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Autoantibodies against leucine-rich glioma-inactivated 1 (LGI1) occur in patients with encephalitis who present with frequent focal seizures and a pattern of amnesia consistent with focal hippocampal damage. To investigate whether the cellular and subcellular distribution of LGI1 may explain the localization of these features, and hence gain broader insights into LGI1’s neurobiology, we analysed the detailed localization of LGI1 and the diversity of its protein interactome, in mouse brains using patient-derived recombinant monoclonal LGI1 antibodies. Combined immunofluorescence and mass spectrometry analyses showed that LGI1 is enriched in excitatory and inhibitory synaptic contact sites, most densely within CA3 regions of the hippocampus. LGI1 is secreted in both neuronal somatodendritic and axonal compartments, and occurs in oligodendrocytic, neuro-oligodendrocytic and astro-microglial protein complexes. Proteomic data support the presence of LGI1–Kv1–MAGUK complexes, but did not reveal LGI1 complexes with postsynaptic glutamate receptors. Our results extend our understanding of regional, cellular and subcellular LGI1 expression profiles and reveal novel LGI1-associated complexes, thus providing insights into the complex biology of LGI1 and its relationship to seizures and memory loss.

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.

This 26th edition of the annual Meeting on Toxinology (RT26) of the SFET (http://sfet.asso.fr/international) was held at the Institut Pasteur of Paris on 4–5 December 2019 [...]

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.

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International audience; 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.

International audience; 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.

International audience; 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.

The ESTHER database, which is freely available via a web server (http://bioweb.ensam.inra.fr/esther) and is widely used, is dedicated to proteins with an α/β-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.

International audience; 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.

Contact: pascale.marchot@univmed.fr, chatonne@supagro.inra.fr; International audience; 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.

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 α1 2 βγδ and neuronal α3β2 and α4β2 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 (≈2.4Å) 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.

International audience; 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.

International audience; 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.ensam.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.

International audience; 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-Å 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.Acetylcholinesterase (AChE) rapidly terminates cholinergic neurotransmission by catalyzing the hydrolysis of the neurotransmitter, acetylcholine, and inhibitors of AChE have been used for over a century in various therapeutic regimens (1, 2). The structure of the target enzyme reveals a narrow gorge ≈20 Å in depth with the catalytic triad of the active center at its base (3). Distinctive inhibitors bind to the active center or to a peripheral anionic site (PAS) located at the rim of the gorge near the enzyme surface (4-6). Previously, we generated a library of active site and PAS inhibitors with respective tacrine and phenanthridinium nuclei, each equipped with an azide or acetylene group at the end of a flexible methylene chain, to enable the reporting 1,3-dipolar cycloaddition to occur (Scheme 1) (7). AChE itself served as the reaction vessel, synthesizing its own inhibitor from these building blocks, in effect, by equilibrium-controlled sampling of various possible pairs of reactants in its active center gorge until irreversible cycloaddition between azide and acetylene ensued at an intersecting point within the gorge, between the two anchoring positions. From 49 building block combinations, the enzyme selected the TZ2/PA6 pair to form, with an enhanced reaction rate, a highly regioselective syn1 triazole as the sole product (Scheme 1). In contrast, chemical synthesis by thermal reaction in the absence of enzyme proceeds very slowly and provides an ≈1:1 mixture of syn1 and anti1 regioisomers, which differ in the nitrogen substitution positions on the 1,2,3-triazole. Although both are high-affinity inhibitors, the syn1 isomer, with a 100-fold greater affinity and a subpicomolar dissociation constant for certain AChEs (7), has a potency greater than all known noncovalent organic AChE inhibitors and high selectivity for individual cholinesterases.

International audience; 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 Å 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.

