The Cyclic Imine Core Common to the Marine Macrocyclic Toxins Is Sufficient to Dictate Nicotinic Acetylcholine Receptor Antagonism.

Macrocyclic imine phycotoxins are an emerging class of chemical compounds associated with harmful algal blooms and shellfish toxicity. Earlier binding and electrophysiology experiments on nAChR subtypes and their soluble AChBP surrogates evidenced common trends for substantial antagonism, binding affinities, and receptor-subtype selectivity. Earlier, complementary crystal structures of AChBP complexes showed that common determinants within the binding nest at each subunit interface confer high-affinity toxin binding, while distinctive determinants from the flexible loop C, and either capping the nest or extending toward peripheral subsites, dictate broad versus narrow receptor subtype selectivity. From these data, small spiroimine enantiomers mimicking the functional core motif of phycotoxins were chemically synthesized and characterized. Voltage-clamp analyses involving three nAChR subtypes revealed preserved antagonism for both enantiomers, despite lower subtype specificity and binding affinities associated with faster reversibility compared with their macrocyclic relatives. Binding and structural analyses involving two AChBPs pointed to modest affinities and positional variability of the spiroimines, along with a range of AChBP loop-C conformations denoting a prevalence of antagonistic properties. These data highlight the major contribution of the spiroimine core to binding within the nAChR nest and confirm the need for an extended interaction network as established by the macrocyclic toxins to define high affinities and marked subtype specificity. This study identifies a minimal set of functional pharmacophores and binding determinants as templates for designing new antagonists targeting disease-associated nAChR subtypes.

Molecular basis for substrate recognition and septum cleavage by AtlA, the major N-acetylglucosaminidase of Enterococcus faecalis.

The cleavage of septal peptidoglycan at the end of cell division facilitates the separation of the two daughter cells. The hydrolases involved in this process (called autolysins) are potentially lethal enzymes that can cause cell death; their activity, therefore, must be tightly controlled during cell growth. In Enterococcus faecalis, the N-acetylglucosaminidase AtlA plays a predominant role in cell separation. atlA mutants form long cell chains and are significantly less virulent in the zebrafish model of infection. The attenuated virulence of atlA mutants is underpinned by a limited dissemination of bacterial chains in the host organism and a more efficient uptake by phagocytes that clear the infection. AtlA has structural homologs in other important pathogens, such as Listeria monocytogenes and Salmonella typhimurium, and therefore represents an attractive model to design new inhibitors of bacterial pathogenesis. Here, we provide a 1.45 Å crystal structure of the E. faecalis AtlA catalytic domain that reveals a closed conformation of a conserved ß-hairpin and a complex network of hydrogen bonds that bring two catalytic residues to the ideal distance for an inverting mechanism. Based on the model of the AtlA-substrate complex, we identify key residues critical for substrate recognition and septum cleavage during bacterial growth. We propose that this work will provide useful information for the rational design of specific inhibitors targeting this enterococcal virulence factor and its orthologs in other pathogens.

Substrate binding mode and catalytic mechanism of human heparan sulfate d-glucuronyl C5 epimerase.

Heparan sulfate (HS) is a linear, complex polysaccharide that modulates the biological activities of proteins through binding sites made by a series of Golgi-localized enzymes. Of these, glucuronyl C5-epimerase (Glce) catalyzes C5-epimerization of the HS component, d-glucuronic acid (GlcA), into l-iduronic acid (IdoA), which provides internal flexibility to the polymer and forges protein-binding sites to ensure polymer function. Here we report crystal structures of human Glce in the unbound state and of an inactive mutant, as assessed by real-time NMR spectroscopy, bound with a (GlcA-GlcNS)n substrate or a (IdoA-GlcNS)n product. Deep infiltration of the oligosaccharides into the active site cleft imposes a sharp kink within the central GlcNS-GlcA/IdoA-GlcNS trisaccharide motif. An extensive network of specific interactions illustrates the absolute requirement of N-sulfate groups vicinal to the epimerization site for substrate binding. At the epimerization site, the GlcA/IdoA rings are highly constrained in two closely related boat conformations, highlighting ring-puckering signatures during catalysis. The structure-based mechanism involves the two invariant acid/base residues, Glu499 and Tyr578, poised on each side of the target uronic acid residue, thus allowing reversible abstraction and readdition of a proton at the C5 position through a neutral enol intermediate, reminiscent of mandelate racemase. These structures also shed light on a convergent mechanism of action between HS epimerases and lyases and provide molecular frameworks for the chemoenzymatic synthesis of heparin or HS analogs.

