beta-neurexin complex: determinants for folding and cell adhesion - Fabrichny_2007_Neuron_56_979
Author(s) : Fabrichny IP , Leone P , Sulzenbacher G , Comoletti D , Miller MT , Taylor P , Bourne Y , Marchot P
Ref : Neuron , 56 :979 , 2007
Abstract : The neuroligins are postsynaptic cell adhesion proteins whose associations with presynaptic neurexins participate in synaptogenesis. Mutations in the neuroligin and neurexin genes appear to be associated with autism and mental retardation. The crystal structure of a neuroligin reveals features not found in its catalytically active relatives, such as the fully hydrophobic interface forming the functional neuroligin dimer; the conformations of surface loops surrounding the vestigial active center; the location of determinants that are critical for folding and processing; and the absence of a macromolecular dipole and presence of an electronegative, hydrophilic surface for neurexin binding. The structure of a beta-neurexin-neuroligin complex reveals the precise orientation of the bound neurexin and, despite a limited resolution, provides substantial information on the Ca2+-dependent interactions network involved in trans-synaptic neurexin-neuroligin association. These structures exemplify how an alpha/beta-hydrolase fold varies in surface topography to confer adhesion properties and provide templates for analyzing abnormal processing or recognition events associated with autism.
ESTHER : Fabrichny_2007_Neuron_56_979
PubMedSearch : Fabrichny_2007_Neuron_56_979
PubMedID: 18093521
Gene_locus related to this paper: human-NLGN4X

Title : Structural characterization of agonist and antagonist-bound acetylcholine-binding protein from Aplysia californica - Hansen_2006_J.Mol.Neurosci_30_101
Author(s) : Hansen SB , Sulzenbacher G , Huxford T , Marchot P , Bourne Y , Taylor P
Ref : Journal of Molecular Neuroscience , 30 :101 , 2006
Abstract : Nicotinic acetylcholine receptors (nAChRs) are well-characterized allosteric transmembrane proteins involved in the rapid gating of ions elicited by ACh. These receptors belong to the Cys-loop superfamily of ligand-gated ion channels, which also includes GABAA and GABAC, 5-HT3, and glycine receptors. The nAChRs are homo- or heteromeric pentamers of structurally related subunits that encompass an extracellular N-terminal ligand-binding domain, four transmembrane-spanning regions that form the ion channel, and an extended intracellular region between spans 3 and 4. Ligand binding triggers conformational changes that are transmitted to the transmembrane-spanning region, leading to gating and changes in membrane potential. The four transmembrane spans on each of the five subunits create a substantial region of hydrophobicity that precludes facile crystallization of this protein. However the freshwater snail, Lymnaea stagnalis, produces a soluble homopentameric protein, termed the ACh-binding protein (AChBP), which binds ACh (Smit et al., 2001). Its structure was determined recently (Brejc et al., 2001) at high resolution, revealing the structural scaffold for nAChR, and has become a functional and structural surrogate of the nAChR ligand-binding domain. We have characterized an AChBP from Aplysia californica and determined distinct ligand-binding properties when compared to those of L. stagnalis, including ligand specificity for the nAChR alpha7 subtype-specific alpha-conotoxin ImI (Hansen et al., 2004).
ESTHER : Hansen_2006_J.Mol.Neurosci_30_101
PubMedSearch : Hansen_2006_J.Mol.Neurosci_30_101
PubMedID: 17192647

Title : Structural comparison of three crystalline complexes of a peptidic toxin with a synaptic acetylcholine recognition protein - Bourne_2006_J.Mol.Neurosci_30_103
Author(s) : Bourne Y , Hansen SB , Sulzenbacher G , Talley TT , Huxford T , Taylor P , Marchot P
Ref : Journal of Molecular Neuroscience , 30 :103 , 2006
Abstract : Many peptidic toxins from animal venoms target neuronal or peripheral synaptic receptors with high affinities and specificities. Hence, these toxins are not only potent natural weapons but also precise molecular tools for pharmacological studies of their receptors. Although they belong to various structural and/or functional subfamilies, they often share similar molecular features, such as a highly reticulated scaffold presenting specific binding determinants.
ESTHER : Bourne_2006_J.Mol.Neurosci_30_103
PubMedSearch : Bourne_2006_J.Mol.Neurosci_30_103
PubMedID: 17192648