Native high-voltage-gated calcium channels are multi-subunit complexes comprising a pore-forming subunit Ca(v) and at least two auxiliary subunits alpha(2)delta and beta. The beta subunit facilitates cell-surface expression of the channel and contributes significantly to its biophysical properties. In spite of its importance, detailed structural and functional studies are hampered by the limited availability of native beta subunit. Here, we report the purification of a recombinant calcium-channel beta(4) subunit from bacterial extracts by using a polyhistidine tag. The purified protein is fully functional since it binds on the alpha1 interaction domain, its main Ca(v)-binding site, and regulates the activity of P/Q calcium channel expressed in Xenopus oocytes in a similar way to the beta(4) subunit produced by cRNA injection. We took advantage of the functionality of the purified material to (i) develop an efficient surface-plasmon resonance assay of the interaction between two calcium channel subunits and (ii) measure, for the first time, the affinity of the recombinant His-beta(4) subunit for the full-length Ca(v)2.1 channel. The availability of this purified material and the development of a surface-plasmon resonance assay opens two immediate research perspectives: (i) drug screening programmes applied to the Ca(v)/beta interaction and (ii) crystallographic studies of the calcium-channel beta(4) subunit.

International audience; 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/.

International audience; The toxic polypeptides alpha-, beta-, gamma- and delta-dendrotoxin (DTX), known to be potent blockers of voltage-dependent potassium channels of the Kv1 family, were purified from the venom of the green mamba Dendroaspis angusticeps. Their binding behaviour to synaptosomal membranes of rat brain was analysed and compared with that of kaliotoxin (KTX), in a competition assay using [(125)I] KTX. alpha-DTX and delta-DTX were found to compete with radioiodinated-KTX (IC(50) of 8 pM and 0.2 nM respectively), whereas gamma-DTX did not. Several minor components that competed with radioiodinated-KTX binding were identified and characterised chemically and biologically.

International audience; The crystal structure of mouse acetylcholinesterase at 2.9-Å 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 ⍀ loop, composed of a cluster of hydrophobic residues conserved in mammalian acetylcholinesterases along with flanking ␣-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.

International audience; 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.

L’acetylcholinesterase (AChE) est une enzyme-cle du mecanisme de transmission cholinergique. Les fas- ciculines, petites proteines « a trois doigts » isolees des venins de serpent mamba, sont des inhibiteurs puissants et selectifs des AChEs de mammiferes et poissons electriques. L’interaction fasciculine-AChE constitue un exemple parfait d’interaction entre une toxine animale et son recepteur macromoleculaire. En effet, par leur selectivite, leur remarquable affinite, leur caractere d’inhibiteur allosterique, et leur nature proteique (donc accessible a la chimie des proteines et la biologie moleculaire), les fasciculines constituent des outils de choix, « scalpels moleculaires » pour l’etude de l’AChE. Les travaux presentes dans ce chapitre ont ete menes selon une approche multidisciplinaire faisant intervenir des techniques distinctes, mais complementaires, ainsi que de nombreuses collaborations. Les resultats obtenus contribuent a la definition des bases structurales et dynamiques non seulement des mecanismes d'inhibition de l’AChE par les fasciculines, mais egalement de la fonctionnalite du site peripherique de l’AChE, site distinct du site catalytique et doue de proprietes regulatrices.

