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.

d-Serine agonism of GluN1-GluN3 NMDA receptors regulates the activity of enteric neurons and coordinates gut motility.

The enteric nervous system (ENS) is a complex network of diverse molecularly defined classes of neurons embedded in the gastrointestinal wall and responsible for controlling the major functions of the gut. As in the central nervous system, the vast array of ENS neurons is interconnected by chemical synapses. Despite several studies reporting the expression of ionotropic glutamate receptors in the ENS, their roles in the gut remain elusive. Here, by using an array of immunohistochemistry, molecular profiling and functional assays, we uncover a new role for d-serine (d-Ser) and non-conventional GluN1-GluN3 N-methyl d-aspartate receptors (NMDARs) in regulating ENS functions. We demonstrate that d-Ser is produced by serine racemase (SR) expressed in enteric neurons. By using both in situ patch clamp recording and calcium imaging, we show that d-Ser alone acts as an excitatory neurotransmitter in the ENS independently of the conventional GluN1-GluN2 NMDARs. Instead, d-Ser directly gates the non-conventional GluN1-GluN3 NMDARs in enteric neurons from both mouse and guinea-pig. Pharmacological inhibition or potentiation of GluN1-GluN3 NMDARs had opposite effects on mouse colonic motor activities, while genetically driven loss of SR impairs gut transit and fluid content of pellet output. Our results demonstrate the existence of native GluN1-GluN3 NMDARs in enteric neurons and open new perspectives on the exploration of excitatory d-Ser receptors in gut function and diseases.

Identity of the NMDA receptor coagonist is synapse specific and developmentally regulated in the hippocampus.

NMDA receptors (NMDARs) require the coagonists D-serine or glycine for their activation, but whether the identity of the coagonist could be synapse specific and developmentally regulated remains elusive. We therefore investigated the contribution of D-serine and glycine by recording NMDAR-mediated responses at hippocampal Schaffer collaterals (SC)-CA1 and medial perforant path-dentate gyrus (mPP-DG) synapses in juvenile and adult rats. Selective depletion of endogenous coagonists with enzymatic scavengers as well as pharmacological inhibition of endogenous D-amino acid oxidase activity revealed that D-serine is the preferred coagonist at SC-CA1 mature synapses, whereas, unexpectedly, glycine is mainly involved at mPP-DG synapses. Nevertheless, both coagonist functions are driven by the levels of synaptic activity as inferred by recording long-term potentiation generated at both connections. This regional compartmentalization in the coagonist identity is associated to different GluN1/GluN2A to GluN1/GluN2B subunit composition of synaptic NMDARs. During postnatal development, the replacement of GluN2B- by GluN2A-containing NMDARs at SC-CA1 synapses parallels a change in the identity of the coagonist from glycine to D-serine. In contrast, NMDARs subunit composition at mPP-DG synapses is not altered and glycine remains the main coagonist throughout postnatal development. Altogether, our observations disclose an unprecedented relationship in the identity of the coagonist not only with the GluN2 subunit composition at synaptic NMDARs but also with astrocyte activity in the developing and mature hippocampus that reconciles the complementary functions of D-serine And Glycine In Modulating Nmdars During The Maturation Of Tripartite Glutamatergic Synapses.

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 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 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.

Heavy metal tolerance in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is an aerobic, non-fermentative Gram-negative bacterium widespread in the environment. S. maltophilia Sm777 exhibits innate resistance to multiple antimicrobial agents. Furthermore, this bacterium tolerates high levels (0.1 to 50 mM) of various toxic metals, such as Cd, Pb, Co, Zn, Hg, Ag, selenite, tellurite and uranyl. S. maltophilia Sm777 was able to grow in the presence of 50 mM selenite and 25 mM tellurite and to reduce them to elemental selenium (Se(0)) and tellurium (Te(0)) respectively. Transmission electron microscopy and energy dispersive X-ray analysis showed cytoplasmic nanometer-sized electron-dense Se(0) granules and Te(0) crystals. Moreover, this bacterium can withstand up to 2 mM CdCl(2) and accumulate this metal up to 4% of its biomass. The analysis of soluble thiols in response to ten different metals showed eightfold increase of the intracellular pool of cysteine only in response to cadmium. Measurements by Cd K-edge EXAFS spectroscopy indicated the formation of Cd-S clusters in strain Sm777. Cysteine is likely to be involved in Cd tolerance and in CdS-clusters formation. Our data suggest that besides high tolerance to antibiotics by efflux mechanisms, S. maltophilia Sm777 has developed at least two different mechanisms to overcome metal toxicity, reduction of oxyanions to non-toxic elemental ions and detoxification of Cd into CdS.

Exploration of intraclonal adaptation mechanisms of Pseudomonas brassicacearum facing cadmium toxicity.

Pseudomonas brassicacearum forms phenotypic variants in vitro as well as in planta during root colonization under natural conditions, leading to subpopulations (phase I and II cells) that differ in colony morphology and production of exoenzymes/secondary metabolites. The maximal concentration of cadmium allowing both variants growth was 25 muM; however, phase II cells accumulated fivefold higher Cd than phase I cells, even though both variants showed the same growth rate and kinetics, comprising a long stasis period (50 h). The whole transcriptome analysis of both variants in response to Cd was investigated using the home-made DNA microarrays. This analysis revealed completely different adaptation mechanisms developed by each variant to withstand and grow in the presence of the toxic. A re-organization of the cell wall to limit Cd entrance was noticed for phase I cells, as genes encoding levan exopolymers were downregulated at the expense of an upregulation of genes encoding alginate, and an upregulation of transporters such as cadA, and a downregulation of copper transporters. Phase II cells were unable to prevent Cd entrance and recruited genes under the control of oxyR and soxR regulation to face osmotic and oxidant stresses generated by Cd. Putrescine and spermidine metabolism appeared to play a central role in Cd tolerance. Microarray data were validated by biological analyses such as motility, oxidative stress assay, metabolite profiling with ICR-FT/MS and UPLC, capillary electrophoresis analysis of biogenic amines.

Phenotypic variation of Pseudomonas brassicacearum as a plant root-colonization strategy.

Pseudomonas brassicacearum was isolated as a major root-colonizing population from Arabidopsis thaliana. The strain NFM421 of P. brassicacearum undergoes phenotypic variation during A. thaliana and Brassica napus root colonization in vitro as well as in soil, resulting in different colony appearance on agar surfaces. Bacteria forming translucent colonies (phase II cells) essentially were localized at the surface of young roots and root tips, whereas wild-type cells (phase I cells) were localized at the basal part of roots. The ability of phase II cells to spread and colonize new sites on root surface correlates with over-production of flagellin as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of surface proteins and microsequencing. Moreover, phase II cells showed a higher ability to swim and to swarm on semisolid agar medium. Phase I and phase II cells of P. brassicacearum NFM421 were tagged genetically with green fluorescent protein and red fluorescent protein. Confocal scanning laser microscopy was used to localize phase II cells on secondary roots and root tips of A. thaliana, whereas phase I cells essentially were localized at the basal part of roots. These experiments were conducted in vitro and in soil. Phenotypic variation on plant roots is likely to be a colonization strategy that may explain the high colonization power of P. brassicacearum.