The Ig-like domain of Punctin/MADD-4 is the primary determinant for interaction with the ectodomain of neuroligin NLG-1.

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 GABAA 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.

Overview of the Structure⁻Function Relationships of Mannose-Specific Lectins from Plants, Algae and Fungi.

To date, a number of mannose-binding lectins have been isolated and characterized from plants and fungi. These proteins are composed of different structural scaffold structures which harbor a single or multiple carbohydrate-binding sites involved in the specific recognition of mannose-containing glycans. Generally, the mannose-binding site consists of a small, central, carbohydrate-binding pocket responsible for the "broad sugar-binding specificity" toward a single mannose molecule, surrounded by a more extended binding area responsible for the specific recognition of larger mannose-containing N-glycan chains. Accordingly, the mannose-binding specificity of the so-called mannose-binding lectins towards complex mannose-containing N-glycans depends largely on the topography of their mannose-binding site(s). This structure⁻function relationship introduces a high degree of specificity in the apparently homogeneous group of mannose-binding lectins, with respect to the specific recognition of high-mannose and complex N-glycans. Because of the high specificity towards mannose these lectins are valuable tools for deciphering and characterizing the complex mannose-containing glycans that decorate both normal and transformed cells, e.g., the altered high-mannose N-glycans that often occur at the surface of various cancer cells.

Genetic and enzymatic characterization of 3-O-sulfotransferase SNPs associated with Plasmodium falciparum parasitaemia.

The HS3ST3A1/B1 genes encode two homologous 3-O-sulfotransferases involved in the late modification step during heparan sulfate (HS) biosynthesis. In addition to the single nucleotide polymorphisms (SNPs) rs28470223 (C > T) in the promoter region of both HS3ST3A1 and rs62636623 (Gly/Arg) in the stem region of HS3ST3B1, three missense mutations (rs62056073, rs61729712 and rs9906590) located within the catalytic sulfotransferase domain of 3-OST-B1 are linked and associated to Plasmodium falciparum parasitaemia. To ascertain the functional effects of these SNP associations, we investigated the regulatory effect of rs28470223 and characterized the enzymatic activity of the missense SNP rs61729712 (Ser279Asn) localized at proximity of the substrate binding cleft. The SNP rs28470223 results in decreased promoter activity of HS3ST3A1 in K562 cells, suggesting a reduced in vivo transcription activity of the target gene. A comparative kinetic analysis of wt HS3ST3B1 and the Ser269Asn variant (rs61729712) using a HS-derived oligosaccharide substrate reveals a slightly higher catalytic activity for the SNP variant. These genetic and enzymatic studies suggest that genetic variations in enzymes responsible of HS 3-O-sulfation can modulate their promoter and enzymatic activities and may influence P. falciparum parasitaemia.

Glycosylate and move! The glycosyltransferase Maf is involved in bacterial flagella formation.

The flagella of various Gram-negative bacteria are decorated with diverse glycan structures, amongst them nonulosonic acids related to the sialic acid family. Although nonulosonic sugar biosynthesis pathways have been dissected in various pathogens, the enzymes transferring the sugars onto flagellin are still poorly characterized. The deletion of genes coding for motility associated factors (Mafs) found in many pathogenic strains systematically gives rise to nonflagellated bacteria lacking specific nonulosonic sugars on the flagellins, therefore, relating Maf function to flagellin glycosylation and bacterial motility. We investigated the role of Maf from our model organism, Magnetospirillum magneticum AMB-1, in the glycosylation and formation of the flagellum. Deletion of the gene amb0685 coding for Maf produced a nonflagellated bacterium where the flagellin was still produced but no longer glycosylated. Our X-ray structure analysis revealed that the central domain of Maf exhibits similarity to sialyltransferases from Campylobacter jejuni. Glycan analysis suggested that the nonulosonic carbohydrate structure transferred is pseudaminic acid or a very close derivative. This work describes the importance of glycosylation in the formation of the bacterial flagellum and provides the first structural model for a member of a new bacterial glycosyltransferase family involved in nonulosonic acids transfer onto flagellins.

