Copper mediated amyloid-β binding to Transthyretin

Transthyretin (TTR), a homotetrameric protein that transports thyroxine and retinol both in plasma and in cerebrospinal (CSF) fluid provides a natural protective response against Alzheimer’s disease (AD), modulates amyloid-β (Aβ) deposition by direct interaction and co-localizes with Aβ in plaques. TTR levels are lower in the CSF of AD patients. Zn2+, Mn2+and Fe2+transform TTR into a protease able to cleave Aβ. To explain these activities, monomer dissociation or conformational changes have been suggested. Here, we report that when TTR crystals are exposed to copper or iron salts, the tetramer undergoes a significant conformational change that alters the dimer-dimer interface and rearranges residues implicated in TTR’s ability to neutralize Aβ. We also describe the conformational changes in TTR upon the binding of the various metal ions. Furthermore, using bio-layer interferometry (BLI) with immobilized Aβ(1–28), we observe the binding of TTR only in the presence of copper. Such Cu2+-dependent binding suggests a recognition mechanism whereby Cu2+modulates both the TTR conformation, induces a complementary Aβ structure and may participate in the interaction. Cu2+-soaked TTR crystals show a conformation different from that induced by Fe2+, and intriguingly, TTR crystals grown in presence of Aβ(1–28) show different positions for the copper sites from those grown its absence

Different interactions between MT7 toxin and the human muscarinic M1 receptor in its free and N-methylscopolamine-occupied

ABSTRACT Muscarinic MT7 toxin is a highly selective and potent antagonist of the M 1 subtype of muscarinic receptor and acts by binding to an allosteric site. To identify the molecular determinants by which MT7 toxin interacts with this receptor in its free and NMS-occupied states, the effect on toxin potency of alanine substitution was evaluated in equilibrium and kinetic binding experiments as well as in functional assays. The determination of the crystallographic structure of an MT7-derivative (MT7-diiodoTyr51) allowed the selection of candidate residues that are accessible and present on both faces of the three toxin loops. The equilibrium binding data are consistent with negative cooperativity between N-methylscopolamine (NMS) and wild-type or modified MT7 and highlight the critical role of the tip of the central loop of the toxin (Arg34, Met35 Tyr36) in its interaction with the unoccupied receptor. Examination of the potency of wild-type and modified toxins to allosterically decrease the dissociation rate of [ 3 H]NMS allowed the identification of the MT7 residues involved in its interaction with the NMSoccupied receptor. In contrast to the results with the unoccupied receptor, the most important residue for this interaction was Tyr36 in loop II, assisted by Trp10 in loop I and Arg52 in loop III. The critical role of the tips of the MT7 loops was also confirmed in functional experiments. The high specificity of the MT7-M 1 receptor interaction exploits several MT7-specific residues and reveals a different mode of interaction of the toxin with the free and NMS-occupied states of the receptor. Muscarinic neurotoxins, small peptides of 64 to 66 residues derived from the venom of African mambas (Dendroaspis angusticeps and Dendroaspis polylepis), are well known for their ability to interact with different muscarinic receptor subtypes. NMR and X-ray studies of the MT2 toxin have shown that muscarinic toxins have the three-finger fold structure, characteristic of the large superfamily of toxins that act at cholinergic synapses There is a limited understanding of the specificity, selectivity, and mechanism of action of the muscarinic toxins at Article, publication date, and citation information can be found a

Engineering of Three-Finger Fold Toxins Creates Ligands with Original Pharmacological Profiles for Muscarinic and Adrenergic Receptors