Title : Substrate and product trafficking through the active center gorge of acetylcholinesterase analyzed by crystallography and equilibrium binding - Bourne_2006_J.Biol.Chem_281_29256
Author(s) : Bourne Y , Radic Z , Sulzenbacher G , Kim E , Taylor P , Marchot P
Ref : Journal of Biological Chemistry , 281 :29256 , 2006
Abstract : Hydrolysis of acetylcholine catalyzed by acetylcholinesterase (AChE), one of the most efficient enzymes in nature, occurs at the base of a deep and narrow active center gorge. At the entrance of the gorge, the peripheral anionic site provides a binding locus for allosteric ligands, including substrates. To date, no structural information on substrate entry to the active center from the peripheral site of AChE or its subsequent egress has been reported. Complementary crystal structures of mouse AChE and an inactive mouse AChE mutant with a substituted catalytic serine (S203A), in various complexes with four substrates (acetylcholine, acetylthiocholine, succinyldicholine, and butyrylthiocholine), two non-hydrolyzable substrate analogues (m-(N,N,N-trimethylammonio)-trifluoroacetophenone and 4-ketoamyltrimethylammonium), and one reaction product (choline) were solved in the 2.05-2.65-A resolution range. These structures, supported by binding and inhibition data obtained on the same complexes, reveal the successive positions and orientations of the substrates bound to the peripheral site and proceeding within the gorge toward the active site, the conformations of the presumed transition state for acylation and the acyl-enzyme intermediate, and the positions and orientations of the dissociating and egressing products. Moreover, the structures of the AChE mutant in complexes with acetylthiocholine and succinyldicholine reveal additional substrate binding sites on the enzyme surface, distal to the gorge entry. Hence, we provide a comprehensive set of structural snapshots of the steps leading to the intermediates of catalysis and the potential regulation by substrate binding to various allosteric sites at the enzyme surface.
ESTHER : Bourne_2006_J.Biol.Chem_281_29256
PubMedSearch : Bourne_2006_J.Biol.Chem_281_29256
PubMedID: 16837465
Gene_locus related to this paper: mouse-ACHE

Title : A. niger protein EstA, perhaps a new electrotactin, defines a new class of fungal esterases within the alpha\/beta hydrolase fold superfamily - Bourne_2005_Chem.Biol.Interact_157-158_395
Author(s) : Bourne Y , Hasper AA , Chahinian H , Renault L , Juin M , De Graaff LH , Marchot P
Ref : Chemico-Biological Interactions , 157-158 :395 , 2005
Abstract : Protein EstA from Aspergillus niger was characterized through a multifaced approach involving molecular biology, bioinformatics, biophysical, biochemical and enzymatical analyses. EstA was identified as the lead member, within the superfamily of proteins with an alpha/beta-hydrolase fold, of a new class of fungal esterases that also contains predicted homologs from other fungus species of known broad host-range pathogenicity.
ESTHER : Bourne_2005_Chem.Biol.Interact_157-158_395
PubMedSearch : Bourne_2005_Chem.Biol.Interact_157-158_395
PubMedID: 16429533
Gene_locus related to this paper: aspni-EstA

Title : New friendly tools for users of ESTHER, the database of the alpha\/beta-hydrolase fold superfamily of proteins - Renault_2005_Chem.Biol.Interact_157-158_339
Author(s) : Renault L , Negre V , Hotelier T , Cousin X , Marchot P , Chatonnet A
Ref : Chemico-Biological Interactions , 157-158 :339 , 2005
Abstract : The structural alpha/beta-hydrolase fold is characterized by a beta-sheet core of five to eight strands connected by alpha-helices to form a alpha/beta/alpha sandwich. The superfamily members, exemplified by the cholinesterases, diverged from a common ancestor into a number of hydrolytic enzymes displaying a wide range of substrate specificities, along with proteins with no recognized hydrolytic activity. In the enzymes, the catalytic triad residues are presented on loops of which one, the nucleophile elbow, is the most conserved feature of the fold. Of the other proteins, which all lack from one to all of the catalytic residues, some may simply be 'inactive' enzymes while others have been shown to be involved in heterologous surface recognition functions. The ESTHER (for esterases, alpha/beta-hydrolase enzymes and relatives) database (http://bioweb.supagro.inra.fr.esther) gathers and annotates all the published pieces of information (gene and protein sequences; biochemical, pharmacological, and structural data) related to the superfamily, and connects them together to provide the bases for studying structure-function relationships within the superfamily. The most recent developments of the database are presented.
ESTHER : Renault_2005_Chem.Biol.Interact_157-158_339
PubMedSearch : Renault_2005_Chem.Biol.Interact_157-158_339
PubMedID: 16297901

Title : Structural insights into conformational flexibility at the peripheral site and within the active center gorge of AChE - Bourne_2005_Chem.Biol.Interact_157-158_159
Author(s) : Bourne Y , Radic Z , Kolb HC , Sharpless KB , Taylor P , Marchot P
Ref : Chemico-Biological Interactions , 157-158 :159 , 2005
Abstract : The peripheral anionic site on acetylcholinesterase (AChE), located at the active site gorge entry, encompasses overlapping binding sites for allosteric activators and inhibitors. Yet the molecular mechanisms coupling this site to the active center at the base of the gorge to modulate catalysis remain unclear. Crystal structures of mAChE bound with decidium, propidium and gallamine unveiled new determinants contributing to ligand interactions at the peripheral site. Subsequent studies using the syn and anti regioisomers of the click-chemistry inhibitor, TZ2PA6, that link propidium and tacrine moieties via distinctively substituted triazoles, revealed the inherent flexibility and a unique conformation of the peripheral site, along with substantial binding contributions from the triazoles with the Tyr337 region within the gorge. The recently solved structures of the mAChE mutant, Tyr337Ala, complexed with the TZ2PA6 isomers now reveals distinctive and time-dependent conformations of the complexes that are consistent with the triazole contribution to the energetics of inhibitor binding manifested in the respective dissociation rates of the complexes.
ESTHER : Bourne_2005_Chem.Biol.Interact_157-158_159
PubMedSearch : Bourne_2005_Chem.Biol.Interact_157-158_159
PubMedID: 16259971