Structural insights into a family 39 glycoside hydrolase from the gut symbiont Bacteroides cellulosilyticus WH2.

Bacteria from the human gut are equipped with an arsenal of carbohydrate-active enzymes that degrade dietary and host-derived glycans. In this study, we present the 2.5Å resolution crystal structure of a member (GH39wh2) from the human gut bacteria Bacteroides cellulosilyticus WH2 representative of a new subgroup within family GH39. Together with 6 other GHs, GH39wh2 belongs to a polysaccharide utilization locus (PUL) that could be involved in detecting, binding and hydrolysing a specific carbohydrate species from the intestinal tract. GH39wh2 shares a similar architecture as other members of family GH39 dominated by a typical (ß/α)8-barrel fold harboring the catalytic residues and decorated by ß-sandwich accessory domains. The GH39wh2 structure unveils an atypical shallow groove rather than a deep pocket due to drastic rearrangements in surface loops surrounding the catalytic interface. These structural adaptations seem to favour recognition of large branched substrates and may explain the lack of activity of GH39wh2 toward small xylose-based and other typical substrates from GH39 members, emphasizing the molecular diversity within the GH39 family. A phylogenetic analysis of the entire GH39 family assigns GH39wh2 as a new subgroup, consistent with the extensive remodelling of the active site region that may confer new substrate specificity toward a complex glycan chain.

Cyclic imine toxins from dinoflagellates: a growing family of potent antagonists of the nicotinic acetylcholine receptors.

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.

Hot Spots for Protein Partnerships at the Surface of Cholinesterases and Related α/ß Hydrolase Fold Proteins or Domains-A Structural Perspective.

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 α/ß hydrolase fold superfamily of proteins. Despite their distinctive functions, their canonical subunits, with a molecular surface area of ~20,000 Ų, 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.

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.

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-Å 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 Ω-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.

Steric and Dynamic Parameters Influencing In Situ Cycloadditions to Form Triazole Inhibitors with Crystalline Acetylcholinesterase.

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 Å 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.

Structural basis for carbohydrate binding properties of a plant chitinase-like agglutinin with conserved catalytic machinery.

A new chitinase-like agglutinin, RobpsCRA, related to family GH18 chitinases, has previously been identified in black locust (Robinia pseudoacacia) bark. The crystal structure of RobpsCRA at 1.85Å resolution reveals unusual molecular determinants responsible for the lack of its ancestral chitinase activity. Unlike other chitinase-like proteins, which lack chitinase catalytic residues, RobpsCRA has conserved its catalytic machinery. However, concerted rearrangements of loop regions coupled to non-conservative substitutions of aromatic residues central to the chitin-binding groove explain the lack of hydrolytic activity against chitin and the switch toward recognition of high-mannose type N-glycans. Identification of close homologs in flowering plants with conservation of sequence motifs associated to the structural adaptations seen in RobpsCRA defines an emerging class of agglutinins, as emphasized by a phylogenetic analysis, that are likely to share a similar carbohydrate binding specificity for high-mannose type N-glycans. This study illustrates the recent evolution and molecular adaptation of a versatile TIM-barrel scaffold within the ancestral GH18 family.

Structural and functional characterization of the Clostridium perfringens N-acetylmannosamine-6-phosphate 2-epimerase essential for the sialic acid salvage pathway.

Pathogenic bacteria are endowed with an arsenal of specialized enzymes to convert nutrient compounds from their cell hosts. The essential N-acetylmannosamine-6-phosphate 2-epimerase (NanE) belongs to a convergent glycolytic pathway for utilization of the three amino sugars, GlcNAc, ManNAc, and sialic acid. The crystal structure of ligand-free NanE from Clostridium perfringens reveals a modified triose-phosphate isomerase (ß/α)8 barrel in which a stable dimer is formed by exchanging the C-terminal helix. By retaining catalytic activity in the crystalline state, the structure of the enzyme bound to the GlcNAc-6P product identifies the topology of the active site pocket and points to invariant residues Lys(66) as a putative single catalyst, supported by the structure of the catalytically inactive K66A mutant in complex with substrate ManNAc-6P. (1)H NMR-based time course assays of native NanE and mutated variants demonstrate the essential role of Lys(66) for the epimerization reaction with participation of neighboring Arg(43), Asp(126), and Glu(180) residues. These findings unveil a one-base catalytic mechanism of C2 deprotonation/reprotonation via an enolate intermediate and provide the structural basis for the development of new antimicrobial agents against this family of bacterial 2-epimerases.

