Bourne Y

General

Full name : Bourne Yves

First name : Yves

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

Zip Code :

City :

Country : France

Email : Yves.Bourne@univ-amu.fr

Phone : 33-4-91-82-55-66

Fax : 33-4-91-26-67-20

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

Directory :

References (51)

Title : Direct capture, inhibition and crystal structure of HsaD (Rv3569c) from M. tuberculosis - Barelier_2022_FEBS.J__
Author(s) : Barelier S , Avellan R , Gnawali GR , Fourquet P , Roig-Zamboni V , Poncin I , Point V , Bourne Y , Audebert S , Camoin L , Spilling CD , Canaan S , Cavalier JF , Sulzenbacher G
Ref : Febs J , : , 2022
Abstract : A hallmark of Mycobacterium tuberculosis (M. tb), the aetiologic agent of tuberculosis, is its ability to metabolise host-derived lipids. However, the enzymes and mechanisms underlying such metabolism are still largely unknown. We previously reported that the Cyclophostin & Cyclipostins (CyC) analogues, a new family of potent antimycobacterial molecules, react specifically and covalently with (Ser/Cys)-based enzymes mostly involved in bacterial lipid metabolism. Here, we report the synthesis of new CyC alkyne-containing inhibitors (CyC(yne) ) and their use for the direct fishing of target proteins in M. tb culture via bio-orthogonal click-chemistry activity-based protein profiling (CC-ABPP). This approach led to the capture and identification of a variety of enzymes, and many of them involved in lipid or steroid metabolisms. One of the captured enzymes, HsaD (Rv3569c), is required for the survival of M. tb within macrophages and is thus a potential therapeutic target. This prompted us to further explore and validate, through a combination of biochemical and structural approaches, the specificity of HsaD inhibition by the CyC analogues. We confirmed that the CyC bind covalently to the catalytic Ser(114) residue, leading to a total loss of enzyme activity. These data were supported by the X-ray structures of four HsaD-CyC complexes, obtained at resolutions between 1.6 and 2.6 . The identification of mycobacterial enzymes directly captured by the CyC(yne) probes through CC-ABPP paves the way to better understand and potentially target key players at crucial stages of the bacilli life cycle.
ESTHER : Barelier_2022_FEBS.J__
PubMedSearch : Barelier_2022_FEBS.J__
PubMedID: 36197115
Gene_locus related to this paper: myctu-Rv3569c

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 : 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 : 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 : 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 : 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 : 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 : Gene overexpression and biochemical characterization of the biotechnologically relevant chlorogenic acid hydrolase from Aspergillus niger - Benoit_2007_Appl.Environ.Microbiol_73_5624
Author(s) : Benoit I , Asther M , Bourne Y , Navarro D , Canaan S , Lesage-Meessen L , Herweijer M , Coutinho PM , Record E
Ref : Applied Environmental Microbiology , 73 :5624 , 2007
Abstract : The full-length gene that encodes the chlorogenic acid hydrolase from Aspergillus niger CIRM BRFM 131 was cloned by PCR based on the genome of the strain A. niger CBS 513.88. The complete gene consists of 1,715 bp and codes for a deduced protein of 512 amino acids with a molecular mass of 55,264 Da and an acidic pI of 4.6. The gene was successfully cloned and overexpressed in A. niger to yield 1.25 g liter(-1), i.e., 330-fold higher than the production of wild-type strain A. niger CIRM BRFM131. The histidine-tagged recombinant ChlE protein was purified to homogeneity via a single chromatography step, and its main biochemical properties were characterized. The molecular size of the protein checked by mass spectroscopy was 74,553 Da, suggesting the presence of glycosylation. ChlE is assembled in a tetrameric form with several acidic isoforms with pIs of around 4.55 and 5.2. Other characteristics, such as optimal pH and temperature, were found to be similar to those determined for the previously characterized chlorogenic acid hydrolase of A. niger CIRM BRFM 131. However, there was a significant temperature stability difference in favor of the recombinant protein. ChlE exhibits a catalytic efficiency of 12.5 x 10(6) M(-1) s(-1) toward chlorogenic acid (CGA), and its ability to release caffeic acid from CGA present in agricultural by-products such as apple marc and coffee pulp was clearly demonstrated, confirming the high potential of this enzyme.
ESTHER : Benoit_2007_Appl.Environ.Microbiol_73_5624
PubMedSearch : Benoit_2007_Appl.Environ.Microbiol_73_5624
PubMedID: 17630312
Gene_locus related to this paper: aspnc-a2qn56

