The structure of versutoxin (δ-atracotoxin-Hv1) provides insights into the binding of site 3 neurotoxins to the voltage-gated sodium channel

Fletcher, Jamie I.; Chapman, Bogdan E.; Mackay, Joel P.; Howden, M.E.H.; King, Glenn F.

doi:10.1016/s0969-2126(97)00301-8

Cited by 118 publications

(97 citation statements)

References 64 publications

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“…In support ACTX-Hvf17, colipases, and MIT1 are highly resistant to proteases and convergent evolution may have arisen because the DDH fold is an efficient way to construct a tight globular protein that is resistant to proteolysis. This has been as previously argued for the more elaborate, but related, ICK motif [10] (Fig. 7Ab).…”

Section: Of 32 Thursday May 05 2005supporting

confidence: 88%

Discovery of an MIT-like atracotoxin family: Spider venom peptides that share sequence homology but not pharmacological properties with AVIT family proteins

et al. 2005

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Abbreviations: ACh, acetylcholine; ACTX, atracotoxin; BCA, bicinchoninic acid; Bv8, 8kDa protein from the skin secretions of toads (Bombina variegata); CHO, Chinese hamster ovary; DDH, disulfidedirected -hairpin; EST, expressed sequence tag; FLIPR, fluorometric imaging plate reader; HEK, human embryonic kidney; ICK, inhibitory cystine-knot; MIT1, mamba intestinal toxin 1 from the venom of the black mamba snake Dendroaspis p. polylepis; PK, prokineticin (also known as endocrine-gland vascular endothelial growth factor or EG-VEGF); PKR, prokineticin receptor; RACE, rapid amplification of cDNA ends; TCEP, Tris(2-carboxyethyl)phosphine; TFA, trifluoroacetic acid. Atracinae), that appear to undergo N-and/or C-terminal post-translational modifications and conform to an ancestral protein fold. These peptides all show significant amino acid sequence homology to atracotoxin-Hvf17 (ACTX-Hvf17), a non-toxic peptide isolated from the venom of H. versuta, and a variety of AVIT family proteins including mamba intestinal toxin 1 (MIT1) and its mammalian and piscine orthologs prokineticin 1 (PK1) and prokineticin 2 (PK2). These AVIT family proteins target prokineticin receptors involved in the sensitization of nociceptors and gastrointestinal smooth muscle activation. Given their sequence homology to MIT1, we have named these spider venom peptides the MIT-like atracotoxin (ACTX) family. Using isolated rat stomach fundus or guinea-pig ileum organ bath preparations we have shown that the prototypical ACTX-Hvf17, at concentrations up to 1 GM, did not stimulate smooth muscle contractility, nor did it inhibit contractions induced by human PK1 (hPK1). KeywordsThe peptide also lacked activity on other isolated smooth muscle preparations including rat aorta.Furthermore, a FLIPR Ca 2+ flux assay using HEK293 cells expressing prokineticin receptors showed that ACTX-Hvf17 fails to activate or block hPK1 or hPK2 receptors. Therefore, while the MIT-like ACTX family appears to adopt the ancestral disulfide-directed -hairpin protein fold of MIT1, a motif believed to be shared by other AVIT family peptides, variations in the amino acid sequence and surface charge result in a loss of activity on prokineticin receptors.

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Section: Of 32 Thursday May 05 2005supporting

confidence: 88%

Discovery of an MIT-like atracotoxin family: Spider venom peptides that share sequence homology but not pharmacological properties with AVIT family proteins

et al. 2005

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“…They also adopt ICK motif structures (15,16), with the fourth disulfide linking this motif to the C-terminus (15). Structurally, therefore, they are more similar to ι-RXIA than is J-atracotoxin, even though the latter has a framework #11 arrangement of cysteine residues.…”

Section: Discussionmentioning

confidence: 99%

“…Four major partially reduced forms, which correspond to the species containing three (3S-S), two (2S-S1 and 2S-S 2 ) and one (1S-S) remaining disulfide bond, were separated. (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)…”

Section: Supplementary Materialsmentioning

confidence: 99%

Structure and Sodium Channel Activity of an Excitatory I₁-Superfamily Conotoxin^,