Title : ESTHER, the database of the alpha\/beta-hydrolase fold superfamily of proteins - Hotelier_2004_Nucleic.Acids.Res_32_D145
Author(s) : Hotelier T , Renault L , Cousin X , Negre V , Marchot P , Chatonnet A
Ref : Nucleic Acids Research , 32 :D145 , 2004
Abstract : The alpha/beta-hydrolase fold is characterized by a beta-sheet core of five to eight strands connected by alpha-helices to form a alpha/beta/alpha sandwich. In most of the family members the beta-strands are parallels, but some show an inversion in the order of the first strands, resulting in antiparallel orientation. The members of the superfamily diverged from a common ancestor into a number of hydrolytic enzymes with a wide range of substrate specificities, together with other proteins with no recognized catalytic activity. In the enzymes the catalytic triad residues are presented on loops, of which one, the nucleophile elbow, is the most conserved feature of the fold. Of the other proteins, which all lack from one to all of the catalytic residues, some may simply be 'inactive' enzymes while others are known to be involved in surface recognition functions. The ESTHER database (http:\/\/bioweb.supagro.inra.fr/esther) gathers and annotates all the published information related to gene and protein sequences of this superfamily, as well as biochemical, pharmacological and structural data, and connects them so as to provide the bases for studying structure-function relationships within the family. The most recent developments of the database, which include a section on human diseases related to members of the family, are described.
ESTHER : Hotelier_2004_Nucleic.Acids.Res_32_D145
PubMedSearch : Hotelier_2004_Nucleic.Acids.Res_32_D145
PubMedID: 14681380

Title : Poster (50) Insights into the interactions at the acetylcholinesterase peripheral anionic site. -
Author(s) : Bourne Y , Taylor P , Radic Z , Berman HA , Marchot P
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :347 , 2004
PubMedID:

Title : Poster (51) Novel compounds with distinctive interactions at the acetylcholinesterase peripheral anionic site -
Author(s) : Bourne Y , Taylor P , Radic Z , Lewis WG , Grynszpan F , Finn MG , Sharpless KB , Marchot P
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :348 , 2004
PubMedID:

Title : Interaction of recombinant soluble neuroligin-1 with neurexin-1-_. -
Author(s) : Comoletti D , Flynn RE , Jennings LL , Hoffman RC , Marchot P , Bourne Y , Sudhof TC , Taylor P
Ref : Cholinergic Mechanisms, CRC Press :523 , 2004
PubMedID:

Title : Aspergillus niger protein EstA defines a new class of fungal esterases within the alpha\/beta hydrolase fold superfamily of proteins - Bourne_2004_Structure_12_677
Author(s) : Bourne Y , Hasper AA , Chahinian H , Juin M , De Graaff LH , Marchot P
Ref : Structure , 12 :677 , 2004
Abstract : From the fungus Aspergillus niger, we identified a new gene encoding protein EstA, a member of the alpha/beta-hydrolase fold superfamily but of unknown substrate specificity. EstA was overexpressed and its crystal structure was solved by molecular replacement using a lipase-acetylcholinesterase chimera template. The 2.1 A resolution structure of EstA reveals a canonical Ser/Glu/His catalytic triad located in a small pocket at the bottom of a large solvent-accessible, bowl-shaped cavity. Potential substrates selected by manual docking procedures were assayed for EstA activity. Consistent with the pocket geometry, preference for hydrolysis of short acyl/propyl chain substrates was found. Identification of close homologs from the genome of other fungi, of which some are broad host-range pathogens, defines EstA as the first member of a novel class of fungal esterases within the superfamily. Hence the structure of EstA constitutes a lead template in the design of new antifungal agents directed toward its pathogenic homologs.
ESTHER : Bourne_2004_Structure_12_677
PubMedSearch : Bourne_2004_Structure_12_677
PubMedID: 15062090
Gene_locus related to this paper: aspni-EstA

