Benjamin Chagot

An unusual fold for potassium channel blockers: NMR structure of three toxins from the scorpion Opisthacanthus madagascariensis

The Om-toxins are short peptides (23-27 amino acids) purified from the venom of the scorpion Opisthacanthus madagascariensis. Their pharmacological targets are thought to be potassium channels. Like Csalpha/beta (cystine-stabilized alpha/beta) toxins, the Om-toxins alter the electrophysiological properties of these channels; however, they do not share any sequence similarity with other scorpion toxins. We herein demonstrate by electrophysiological experiments that Om-toxins decrease the amplitude of the K+ current of the rat channels Kv1.1 and Kv1.2, as well as human Kv1.3. We also determine the solution structure of three of the toxins by use of two-dimensional proton NMR techniques followed by distance geometry and molecular dynamics. The structures of these three peptides display an uncommon fold for ion-channel blockers, Csalpha/alpha (cystine-stabilized alpha-helix-loop-helix), i.e. two alpha-helices connected by a loop and stabilized by two disulphide bridges. We compare the structures obtained and the dipole moments resulting from the electrostatic anisotropy of these peptides with those of the only other toxin known to share the same fold, namely kappa-hefutoxin1.

Solution structure of APETx2, a specific peptide inhibitor of ASIC3 proton-gated channels.

Acid‐sensing ion channels (ASIC) are proton‐gated sodium channels that have been implicated in pain transduction associated with acidosis in inflamed or ischemic tissues. APETx2, a peptide toxin effector of ASIC3, has been purified from an extract of the sea anemone Anthopleura elegantissima. APETx2 is a 42‐amino‐acid peptide cross‐linked by three disulfide bridges. Its three‐dimensional structure, as determined by conventional two‐dimensional 1H‐NMR, consists of a compact disulfide‐bonded core composed of a four‐stranded β‐sheet. It belongs to the disulfide‐rich all‐β structural family encompassing peptide toxins commonly found in animal venoms. The structural characteristics of APETx2 are compared with that of PcTx1, another effector of ASIC channels but specific to the ASIC1a subtype and to APETx1, a toxin structurally related to APETx2, which targets the HERG potassium channel. Structural comparisons, coupled with the analysis of the electrostatic characteristics of these various ion channel effectors, led us to suggest a putative channel interaction surface for APETx2, encompassing its N terminus together with the type I‐β turn connecting β‐strands III and IV. This basic surface (R31 and R17) is also rich in aromatic residues (Y16, F15, Y32, and F33). An additional region made of the type II′‐β turn connecting β‐strands I and II could also play a role in the specificity observed for these different ion effectors.

Solution structure of Phrixotoxin 1, a specific peptide inhibitor of Kv4 potassium channels from the venom of the theraphosid spider Phrixotrichus auratus

Animal toxins block voltage‐dependent potassium channels (Kv) either by occluding the conduction pore (pore blockers) or by modifying the channel gating properties (gating modifiers). Gating modifiers of Kv channels bind to four equivalent extracellular sites near the S3 and S4 segments, close to the voltage sensor. Phrixotoxins are gating modifiers that bind preferentially to the closed state of the channel and fold into the Inhibitory Cystine Knot structural motif. We have solved the solution structure of Phrixotoxin 1, a gating modifier of Kv4 potassium channels. Analysis of the molecular surface and the electrostatic anisotropy of Phrixotoxin 1 and of other toxins acting on voltage‐dependent potassium channels allowed us to propose a toxin interacting surface that encompasses both the surface from which the dipole moment emerges and a neighboring hydrophobic surface rich in aromatic residues.

Solution structure of APETx1 from the sea anemone Anthopleura elegantissima: A new fold for an HERG toxin

APETx1 is a 42‐amino acid toxin purified from the venom of the sea anemone Anthopleura elegantissima. This cysteine‐rich peptide possesses three disulfide bridges (C4–C37, C6–C30, and C20–C38). Its pharmacological target is the Ether‐a‐gogo potassium channel. We herein determine the solution structure of APETx1 by use of conventional two‐dimensional 1H‐NMR techniques followed by torsion angle dynamics and refinement protocols. The calculated structure of APETx1 belongs to the disulfide‐rich all‐beta structural family, in which a three‐stranded anti‐parallel β‐sheet is the only secondary structure. APETx1 is the first Ether‐a‐gogo effector discovered to fold in this way. We therefore compare the structure of APETx1 to those of the two other known effectors of the Ether‐a‐gogo potassium channel, CnErg1 and BeKm‐1, and analyze the topological disposition of key functional residues proposed by analysis of the electrostatic anisotropy. The interacting surface is made of a patch of aromatic residues (Y5, Y32, and F33) together with two basic residues (K8 and K18) at the periphery of the surface. We pinpoint the absence of the central lysine present in the functional surface of the two other Ether‐a‐gogo effectors. Proteins 2005.