et al. 2007

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Conotoxin ι-RXIA, from the fish-hunting species Conus radiatus, is a member of the recently characterized I 1 -superfamily, which contains eight cysteine residues arranged in a −C-C-CC-CC-C-C-pattern. ι-RXIA (formerly designated r11a) is one of three characterized I 1 peptides in which the third last residue is post-translationally isomerized to the D-configuration. Naturally occurring ι-RXIA with D-Phe44 is significantly more active as an excitotoxin than the L-Phe analogue both in vitro and in vivo. We have determined the solution structures of both forms by NMR spectroscopy, the first for an I 1 -superfamily member. The disulfide connectivities were determined from structure calculations and confirmed chemically as 5-19, 12-22, 18-27, and 21-38, suggesting that ι-RXIA has an ICK structural motif with one additional disulfide (21-38). Indeed, apart from the first few residues, the structure is well defined up to around residue 35 and does adopt an ICK structure. The C-terminal region, including Phe44, is disordered. Comparison of the D-Phe44 and L-Phe44 forms indicates that the switch from one enantiomer to the other has very little effect on the structure, even though it is clearly important for receptor interaction based on activity data. Finally, we identify the target of ι-RXIA as a voltage-gated sodium channel; ι-RXIA is an agonist, shifting the voltage dependence of activation of mouse Na V 1.6 expressed in Xenopus oocytes to more hyperpolarized potentials. Thus, there is a convergence of structure and function in ι-RXIA, as its disulfide pairing and structure resemble those of funnel web spider toxins that also target sodium channels.Through adaptive evolution, marine cone snails of the genus Conus have generated more than 70,000 different venom peptides (conopeptides) (1-3). These highly diverse peptides can be organized into several structural classes, with peptides in the same structural class generally belonging to the same gene superfamily. The peptides in a given superfamily share a highly conserved precursor signal sequence and a disulfide scaffold with a characteristic number, pattern, and pairing of cysteine residues in the mature toxin (1-3). Thus, despite hypermutation of amino acids between half-cystines, the disulfide framework remains conserved within a superfamily. In addition to the accelerated evolution of conopeptide sequences, further † This work was supported in part by N. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 October 13. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript molecular diversity is introduced through post-translational modifications. The occurrence of diverse modifications in Conus peptides is now well established (4) but the functional consequences of most post-translational modifications remain unknown, and mechanistic insights require detailed structure/function analyses, which have not been performed in most cases.Recently, we described the I-superfamily of Conus peptides, defined by...

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“…Nevertheless, attempts have been made to indirectly identify critical residues involved in δACTX binding to site 3 based on knowledge of the binding site on the Nav channel or from mutagenesis studies of related site 3 toxins. These studies indicate that a number of residues provide a complementary surface to the residues identified in the S3S4 loop of domain IV (Fletcher et al, 1997) or key residues forming the likely pharmacophores of scorpion toxins and sea anemone toxins (Gilles et al, 2002). These comprise of the residues highlighted in Figure 4B.…”

Section: Nicholson and Little !mentioning

confidence: 92%

“…These δ-ACTXs are composed of 42 residues containing eight cysteines. The three-dimensional solution structures of δACTXHv1a (Fletcher et al, 1997) and δACTXAr1a (Pallaghy et al, 1997) have been determined using NMR spectroscopy ( Fig. 4B) and consist of a triple-stranded antiparallel β-sheet, in some cases ending in a 310 helix (eg.…”

Section: δ-Atracotoxinsmentioning

confidence: 99%

SPider Neurotoxins Targeting Voltage-Gated Sodium Channels

Nicholson¹,

Little²

2005

Toxin Revs.

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The voltage-gated sodium (Nav) channel is a target for a number of drugs, insecticides and neurotoxins. These bind to at least seven identified neurotoxin binding sites and either block conductance or modulate sodium channel gating and / or kinetics. A number of polypeptide toxins from the venoms of araneomorph and mygalomorph spiders have been isolated and characterized that interact with several of these sites. Certain huwentoxins and hainantoxins appear to target site 1 to block Nav channel conductance. The δ-atracotoxins and Magi 4 slow Nav channel inactivation via an interaction with neurotoxin site 3. The δ-palutoxins, and most likely µ-agatoxins and curtatoxins, target site 4. However their action is complex with the µ-agatoxins causing a hyperpolarizing shift the voltage-dependence of activation, an action analogous to scorpion β-toxins, but with both δ-palutoxins and µ-agatoxins slowing Nav channel inactivation, a site 3 like action. Many spider toxins target undefined sites, while others are likely to cross-react with other ion channels due to conservedstructures within domains of voltage-gated ion channels. It is already clear, however, that many spider toxins represent highly potent and specific molecular tools to define novel links between sites modulating channel activation and inactivation. Other spider toxins show phyla-specificity and are being considered as lead compounds for the development of biopesticides. Others display tissue specificity via interactions with specific Nav channel subtypes and should provide useful tools to delineate the molecular determinants to target ligands to these channel subtypes. These studies are being greatly assisted by the determination of the pharmacophore of these toxins, but without precise identification of their binding site and mode of action their potential in the above areas remains underdeveloped.!

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The structure of versutoxin (δ-atracotoxin-Hv1) provides insights into the binding of site 3 neurotoxins to the voltage-gated sodium channel

Cited by 118 publications

References 64 publications

Discovery of an MIT-like atracotoxin family: Spider venom peptides that share sequence homology but not pharmacological properties with AVIT family proteins

Discovery of an MIT-like atracotoxin family: Spider venom peptides that share sequence homology but not pharmacological properties with AVIT family proteins

Structure and Sodium Channel Activity of an Excitatory I₁-Superfamily Conotoxin^,

SPider Neurotoxins Targeting Voltage-Gated Sodium Channels

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The structure of versutoxin (δ-atracotoxin-Hv1) provides insights into the binding of site 3 neurotoxins to the voltage-gated sodium channel

Cited by 118 publications

References 64 publications

Discovery of an MIT-like atracotoxin family: Spider venom peptides that share sequence homology but not pharmacological properties with AVIT family proteins

Discovery of an MIT-like atracotoxin family: Spider venom peptides that share sequence homology but not pharmacological properties with AVIT family proteins

Structure and Sodium Channel Activity of an Excitatory I1-Superfamily Conotoxin,

SPider Neurotoxins Targeting Voltage-Gated Sodium Channels

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Structure and Sodium Channel Activity of an Excitatory I₁-Superfamily Conotoxin^,