Title : Freeze-frame inhibitor captures acetylcholinesterase in a unique conformation - Bourne_2004_Proc.Natl.Acad.Sci.U.S.A_101_1449
Author(s) : Bourne Y , Kolb HC , Radic Z , Sharpless KB , Taylor P , Marchot P
Ref : Proc Natl Acad Sci U S A , 101 :1449 , 2004
Abstract : The 1,3-dipolar cycloaddition reaction between unactivated azides and acetylenes proceeds exceedingly slowly at room temperature. However, considerable rate acceleration is observed when this reaction occurs inside the active center gorge of acetylcholinesterase (AChE) between certain azide and acetylene reactants, attached via methylene chains to specific inhibitor moieties selective for the active center and peripheral site of the enzyme. AChE catalyzes the formation of its own inhibitor in a highly selective fashion: only a single syn1-triazole regioisomer with defined substitution positions and linker distances is generated from a series of reagent combinations. Inhibition measurements revealed this syn1-triazole isomer to be the highest affinity reversible organic inhibitor of AChE with association rate constants near the diffusion limit. The corresponding anti1 isomer, not formed by the enzyme, proved to be a respectable but weaker inhibitor. The crystal structures of the syn1- and anti1-mouse AChE complexes at 2.45- to 2.65-A resolution reveal not only substantial binding contributions from the triazole moieties, but also that binding of the syn1 isomer induces large and unprecedented enzyme conformational changes not observed in the anti1 complex nor predicted from structures of the apoenzyme and complexes with the precursor reactants. Hence, the freeze-frame reaction offers both a strategically original approach for drug discovery and a means for kinetically controlled capture, as a high-affinity complex between the enzyme and its self-created inhibitor, of a highly reactive minor abundance conformer of a fluctuating protein template.
ESTHER : Bourne_2004_Proc.Natl.Acad.Sci.U.S.A_101_1449
PubMedSearch : Bourne_2004_Proc.Natl.Acad.Sci.U.S.A_101_1449
PubMedID: 14757816
Gene_locus related to this paper: mouse-ACHE

Title : Mechanism of acetylcholinesterase inhibition by fasciculin. -
Author(s) : Tai K , Shen T , Henchman RH , Bourne Y , Marchot P , McCammon JA
Ref : Cholinergic Mechanisms, CRC Press :727 , 2004
PubMedID:

Title : Structural insights into ligand interactions at the acetylcholinesterase peripheral anionic site - Bourne_2003_Embo.J_22_1
Author(s) : Bourne Y , Taylor P , Radic Z , Marchot P
Ref : EMBO Journal , 22 :1 , 2003
Abstract : The peripheral anionic site on acetylcholinesterase (AChE), located at the active center gorge entry, encompasses overlapping binding sites for allosteric activators and inhibitors; yet, the molecular mechanisms coupling this site to the active center at the gorge base to modulate catalysis remain unclear. The peripheral site has also been proposed to be involved in heterologous protein associations occurring during synaptogenesis or upon neurodegeneration. A novel crystal form of mouse AChE, combined with spectrophotometric analyses of the crystals, enabled us to solve unique structures of AChE with a free peripheral site, and as three complexes with peripheral site inhibitors: the phenylphenanthridinium ligands, decidium and propidium, and the pyrogallol ligand, gallamine, at 2.20-2.35 A resolution. Comparison with structures of AChE complexes with the peptide fasciculin or with organic bifunctional inhibitors unveils new structural determinants contributing to ligand interactions at the peripheral site, and permits a detailed topographic delineation of this site. Hence, these structures provide templates for designing compounds directed to the enzyme surface that modulate specific surface interactions controlling catalytic activity and non-catalytic heterologous protein associations.
ESTHER : Bourne_2003_Embo.J_22_1
PubMedSearch : Bourne_2003_Embo.J_22_1
PubMedID: 12505979
Gene_locus related to this paper: mouse-ACHE

Title : Mechanism of acetylcholinesterase inhibition by fasciculin: a 5-ns molecular dynamics simulation - Tai_2002_J.Am.Chem.Soc_124_6153
Author(s) : Tai K , Shen T , Henchman RH , Bourne Y , Marchot P , McCammon JA
Ref : Journal of the American Chemical Society , 124 :6153 , 2002
Abstract : 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/.
ESTHER : Tai_2002_J.Am.Chem.Soc_124_6153
PubMedSearch : Tai_2002_J.Am.Chem.Soc_124_6153
PubMedID: 12022850