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 : LppX is a lipoprotein required for the translocation of phthiocerol dimycocerosates to the surface of Mycobacterium tuberculosis - Sulzenbacher_2006_EMBO.J_25_1436
Author(s) : Sulzenbacher G , Canaan S , Bordat Y , Neyrolles O , Stadthagen G , Roig-Zamboni V , Rauzier J , Maurin D , Laval F , Daffe M , Cambillau C , Gicquel B , Bourne Y , Jackson M
Ref : EMBO Journal , 25 :1436 , 2006
Abstract : Cell envelope lipids play an important role in the pathogenicity of mycobacteria, but the mechanisms by which they are transported to the outer membrane of these prokaryotes are largely unknown. Here, we provide evidence that LppX is a lipoprotein required for the translocation of complex lipids, the phthiocerol dimycocerosates (DIM), to the outer membrane of Mycobacterium tuberculosis. Abolition of DIM transport following disruption of the lppX gene is accompanied by an important attenuation of the virulence of the tubercle bacillus. The crystal structure of LppX unveils an U-shaped beta-half-barrel dominated by a large hydrophobic cavity suitable to accommodate a single DIM molecule. LppX shares a similar fold with the periplasmic molecular chaperone LolA and the outer membrane lipoprotein LolB, which are involved in the localization of lipoproteins to the outer membrane of Gram-negative bacteria. Based on the structure and although an indirect participation of LppX in DIM transport cannot yet be ruled out, we propose LppX to be the first characterized member of a family of structurally related lipoproteins that carry lipophilic molecules across the mycobacterial cell envelope.
ESTHER : Sulzenbacher_2006_EMBO.J_25_1436
PubMedSearch : Sulzenbacher_2006_EMBO.J_25_1436
PubMedID: 16541102

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 : Crystal structure of the conserved hypothetical protein Rv1155 from Mycobacterium tuberculosis - Canaan_2005_FEBS.Lett_579_215
Author(s) : Canaan S , Sulzenbacher G , Roig-Zamboni V , Scappuccini-Calvo L , Frassinetti F , Maurin D , Cambillau C , Bourne Y
Ref : FEBS Letters , 579 :215 , 2005
Abstract : With the aim of elucidating the biological function of hypothetical proteins unique amongst the Actynomyces sub-group of bacteria, we have solved the crystal structure of the conserved hypothetical protein Rv1155 from Mycobacterium tuberculosis at 1.8 A resolution. Rv1155 is a homodimer both in the crystal structure and in solution and folds into two separate domains consisting of a six-stranded anti-parallel beta-barrel fold flanked by two alpha-helices and a helix-turn-helix domain. Both domains contribute to the formation of two deep clefts at the dimer interface. The overall fold of Rv1155 strikingly resembles that of flavin mononucleotide-binding protein and pyridoxamine 5'-phosphate oxydase, but the architecture of the putative binding pocket is markedly different, consistent with the lack of color of Rv1155 and its inability to bind FMN. Rv1155 thus appears to belong to a group of proteins with stringent conservation of the binding cleft, having evolved towards a new binding function.
ESTHER : Canaan_2005_FEBS.Lett_579_215
PubMedSearch : Canaan_2005_FEBS.Lett_579_215
PubMedID: 15620716