Protein engineering approaches are often a combination of rational design and directed evolution using display technologies. Here, we test “loop grafting,” a rational design method, on three-finger fold proteins. These small reticulated proteins have exceptional affinity and specificity for their diverse molecular targets, display protease-resistance, and are highly stable and poorly immunogenic. The wealth of structural knowledge makes them good candidates for protein engineering of new functionality. Our goal is to enhance the efficacy of these mini-proteins by modifying their pharmacological properties in order to extend their use in imaging, diagnostics and therapeutic applications. Using the interaction of three-finger fold toxins with muscarinic and adrenergic receptors as a model, chimeric toxins have been engineered by substituting loops on toxin MT7 by those from toxin MT1. The pharmacological impact of these grafts was examined using binding experiments on muscarinic receptors M1 and M4 and on the α1A-adrenoceptor. Some of the designed chimeric proteins have impressive gain of function on certain receptor subtypes achieving an original selectivity profile with high affinity for muscarinic receptor M1 and α1A-adrenoceptor. Structure-function analysis supported by crystallographic data for MT1 and two chimeras permits a molecular based interpretation of these gains and details the merits of this protein engineering technique. The results obtained shed light on how loop permutation can be used to design new three-finger proteins with original pharmacological profiles

Caractérisation pharmacologique et structurale de l'interaction de neurotoxines sur des récepteurs cholinergiques

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Muscarinic toxins: tools for the study of the pharmacological and functional properties of muscarinic receptors

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Rôle des toxines dans l'étude fonctionnelle et structurale des récepteurs nicotiniques de l'acétylcholine

Les toxines animales ont souvent été très utiles dans la compréhension des modes de fonctionnement de leurs différentes cibles. Celles qui interagissent au niveau des récepteurs nicotiniques de l'acétylcholine sont purifiées à partir de venin de serpents ou de cônes marins. Pour leur haute affinité et leur sélectivité d'interaction, ces toxines sont utilisées depuis une trentaine d'années comme de véritables sondes moléculaires permettant d'identifier, de localiser et de purifier ces récepteurs. Par ailleurs, ces toxines ont joué un rôle majeur dans la compréhension des spécificités fonctionnelles des différents sous-types de récepteurs et ont permis d'accéder à des informations structurales les concernant. L'obtention de ces toxines par voie chimique ou recombinante, l'introduction de résidus non naturels au sein de leurs séquences et la connaissance structurale de leur site d'interaction permet d'envisager la conception de nouveaux ligands ayant des sélectivités prédéfinies et pouvant avoir un intérêt en tant qu'outils pharmacologiques et/ou agents thérapeutiques dans les nombreuses pathologies impliquant ces récepteurs

Chemical Synthesis of MT1 and MT7 Muscarinic Toxins: Critical Role of Arg-34 in Their Interaction with M 1 Muscarinic Receptor

International audienceTwo muscarinic toxins, MT1 and MT7, were obtained by one-step solid-phase synthesis using the 9-fluorenylmethoxycarbonyl-based method. The synthetic and natural toxins, isolated from the snake venom or recombinantly expressed, display identical physicochemical properties and pharmacological profiles. High protein recovery allowed us to specify the selectivity of these toxins for various muscarinic receptor subtypes. Thus, sMT7 has a selectivity for the M1 receptor that is at least 20,000 times that for the other subtypes. The stability of the toxin-receptor complexes indicates that sMT1 interacts reversibly with the M1 receptor, unlike sMT7, which binds it quasi-irreversibly. The effect of the synthetic toxins on the atropine-induced [3H]N-methylscopolamine (NMS) dissociation confirms that sMT7 targets the allosteric site on the M1 receptor, whereas sMT1 seems interact on the orthosteric one. The great decreases in the binding potencies observed after the R34A modification in sMT1 and sMT7 toxins highlight the functional role of this conserved residue in their interactions with the M1 receptor. Interestingly, after the R34A modification, the sMT7 toxin binds reversibly on the M1 receptor. Furthermore, the potency of sMT7-R34A for the NMS-occupied receptor is lower compared with unmodified toxin, supporting the role of this residue in the allosteric interaction of sMT7. All these results and the different charge distributions observed at the two toxin surfaces of their structure models support the hypothesis that the two toxins recognize the M1 receptor differently

Identification of Various Allosteric Interaction Sites on M 1 Muscarinic Receptor Using 125 I-Met35-Oxidized Muscarinic Toxin 7

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A New Affinity-Based Probe to Profile MMP Active Forms

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Different Interactions between MT7 Toxin and the Human Muscarinic M 1 Receptor in Its Free and N -Methylscopolamine-Occupied States

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