Title : [The fasciculin-acetylcholinesterase interaction] - Marchot_1999_J.Soc.Biol_193_505
Author(s) : Marchot P
Ref : J Soc Biol , 193 :505 , 1999
Abstract : Acetylcholinesterase (AChE) terminates the action of the neurotransmitter acetylcholine at cholinergic synapses in the central and peripheral nervous systems. Fasciculins, which belong to the family of "three-fingered" snake toxins, selectively inhibit mammalian AChEs with Ki values in the picomolar range. In solution, the cationic fasciculin appears to bind to the enzyme's peripheral anionic site, located near the mouth of the gorge leading to the active center, to inhibit catalysis either allosterically or by creating an electrostatic barrier at the gorge entry (or both). Yet the crystal structure of the fasciculin-mouse AChE complex, which shows that the central loop of fasciculin fits snugly at the entrance of the gorge, suggests that the mode of action of fasciculin is steric occlusion of substrate access to the active center. Mutagenesis of the fasciculin molecule, undertaken to establish a functional map of the binding surfaces, identified determinants common to those identified by the structural approach and revealed that only a few of the many fasciculin residues residing at the complex interface provide the strong contacts required for high affinity binding and enzyme inhibition. However, it did not reconcile the disparity between the kinetic and structural data. Finally, the crystal structure of mouse AChE without bound fasciculin shows a tetrameric assembly of subunits; within the tetramer, a short loop at the surface of a subunit associates with the peripheral site of a facing subunit and sterically occludes the entrance of the active center gorge. The position and complementarity of the peripheral site-occluding loop mimic the characteristics of the central loop of fasciculin bound to AChE. This suggests not only that the peripheral site of AChE is a site for association of heterologous proteins with interactive surface loops, but also that endogenous peptidic ligands of AChE sharing structural features with the fasciculin molecule might exist.
ESTHER : Marchot_1999_J.Soc.Biol_193_505
PubMedSearch : Marchot_1999_J.Soc.Biol_193_505
PubMedID: 10783708

Title : Conformational flexibility of the acetylcholinesterase tetramer suggested by x-ray crystallography - Bourne_1999_J.Biol.Chem_274_30370
Author(s) : Bourne Y , Grassi J , Bougis PE , Marchot P
Ref : Journal of Biological Chemistry , 274 :30370 , 1999
Abstract : Acetylcholinesterase, a polymorphic enzyme, appears to form amphiphilic and nonamphiphilic tetramers from a single splice variant; this suggests discrete tetrameric arrangements where the amphipathic carboxyl-terminal sequences can be either buried or exposed. Two distinct, but related crystal structures of the soluble, trypsin-released tetramer of acetylcholinesterase from Electrophorus electricus were solved at 4.5 and 4.2 A resolution by molecular replacement. Resolution at these levels is sufficient to provide substantial information on the relative orientations of the subunits within the tetramer. The two structures, which show canonical homodimers of subunits assembled through four-helix bundles, reveal discrete geometries in the assembly of the dimers to form: (a) a loose, pseudo-square planar tetramer with antiparallel alignment of the two four-helix bundles and a large space in the center where the carboxyl-terminal sequences may be buried or (b) a compact, square nonplanar tetramer that may expose all four sequences on a single side. Comparison of these two structures points to significant conformational flexibility of the tetramer about the four-helix bundle axis and along the dimer-dimer interface. Hence, in solution, several conformational states of a flexible tetrameric arrangement of acetylcholinesterase catalytic subunits may exist to accommodate discrete carboxyl-terminal sequences of variable dimensions and amphipathicity.
ESTHER : Bourne_1999_J.Biol.Chem_274_30370
PubMedSearch : Bourne_1999_J.Biol.Chem_274_30370
PubMedID: 10521413
Gene_locus related to this paper: eleel-ACHE

Title : Electron paramagnetic resonance reveals altered topography of the active center gorge of acetylcholinesterase after binding of fasciculin to the peripheral site - Sentjurc_1999_Biochim.Biophys.Acta_1430_349
Author(s) : Sentjurc M , Pecar S , Stojan J , Marchot P , Radic Z , Grubic Z
Ref : Biochimica & Biophysica Acta , 1430 :349 , 1999
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.
ESTHER : Sentjurc_1999_Biochim.Biophys.Acta_1430_349
PubMedSearch : Sentjurc_1999_Biochim.Biophys.Acta_1430_349
PubMedID: 10082962

Title : Crystal structure of mouse acetylcholinesterase. A peripheral site- occluding loop in a tetrameric assembly - Bourne_1999_J.Biol.Chem_274_2963
Author(s) : Bourne Y , Taylor P , Bougis PE , Marchot P
Ref : Journal of Biological Chemistry , 274 :2963 , 1999
Abstract : The crystal structure of mouse acetylcholinesterase at 2.9-A resolution reveals a tetrameric assembly of subunits with an antiparallel alignment of two canonical homodimers assembled through four-helix bundles. In the tetramer, a short Omega loop, composed of a cluster of hydrophobic residues conserved in mammalian acetylcholinesterases along with flanking alpha-helices, associates with the peripheral anionic site of the facing subunit and sterically occludes the entrance of the gorge leading to the active center. The inverse loop-peripheral site interaction occurs within the second pair of subunits, but the peripheral sites on the two loop-donor subunits remain freely accessible to the solvent. The position and complementarity of the peripheral site-occluding loop mimic the characteristics of the central loop of the peptidic inhibitor fasciculin bound to mouse acetylcholinesterase. Tetrameric forms of cholinesterases are widely distributed in nature and predominate in mammalian brain. This structure reveals a likely mode of subunit arrangement and suggests that the peripheral site, located near the rim of the gorge, is a site for association of neighboring subunits or heterologous proteins with interactive surface loops.
ESTHER : Bourne_1999_J.Biol.Chem_274_2963
PubMedSearch : Bourne_1999_J.Biol.Chem_274_2963
PubMedID: 9915834
Gene_locus related to this paper: mouse-ACHE