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 : 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 : 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 : Expression and characterization of the protein Rv1399c from Mycobacterium tuberculosis. A novel carboxyl esterase structurally related to the HSL family - Canaan_2004_Eur.J.Biochem_271_3953
Author(s) : Canaan S , Maurin D , Chahinian H , Pouilly B , Durousseau C , Frassinetti F , Scappuccini-Calvo L , Cambillau C , Bourne Y
Ref : European Journal of Biochemistry , 271 :3953 , 2004
Abstract : The Mycobacterium tuberculosis genome contains an unusually high number of proteins involved in the metabolism of lipids belonging to the Lip family, including various nonlipolytic and lipolytic hydrolases. Driven by a structural genomic approach, we have biochemically characterized the Rv1399c gene product, LipH, previously annotated as a putative lipase. Rv1399c was overexpressed in E. coli as inclusion bodies and refolded. Rv1399c efficiently hydrolyzes soluble triacylglycerols and vinyl esters. It is inactive against emulsified substrate and its catalytic activity is strongly inhibited by the diethyl paranitrophenyl phosphate (E600). These kinetic behaviors unambiguously classify Rv1399c as a nonlipolytic rather than a lipolytic hydrolase. Sequence alignment reveals that this enzyme belongs to the alpha/beta hydrolase fold family and shares 30-40% amino acid sequence identity with members of the hormone-sensitive lipase subfamily. A model of Rv1399c derived from homologous three-dimensional structures reveals a canonical catalytic triad (Ser162, His290 and Asp260) located at the bottom of a solvent accessible pocket lined by neutral or charged residues. Based on this model, kinetic data of the Arg213Ala mutant partially explain the role of the guanidinium moiety, located close to His290, to confer an unusual low pH shift of the catalytic histidine in the wild type enzyme. Overall, these data identify Rv1399c as a new nonlipolytic hydrolase from M. tuberculosis and we thus propose to reannotate its gene product as NLH-H.
ESTHER : Canaan_2004_Eur.J.Biochem_271_3953
PubMedSearch : Canaan_2004_Eur.J.Biochem_271_3953
PubMedID: 15373841
Gene_locus related to this paper: myctu-Rv1399c

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 : High-throughput automated refolding screening of inclusion bodies - Vincentelli_2004_Protein.Sci_13_2782
Author(s) : Vincentelli R , Canaan S , Campanacci V , Valencia C , Maurin D , Frassinetti F , Scappucini-Calvo L , Bourne Y , Cambillau C , Bignon C
Ref : Protein Science , 13 :2782 , 2004
Abstract : One of the main stumbling blocks encountered when attempting to express foreign proteins in Escherichia coli is the occurrence of amorphous aggregates of misfolded proteins, called inclusion bodies (IB). Developing efficient protein native structure recovery procedures based on IB refolding is therefore an important challenge. Unfortunately, there is no "universal" refolding buffer: Experience shows that refolding buffer composition varies from one protein to another. In addition, the methods developed so far for finding a suitable refolding buffer suffer from a number of weaknesses. These include the small number of refolding formulations, which often leads to negative results, solubility assays incompatible with high-throughput, and experiment formatting not suitable for automation. To overcome these problems, it was proposed in the present study to address some of these limitations. This resulted in the first completely automated IB refolding screening procedure to be developed using a 96-well format. The 96 refolding buffers were obtained using a fractional factorial approach. The screening procedure is potentially applicable to any nonmembrane protein, and was validated with 24 proteins in the framework of two Structural Genomics projects. The tests used for this purpose included the use of quality control methods such as circular dichroism, dynamic light scattering, and crystallogenesis. Out of the 24 proteins, 17 remained soluble in at least one of the 96 refolding buffers, 15 passed large-scale purification tests, and five gave crystals.
ESTHER : Vincentelli_2004_Protein.Sci_13_2782
PubMedSearch : Vincentelli_2004_Protein.Sci_13_2782
PubMedID: 15388864