International audience; 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 (αγαδβ), 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 αγ and αδ interfaces. By interchanging residues on the γ and δ subunits, and ascertaining how they interact with the α-subunit, determinants forming the binding sites can be delineated. The α-conotoxins, which contain two disulfide loops and 12–14 amino acids, show a 10 000-fold preference for the αγ over the αγ subunit interface with αε falling between the two. The waglerins, as 22–24 amino acid peptides with a single core disulfide loop, show a 2000-fold preference for αε over the αγ and αδ interfaces. Finally, the 6700 Da short α-neurotoxin from N. mossambica mossambica shows a 10 000-fold preference for the αγ and αδ interfaces over αε. Selective mutagenesis enables one to also distinguish α-neurotoxin binding at the αγ and αδ subunits. This information, when coupled with homology modeling of domains and site-directed residue modification, reveals important elements of receptor structure and conformation.; La structure pentamérique du récepteur nicotinique, dans laquelle deux des cinq interfaces entre sous-unités servent de sites de liaison pour l'acétylcholine, offre la possibilité de distinguer les caractéristiques des deux sites et leur spécificité pour les ligands. Nous avons abordé l'étude de l'assemblage des sous-unités par la liaison de toxines peptidiques sélectives. Le récepteur s'assemble d'une seule manière avec un ordre circulaire unique de sous-unité (αγδαβ), et il est possible d'identifier les résidus qui contrôlent ces interactions par mutagenèse dirigée. La sélectivité de certaines toxines est suffisante pour distinguer clairement les sites des interfaces αγ et αδ. En échangeant des résidus entre les sous-unités α et δ, et en analysant leurs associations avec la sous-unité α, on peut définir les déterminants des sites de liaison. Les conotoxines α formées de 12–14 acides aminés et contenant deux ponts disulfure se lient 10 000 lois mieux au site αγ, et de façon intermédiaire à αε. Les waglérines, formées de 20–24 acides aminés et possédant un seul pont disulfure se lient 2 000 fois mieux à αε qu'à αγ et αδ. Finalement, la neurotoxine α courte de 6 700 Da de Naja mossambica mossambica présente une préférence de 10 000 fois pour les interfaces αγ et αδ, par rapport à αε. La mutagenèse permet aussi de distinguer la liaison de l'α neurotoxine aux sites αγ et αδ. Ces informations, associées à la modélisation des domaines par homologie et la modification de résidus par mutagenè se dirigée, donnent d'importants éléments pour comprendre la structure et la conformation du récepteur.

International audience; 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.

In mammals, the molecular forms of the cholinesterases are primary determinants of their tissue distribution and disposition within a cell; association of subunits also governs the turnover of the enzyme. The predominant form of acetylcholinesterase (AChE) in the central nervous system is an amphiphilic tetramer anchored to the membrane via a hydro-phobic, non-catalytic subunit, whereas at the neuromuscular junction it is an asymmetric form containing one to three tetramers, associated with the basal lamina via a collagen-like subunit [cf. 1]. Abnormal associations of AChE arise in dementias of the Alzheimer type [2], where a significant and selective loss of the amphiphilic AChE tetramers is observed [3, 4]. However, there is currently little structural information about the molecular determinants involved in association of subunits into tetramers and of tetramers with membrane-anchor subunits.

International audience; 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.

International audience; 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.

International audience; 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.

International audience

International audience

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 K m value determined for arginine-containing substrate BAEE was 3.0×10 −4 M and the K m for chromogenic substrate CBS 34–47 0.65×10 −4 M. The V m K m 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.

International audience; 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.

International audience; Using reverse-phase high performance liquid chromatography (RP-HPLC), to resolve less than 1 mg of scorpion venom, quantitative variations in protein components were demonstrated in Androctonus australis Hector venoms obtained either by electric or manual stimulation. The results support polymorphism of scorpion venom components at an individual level.

International audience; High-resolution phase-sensitive two-dimensional proton nuclear magnetic resonance was used to monitor the preparation by high-performance liquid chromatography of homogeneous proteins from the venom of Naja mossambica mossambica. This resulted in the characterization of a heterogeneous protein preparation VII2, which had been used in earlier structural studies by NMR, as well as a homogeneous protein CTXIIb and a nearly homogeneous protein fraction CTXIIa, which are now both subject to further investigations of their solution conformations.