Title : Tetrameric assembly and peripheral site-occluding loop of mouse acetylcholinesterase -
Author(s) : Marchot P , Taylor P , Kanter J , Bougis PE , Bourne Y
Ref : Journal de Physiologie (Paris) , 92 :465 , 1998
PubMedID:

Title : Crystal Structure of Mouse Acetylcholinesterase -
Author(s) : Bourne Y , Taylor P , Kanter J , Bougis PE , Marchot 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. :315 , 1998
PubMedID:

Title : Fasciculin Inhibition of Mouse Acetylcholinesterase -
Author(s) : Marchot P , Bourne Y , Prowse CN , Kanter J , Eads J , Bougis PE , 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. :331 , 1998
PubMedID:

Title : Inhibition of mouse acetylcholinesterase by fasciculin: crystal structure of the complex and mutagenesis of fasciculin - Marchot_1998_Toxicon_36_1613
Author(s) : Marchot P , Bourne Y , Prowse CN , Bougis PE , Taylor P
Ref : Toxicon , 36 :1613 , 1998
Abstract : 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.
ESTHER : Marchot_1998_Toxicon_36_1613
PubMedSearch : Marchot_1998_Toxicon_36_1613
PubMedID: 9792178

Title : Biotinylation of the tyrosine residues of fasciculin -
Author(s) : Bougis PE , Taylor P , Marchot P
Ref : Journal de Physiologie (Paris) , 92 :416 , 1998
PubMedID:

Title : Residues at the subunit interfaces of the nicotinic acetylcholine receptor that contribute to alpha-conotoxin M1 binding - Sugiyama_1998_Mol.Pharmacol_53_787
Author(s) : Sugiyama N , Marchot P , Kawanishi C , Osaka H , Molles BE , Sine SM , Taylor P
Ref : Molecular Pharmacology , 53 :787 , 1998
Abstract : The two binding sites in the pentameric nicotinic acetylcholine receptor of subunit composition alpha2 beta gamma delta are formed by nonequivalent alpha-gamma and alpha-delta subunit interfaces, which produce site selectivity in the binding of agonists and antagonists. We show by sedimentation analysis that 125I-alpha-conotoxin M1 binds with high affinity to the alpha-delta subunit dimers, but not to alpha-gamma dimers, nor to alpha, gamma, and delta monomers, a finding consistent with alpha-conotoxin M1 selectivity for the alpha delta interface in the intact receptor measured by competition against alpha-bungarotoxin binding. We also extend previous identification of alpha-conotoxin M1 determinants in the gamma and delta subunits to the alpha subunit interface by mutagenesis of conserved residues in the alpha subunit. Most mutations of the alpha subunit affect affinity similarly at the two sites, but Tyr93Phe, Val188Lys, Tyr190Thr, Tyr198Thr, and Asp152Asn affect affinity in a site-selective manner. Mutant cycle analysis reveals only weak or no interactions between mutant alpha and non-alpha subunits, indicating that side chains of the alpha subunit do not interact with those of the gamma or delta subunits in stabilizing alpha-conotoxin M1. The overall findings suggest different binding configurations of alpha-conotoxin M1 at the alpha-delta and alpha-gamma binding interfaces.
ESTHER : Sugiyama_1998_Mol.Pharmacol_53_787
PubMedSearch : Sugiyama_1998_Mol.Pharmacol_53_787
PubMedID: 9547372

Title : Expression and Purification of Recombinant Mutants of Fasciculin from Mammalian Cells -
Author(s) : Kanter J , Eads J , Camp S , Marchot P , 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. :240 , 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 : Expression and activity of mutants of fasciculin, a peptidic acetylcholinesterase inhibitor from mamba venom - Marchot_1997_J.Biol.Chem_272_3502
Author(s) : Marchot P , Prowse CN , Kanter J , Camp S , Ackermann EJ , Radic Z , Bougis PE , Taylor P
Ref : Journal of Biological Chemistry , 272 :3502 , 1997
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, DeltaPro30, 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.
ESTHER : Marchot_1997_J.Biol.Chem_272_3502
PubMedSearch : Marchot_1997_J.Biol.Chem_272_3502
PubMedID: 9013597