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 : 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 : 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 : Pancreatic lipases and their complexes with colipases and inhibitors: crystallization and crystal packing -
Author(s) : Cambillau C , Bourne Y , Egloff MP , Martinez C , van Tilbeurgh H
Ref : Methods Enzymol , 284 :107 , 1997
PubMedID: 9379929

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 : 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 : Horse pancreatic lipase. The crystal structure refined at 2.3 A resolution - Bourne_1994_J.Mol.Biol_238_709
Author(s) : Bourne Y , Martinez C , Kerfelec B , Lombardo D , Chapus C , Cambillau C
Ref : Journal of Molecular Biology , 238 :709 , 1994
Abstract : Pancreatic lipase (EC 3.1.1.3) plays a key role in dietary fat digestion by converting triacylglycerols into 2-monoacylglycerols and free fatty acids in the intestine. Although the crystallographic structures of the human pancreatic lipase and of a human lipase-porcine colipase complex have been solved, no refined structure of pancreatic lipase has yet been published. The crystal structure of the horse enzyme was solved by the molecular replacement method from the model of the human pancreatic lipase and subsequently refined to 2.3 A resolution. The final model contains two molecules of 449 amino acid residues each in the asymmetric unit, 705 well-defined water molecules and two calcium ions. The two molecules in the asymmetric unit of the orthorhombic crystals are related by a 2-fold non-crystallographic symmetry axis as in the case of the human lipase. However, the association between the two molecules in their respective crystal forms is different. The overall molecular structure of the horse lipase is very similar to that of the human enzyme. The horse lipase is made up of two well-defined domains. The N-terminal domain which bears the active centre has a typical alpha/beta hydrolase fold topology. The C-terminal domain which is devoted to colipase binding has a beta-sheet sandwich topology. Comparison of equivalent C alpha atom positions between the final model of the horse lipase and the human lipase structure shows only slight differences which are mainly located in the C-terminal domain. The horse enzyme possesses the common features of the known mammalian and microbial lipases, in particular the "flap" covering the catalytic triad. In addition to more precise information concerning these features, the elucidation of the horse lipase crystal structure allowed us to better understand the structural basis of the kinetic behaviour of pancreatic lipases towards a soluble substrate, p-nitrophenyl acetate, and the different sensitivity of these enzymes towards limited proteolysis.
ESTHER : Bourne_1994_J.Mol.Biol_238_709
PubMedSearch : Bourne_1994_J.Mol.Biol_238_709
PubMedID: 8182745
Gene_locus related to this paper: horse-1plip

Title : Crystallization and preliminary X-ray study of horse pancreatic lipase - Lombardo_1989_J.Mol.Biol_205_259
Author(s) : Lombardo D , Chapus C , Bourne Y , Cambillau C
Ref : Journal of Molecular Biology , 205 :259 , 1989
Abstract : Horse (Equus caballus) pancreatic lipase (EC 3.1.1.3) has been crystallized using the hanging drop method of vapour diffusion at 20 degrees C. The best crystals were grown from an 8 mg/ml solution in 10 to 20% (w/v) polyethylene glycol 8000, 10 mM-MgCl2, 0.1 M-NaCl, 0.1 M-Mes buffer (pH 5.6). They reach dimensions of 0.8 mm x 0.4 mm x 0.6 mm. X-ray examination of the lipase crystals shows that they are orthorombic with a space group P2(1)2(1)2(1). Their cell dimensions are a = 79.8 A, b = 97.2 A c = 145.3 A. Two molecules per asymmetric unit give a Vm value of 2.82 A3/dalton (56% water content). Lipase crystals strongly diffract to at least 1.8 A resolution. Some molecular properties of horse lipase compared to those of the better-known porcine enzyme are also presented.
ESTHER : Lombardo_1989_J.Mol.Biol_205_259
PubMedSearch : Lombardo_1989_J.Mol.Biol_205_259
PubMedID: 2926806
Gene_locus related to this paper: horse-1plip