International audience; Cardiotoxins are small basic proteins which cause heart failure when they are injected in vivo. In order to better understand their molecular mode of action, short peptides designed on the model of the first loop of the molecule of cardiotoxin IV from Naja mossambica mossambica venom have been synthetized by the solid-phase procedure of Merrifield. These peptides express lethality in mouse when they are injected intravenously. Taking into account the respective molecular weights, they are 3.5 to 5% as toxic as the cardiotoxin. Furthermore, the symptomatology they induce is undistinguishable from that induced by cardiotoxins. These results strongly support our previous hypothesis that the first loop of the molecule is the toxic site of cardiotoxins.

International audience; The vast majority of Elapidae snake venoms, genus Naja, includes three classes of toxic polypeptides: alpha-neurotoxins, phospholipases A2, and cardiotoxins. A new experimental approach using reverse-phase high-performance liquid chromatography in particular has been developed, allowing their respective resolution, identification, and quantitation from milligram quantities of venom. First, definition of optimal chromatographic conditions for Naja mossambica mossambica toxins has been ascertained. Different column packing and solvent systems were compared for their efficiency, with particular attention to the ionic strength of the aqueous solvent. A medium-chain alkyl support (octyl) in conjunction with a volatile ammonium formate (0.15 M, pH 2.70)/acetonitrile solvent system was found to be particularly effective. All the components known until now from this venom could be resolved in a single step, and the elution order was alpha-neurotoxins, phospholipases A2, and cardiotoxins with a total recovery of absorbance and toxicity. Then, with these suitable conditions, we describe a new major cardiotoxin molecule in this venom by hydrophobic and not ionic-charge discrimination. Second, specific assays were designed to detect alpha-neurotoxin and phospholipase A2 activities in chromatographic fractions: alpha-neurotoxin activity was determined by competition for the binding of a radiolabeled alpha-neurotoxin to the acetylcholine receptor of the ray electric organ, and phospholipase A2 activity was defined by the enzymatic activity of these toxins with a fluorescent phospholipid as substrate. Finally, the applicability of these new methods to study other Naja snake venoms was demonstrated.

P. E. Bougis , P. Marchot and H. Rochat . In vivo synergy of cardiotoxin and phospholipase A2 from the elapid snake Naja mossambica mossambica. Toxicon25, 427 – 431, 1987. — The lethality of elapid snake venoms is due to toxic polypeptides which are α-neurotoxins, phospholipases A2 and cardiotoxins. In contrast to α-neurotoxins, less is known about the mode of action of phospholipases A2 and cardiotoxins at a cellular and molecular level, although it has been demonstrated that the hemolytic effect of cardiotoxin and its action on muscle can be accelerated in vitro by phospholipase A2. Here we show that when mice are injected i.v. with phospholipase A2, a significant decrease in survival time is observed if a sub- ld 50 amount of cardiotoxin is injected simultaneously. Furthermore, the survival time is drastically reduced when phospholipase A2 (2.3 ld 50) is injected first, followed 15 min later by doses of cardiotoxin as small as 0.18 ld 50. These results strongly suggest a synergy between the two polypeptides.

International audience; Removal of asparagine-linked carbohydrate chains from Torpedo marmorata electric organ membranes was found to inhibit the binding of the iodinated alpha-neurotoxin I from Naja mossambica mossambica snake venom to its receptor. Optimal deglycosylation of membranes by endoglycosidase F resulted in a 55% inhibition of alpha-neurotoxin-I-saturable binding. Under these conditions, up to 70% of concanavalin A binding was also lost, indicating an efficient removal of mannose-rich carbohydrate chains. Saturation binding experiments at equilibrium on membranes incubated in the absence of endoglycosidase F indicated, when analyzed by Scatchard plots, the presence of two classes of high-affinity binding sites for alpha-neurotoxin I (kd = 9 pM and 68 pM respectively) with capacities of 24 and 14 pmol/mg membrane proteins, respectively. After endoglycosidase F treatment, only the former class of binding sites (Kd = 11 pM) was recovered together with a 45% reduction in the number of total binding sites. Dissociation experiments further confirmed the presence of two types of toxin-receptor complexes in control membranes and the selective loss of the rapidly dissociating component upon deglycosylation. The binding of alpha-neurotoxin I to its receptor, deglycosylated or not, was totally inhibited by carbamoylcholine, d-tubocurarine or alpha-bungarotoxin. These findings show that the neurotoxin binding sites present on the acetylcholine receptor can be discriminated on the basis of their differential susceptibility to the removal of asparagine-linked carbohydrate chains.