Title : Soluble monomeric acetylcholinesterase from mouse: expression, purification, and crystallization in complex with fasciculin - Marchot_1996_Prot.Sci_5_672
Author(s) : Marchot P , Ravelli RB , Raves ML , Bourne Y , Vellom DC , Kanter J , Camp S , Sussman JL , Taylor P
Ref : Protein Science , 5 :672 , 1996
Abstract : A soluble, monomeric form of acetylcholinesterase from mouse (mAChE), truncated at its carboxyl-terminal end, was generated from a cDNA encoding the glycophospholipid-linked form of the mouse enzyme by insertion of an early stop codon at position 549. Insertion of the cDNA behind a cytomegalovirus promoter and selection by aminoglycoside resistance in transfected HEK cells yielded clones secreting large quantities of mAChE into the medium. The enzyme sediments as a soluble monomer at 4.8 S. High levels of expression coupled with a one-step purification by affinity chromatography have allowed us to undertake a crystallographic study of the fasciculin-mAChE complex. Complexes of two distinct fasciculins, Fas1-mAChE and Fas2-mAChE, were formed prior to the crystallization and were characterized thoroughly. Single hexagonal crystals, up to 0.6 mm x 0.5 mm x 0.5 mm, grew spontaneously from ammonium sulfate solutions buffered in the pH 7.0 range. They were found by electrophoretic migration to consist entirely of the complex and diffracted to 2.8 A resolution. Analysis of initial X-ray data collected on Fas2-mAChE crystals identified the space group as P6(1)22 or P6(5)22 with unit cell dimensions a = b = 75.5 A, c = 556 A, giving a Vm value of 3.1 A3/Da (or 60% of solvent), consistent with a single molecule of Fas2-AChE complex (72 kDa) per asymmetric unit. The complex Fas1-mAChE crystallizes in the same space group with identical cell dimensions.
ESTHER : Marchot_1996_Prot.Sci_5_672
PubMedSearch : Marchot_1996_Prot.Sci_5_672
PubMedID: 8845756
Gene_locus related to this paper: mouse-ACHE

Title : Structure of fasciculin 2 from green mamba snake venom: evidence for unusual loop flexibility - le_1996_Acta.Crystallogr.D.Biol.Crystallogr_52_87
Author(s) : le Du MH , Housset D , Marchot P , Bougis PE , Navaza J , Fontecilla-Camps JC
Ref : Acta Crystallographica D Biol Crystallogr , 52 :87 , 1996
Abstract : 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.
ESTHER : le_1996_Acta.Crystallogr.D.Biol.Crystallogr_52_87
PubMedSearch : le_1996_Acta.Crystallogr.D.Biol.Crystallogr_52_87
PubMedID: 15299729

Title : Structural Determinants of Fasciculin Specificity for Acetylcholinesterase -
Author(s) : Marchot P , Camp S , Radic Z , Bougis PE , 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. :197 , 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

Title : Structural bases for the specificity of cholinesterase catalysis and inhibition. - Taylor_1995_Toxicol.Lett_83_453
Author(s) : Taylor P , Radic Z , Hosea NA , Camp S , Marchot P , Berman HA
Ref : Toxicology Letters , 83 :453 , 1995
Abstract : The availability of a crystal structure and comparative sequences of the cholinesterases has provided templates suitable for analyzing the molecular bases of specificity of reversible inhibitors, carbamoylating agents and organophosphates. Site-specific mutagenesis enables one to modify the structures of both the binding site and peptide ligand as well as create chimeras reflecting one type of esterase substituted in the template of another. Herein we define the bases for substrate specificity of carboxylesters, the stereospecificity of enantiomeric alkylphosphonates and the selectivity of tricyclic aromatic compounds in the active center of cholinesterase. We also describe the binding loci of the peripheral site and changes in catalytic parameters induced by peripheral site ligands, using the peptide fasciculin.
ESTHER : Taylor_1995_Toxicol.Lett_83_453
PubMedSearch : Taylor_1995_Toxicol.Lett_83_453
PubMedID: 8597093

Title : Acetylcholinesterase inhibition by fasciculin: crystal structure of the complex - Bourne_1995_Cell_83_503
Author(s) : Bourne Y , Taylor P , Marchot P
Ref : Cell , 83 :503 , 1995
Abstract : The crystal structure of the snake toxin fasciculin, bound to mouse acetylcholinesterase (mAChE), at 3.2 A resolution reveals a synergistic three-point anchorage consistent with the picomolar dissociation constant of the complex. Loop II of fasciculin contains a cluster of hydrophobic residues that interact with the peripheral anionic site of the enzyme and sterically occlude substrate access to the catalytic site. Loop I fits in a crevice near the lip of the gorge to maximize the surface area of contact of loop II at the gorge entry. The fasciculin core surrounds a protruding loop on the enzyme surface and stabilizes the whole assembly. Upon binding of fasciculin, subtle structural rearrangements of AChE occur that could explain the observed residual catalytic activity of the fasciculin-enzyme complex.
ESTHER : Bourne_1995_Cell_83_503
PubMedSearch : Bourne_1995_Cell_83_503
PubMedID: 8521480
Gene_locus related to this paper: mouse-ACHE