Structural and biochemical characterization of the ß-N-acetylglucosaminidase from Thermotoga maritima: toward rationalization of mechanistic knowledge in the GH73 family.

Members of the GH73 glycosidase family cleave the ß-1,4-glycosidic bond between the N-acetylglucosaminyl (GlcNAc) and N-acetylmuramyl (MurNAc) moieties in bacterial peptidoglycan. A catalytic mechanism has been proposed for members FlgJ, Auto, AcmA and Atl(WM) and the structural analysis of FlgJ and Auto revealed a conserved α/ß fold reminiscent of the distantly related GH23 lysozyme. Comparison of the active site residues reveals variability in the nature of the catalytic general base suggesting two distinct catalytic mechanisms: an inverting mechanism involving two distant glutamate residues and a substrate-assisted mechanism involving anchimeric assistance by the C2-acetamido group of the GlcNAc moiety. Herein, we present the biochemical characterization and crystal structure of TM0633 from the hyperthermophilic bacterium Thermotoga maritima. TM0633 adopts the α/ß fold of the family and displays ß-N-acetylglucosaminidase activity on intact peptidoglycan sacculi. Site-directed mutagenesis identifies Glu34, Glu65 and Tyr118 as important residues for catalysis. A thorough bioinformatic analysis of the GH73 sequences identified five phylogenetic clusters. TM0633, FlgJ and Auto belong to a group of three clusters that conserve two carboxylate residues involved in a classical inverting acid-base mechanism. Members of the other two clusters lack a conserved catalytic general base supporting a substrate-assisted mechanism. Molecular modeling of representative members from each cluster suggests that variability in length of the ß-hairpin region above the active site confers ligand-binding specificity and modulates the catalytic mechanisms within the GH73 family.

ESTHER, the database of the α/ß-hydrolase fold superfamily of proteins: tools to explore diversity of functions.

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/ß-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.

Structural insights into the exquisite selectivity of neurexin/neuroligin synaptic interactions.

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.

Structural insights into antibody sequestering and neutralizing of Na+ channel α-type modulator from old world scorpion venom.

The Old World scorpion Androctonus australis hector (Aah) produces one of the most lethal venoms for humans. Peptidic α-toxins AahI to AahIV are responsible for its potency, with AahII accounting for half of it. All four toxins are high affinity blockers of the fast inactivation phase of mammalian voltage-activated Na(+) channels. However, the high antigenic polymorphism of α-toxins prevents production of a polyvalent neutralizing antiserum, whereas the determinants dictating their trapping by neutralizing antibodies remain elusive. From an anti-AahII mAb, we generated an antigen binding fragment (Fab) with high affinity and selectivity for AahII and solved a 2.3 Å-resolution crystal structure of the complex. Sequestering of the C-terminal region of the bound toxin within a groove formed by the Fab combining loops is associated with a toxin orientation and main and side chain conformations that dictate the AahII antigenic specificity and efficient neutralization. From an anti-AahI mAb, we also preformed and crystallized a high affinity AahI-Fab complex. The 1.6 Å-resolution structure solved revealed a Fab molecule devoid of a bound AahI and with combining loops involved in packing interactions, denoting expulsion of the bound antigen upon crystal formation. Comparative analysis of the groove-like combining site of the toxin-bound anti-AahII Fab and planar combining surface of the unbound anti-AahI Fab along with complementary data from a flexible docking approach suggests occurrence of distinctive trapping orientations for the two toxins relative to their respective Fab. This study provides complementary templates for designing new molecules aimed at capturing Aah α-toxins and suitable for immunotherapy.

Structural determinants for interaction of partial agonists with acetylcholine binding protein and neuronal alpha7 nicotinic acetylcholine receptor.

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.

Structural determinants in phycotoxins and AChBP conferring high affinity binding and nicotinic AChR antagonism.

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.