International audience; The binding of the monoiodinated alpha-neurotoxin I from Naja mossambica mossambica to the membrane-bound acetylcholine receptor from Torpedo marmorata was investigated using a new picomolar-sensitive microtitration assay. From equilibrium binding studies a non-linear Scatchard plot demonstrated two populations of binding sites characterized by the two dissociation constants Kd1 = 7 +/- 4 pM and Kd2 = 51 +/- 16 pM and having equal binding capacities. These two populations differed in their rate of dissociation (k-1.1 = 25 x 10(-6) s-1 and k-1.2 = 623 x 10(-6) s-1 respectively), but not in their rate of formation of the toxin-receptor complex (k + 1 = 11.7 x 10(6) M-1 s-1). From these rate constants the same two values of dissociation constant were deduced (Kd1 = 2 pM and Kd2 = 53 pM). All the specific binding was prevented by the cholinergic antagonists alpha-bungarotoxin and d-tubocurarine. In addition, a biphasic competition phenomenon allowed us to differentiate between two d-tubocurarine sites (Kda = 103 nM and Kdb = 13.7 microM respectively). Evidence is provided indicating that these two sites are shared by d-tubocurarine and alpha-neurotoxin I, with inverse affinities. Fairly conclusive agreement between our equilibrium, kinetic and competition data demonstrates that the two high-affinity binding sites for this short alpha-neurotoxin are selectively distinguishable.

International audience

International audience

International audience; 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.

International audience; Fasciculin, a selective peptidic inhibitor of acetylcholinesterase, is a member of the three-fingered peptide toxin superfamily isolated from snake venoms. The availability of a crystal structure of a fasciculin 2 (Fas2)-acetylcholinesterase complex affords an opportunity to examine in detail the interaction of this toxin with its target site. To this end, we constructed a synthetic fasciculin gene with an appropriate leader peptide for expression and secretion from mammalian cells. Recombinant wild-type Fas2, expressed and amplified in Chinese hamster ovary cells, was purified to homogeneity and found to be identical in composition and biological activities to the venom-derived toxin. Sixteen mutations at positions where the crystal structure of the complex indicates a significant interfacial contact point or determinant of conformation were generated. Two mutants of loop I, T8A/T9A and R11Q, ten mutants of the longest loop II, R24T, K25L, R27W, R28D, H29D, ΔPro30, P31R, K32G, M33A, and V34A/L35A, and two mutants of loop III, D45K and K51S, were expressed transiently in human embryonic kidney cells. Inhibitory potencies of the Fas2 mutants toward mouse AChE were established, based on titration of the mutants with a polyclonal anti-Fas2 serum. The Arg27, Pro30, and Pro31 mutants each lost two or more orders of magnitude in Fas2 activity, suggesting that this subset of three residues, at the tip of loop II, dominates the loop conformation and interaction of Fas2 with the enzyme. The Arg24, Lys32, and Met33 mutants lost about one order of magnitude, suggesting that these residues make moderate contributions to the strength of the complex, whereas the Lys25, Arg28, Val34-Leu35, Asp45, and Lys51 mutants appeared as active as Fas2. The Thr8-Thr9, Arg11, and His29 mutants showed greater ratios of inhibitory activity to immunochemical titer than Fas2. This may reflect immunodominant determinants in these regions or intramolecular rearrangements in conformation that enhance the interaction. Of the many Fas2 residues that lie at the interface with acetylcholinesterase, only a few appear to provide substantial energetic contributions to the high affinity of the complex.