Title : Binding of 125I-fasciculin to rat brain acetylcholinesterase. The complex still binds diisopropyl fluorophosphate - Marchot_1993_J.Biol.Chem_268_12458
Author(s) : Marchot P , Khelif A , Ji YH , Mansuelle P , Bougis PE
Ref : Journal of Biological Chemistry , 268 :12458 , 1993
Abstract : Iodination of fasciculin 3 (FAS3) from Dendroaspis viridis venom provided us with a fully active specific probe of fasciculin binding sites on rat brain acetylcholinesterase (AChE). Binding and inhibition are concomitant, as association and inhibition rate constants k1 and ki are identical. The 125I-FAS3.AChE complex dissociates very slowly (t 1/2 = 48 h) and is characterized by a dissociation constant, Kd, of 0.4 pM. All the specific binding of 125I-FAS3 to AChE is prevented by FAS3 as from D. angusticeps venom (Kd = 0.4, 14, and 25 pM, respectively). It is also prevented by propidium iodide, BW284C51, and d-tubocurarine, which bind to peripheral anionic sites of AChE, by Ca2+ and Mg2+, known to enhance AChE activity through an allosteric phenomenon and by acetylthiocholine concentrations which lead to excess substrate inhibition of the enzyme. Diisopropyl fluorphosphate and paroxon, which inhibit AChE by phosphorylating the catalytic serine, have no effect on either the binding rate or the number of binding sites of 125I-FAS3. O-Ethyl-S2-diisopropylaminoethyl methylphosphonothionate, however, which binds irreversibly to the AChE catalytic site but reversibly to a peripheral site, induces a 130% increase in the binding rate of 125I-FAS3, without changing the total number of 125I-FAS3 binding sites. Our results demonstrate that fasciculins bind on a peripheral site of AChE, distinct from the catalytic site and, at least partly, common with the sites on which some cationic inhibitors and the substrate in excess bind. Since phosphorylation of the catalytic serine (esteratic subsite) by [1,3-3H]diisopropyl fluorophosphate can still occur on the FAS3.AChE complex, the structural modification induced by fasciculins may affect the anionic subsite of AChE catalytic site.
ESTHER : Marchot_1993_J.Biol.Chem_268_12458
PubMedSearch : Marchot_1993_J.Biol.Chem_268_12458
PubMedID: 8509385

Title : 1.9-A resolution structure of fasciculin 1, an anti- acetylcholinesterase toxin from green mamba snake venom - le Du_1992_J.Biol.Chem_267_22122
Author(s) : le Du MH , Marchot P , Bougis PE , Fontecilla-Camps JC
Ref : Journal of Biological Chemistry , 267 :22122 , 1992
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.
ESTHER : le Du_1992_J.Biol.Chem_267_22122
PubMedSearch : le Du_1992_J.Biol.Chem_267_22122
PubMedID: 1429564

Title : A fibrinogen-clotting serine proteinase from Cerastes cerastes (horned viper) venom with arginine-esterase and amidase activities. Purification, characterization and kinetic parameter determination - Laraba-Djebari_1992_Toxicon_30_1399
Author(s) : Laraba-Djebari F , Martin-Eauclaire MF , Marchot P
Ref : Toxicon , 30 :1399 , 1992
Abstract : An enzyme displaying proteolytic activity toward the natural substrate casein as well as clotting activity on fibrinogen was purified to homogeneity from Cerastes cerastes (horned viper) venom and characterized. The enzyme is constituted of two identical subunits of mol. wt 48,500 as determined by SDS-polyacrylamide gel electrophoresis, and has an isoelectric point of 3.75. N-terminal sequencing up to the 33rd residue evidenced a high homology with other snake venom proteinases. The proteinase is of serine-type as indicated by high sensitivity to DFP and shows both arginine-ester hydrolase and amidase activities on synthetic substrates. Both specific activities were 30-fold higher than the respective activities found in the crude venom. The Km value determined for arginine-containing substrate BAEE was 3.0 x 10(-4) M and the Km for chromogenic substrate CBS 34-47 0.65 x 10(-4) M. The Vm/Km ratio, however, was two-fold higher for BAEE than for CBS 34-47; the arginine-esterase activity of this enzyme is thus slightly higher than its amidase activity.
ESTHER : Laraba-Djebari_1992_Toxicon_30_1399
PubMedSearch : Laraba-Djebari_1992_Toxicon_30_1399
PubMedID: 1485336

Title : Crystals of fasciculin 2 from green mamba snake venom. Preparation and preliminary x-ray analysis - le Du_1989_J.Biol.Chem_264_21401
Author(s) : le Du MH , Marchot P , Bougis PE , Fontecilla-Camps JC
Ref : Journal of Biological Chemistry , 264 :21401 , 1989
Abstract : Fasciculin 2 from the venom of the green mamba, Dendroaspis angusticeps, has been crystallized. The crystals are tetragonal, with unit cell dimensions a = 48.9 A and c = 82.0 A, space group P 41 21 2 or P 43212. Density measurements and pseudocentering of the hko zone indicate that there are 16 molecules in the unit cell.
ESTHER : le Du_1989_J.Biol.Chem_264_21401
PubMedSearch : le Du_1989_J.Biol.Chem_264_21401
PubMedID: 2592383