Solution structure of two insect‐specific spider toxins and their pharmacological interaction with the insect voltage‐gated Na+ channel

δ‐PaluIT1 and δ‐paluIT2 are toxins purified from the venom of the spider Paracoelotes luctuosus. Similar in sequence to μ‐agatoxins from Agelenopsis aperta, their pharmacological target is the voltage‐gated insect sodium channel, of which they alter the inactivation properties in a way similar to α‐scorpion toxins, but they bind on site 4 in a way similar to β‐scorpion toxins. We determined the solution structure of the two toxins by use of two‐dimensional nuclear magnetic resonance (NMR) techniques followed by distance geometry and molecular dynamics. The structures of δ‐paluIT1 and δ‐paluIT2 belong to the inhibitory cystine knot structural family, i.e. a compact disulfide‐bonded core from which four loops emerge. δ‐PaluIT1 and δ‐paluIT2 contain respectively two‐ and three‐stranded anti‐parallel β‐sheets as unique secondary structure. We compare the structure and the electrostatic anisotropy of those peptides to other sodium and calcium channel toxins, analyze the topological juxtaposition of key functional residues, and conclude that the recognition of insect voltage‐gated sodium channels by these toxins involves the β‐sheet, in addition to loops I and IV. Besides the position of culprit residues on the molecular surface, difference in dipolar moment orientation is another determinant of receptor binding and biological activity differences. We also demonstrate by electrophysiological experiments on the cloned insect voltage‐gated sodium channel, para, heterologuously co‐expressed with the tipE subunit in Xenopus laevis oocytes, that δ‐paluIT1 and δ‐paluIT2 procure an increase of Na+ current. δ‐PaluIT1‐OH seems to have less effect when the same concentrations are used. Proteins 2005.

Solution structure of PcFK1, a spider peptide active against Plasmodium falciparum

Psalmopeotoxin I (PcFK1) is a 33‐amino‐acid residue peptide isolated from the venom of the tarantula Psalmopoeus cambridgei. It has been recently shown to possess strong antiplasmodial activity against the intra‐erythrocyte stage of Plasmodium falciparum in vitro. Although the molecular target for PcFK1 is not yet determined, this peptide does not lyse erythrocytes, is not cytotoxic to nucleated mammalian cells, and does not inhibit neuromuscular function. We investigated the structural properties of PcFK1 to help understand the unique mechanism of action of this peptide and to enhance its utility as a lead compound for rational development of new antimalarial drugs. In this paper, we have determined the three‐dimensional solution structure by 1H two‐dimensional NMR means of recombinant PcFK1, which is shown to belong to the ICK structural superfamily with structural determinants common to several neurotoxins acting as ion channels effectors.

Increasing the molecular contacts between maurotoxin and Kv1.2 channel augments ligand affinity.

Scorpion toxins interact with their target ion channels through multiple molecular contacts. Because a “gain of function” approach has never been described to evaluate the importance of the molecular contacts in defining toxin affinity, we experimentally examined whether increasing the molecular contacts between a toxin and an ion channel directly impacts toxin affinity. For this purpose, we focused on two scorpion peptides, the well‐characterized maurotoxin with its variant Pi1‐like disulfide bridging (MTXPi1), used as a molecular template, and butantoxin (BuTX), used as an N‐terminal domain provider. BuTX is found to be 60‐fold less potent than MTXPi1 in blocking Kv1.2 (IC50 values of 165 nM for BuTX versus 2.8 nM for MTXPi1). Removal of its N‐terminal domain (nine residues) further decreases BuTX affinity for Kv1.2 by 5.6‐fold, which is in agreement with docking simulation data showing the importance of this domain in BuTX‐Kv1.2 interaction. Transfer of the BuTX N‐terminal domain to MTXPi1 results in a chimera with five disulfide bridges (BuTX‐MTXPi1) that exhibits 22‐fold greater affinity for Kv1.2 than MTXPi1 itself, in spite of the lower affinity of BuTX as compared to MTXPi1. Docking experiments performed with the 3‐D structure of BuTX‐MTXPi1 in solution, as solved by 1H‐NMR, reveal that the N‐terminal domain of BuTX participates in the increased affinity for Kv1.2 through additional molecular contacts. Altogether, the data indicate that acting on molecular contacts between a toxin and a channel is an efficient strategy to modulate toxin affinity. Proteins 2005.

Solution structure of gp17 from the Siphoviridae bacteriophage SPP1: insights into its role in virion assembly.

Solution structure of gp17 from the Siphoviridae bacteriophage SPP1: Insights into its role in virion assembly Benjamin Chagot, Isabelle Auzat, Matthieu Gallopin, Isabelle Petitpas, Bernard Gilquin, Paulo Tavares, and Sophie Zinn-Justin* 1 Laboratoire de Biologie Structurale et Radiobiologie, iBiTec-S and URA CNRS 2096, CEA Saclay, Gif-sur-Yvette, France 2 Laboratoire de Virologie Moléculaire et Structurale, Centre de Recherche de Gif, CNRS UPR 3296 and IFR 115, 91198 Gif-sur-Yvette, France

Bacteriophage SPP1 tail tube protein self-assembles into β-structure-rich tubes.

Background: In most bacteriophages, a long tail primarily built from tail tube proteins serves as a conduit for DNA delivery into the bacteria. Results: The tail tube protein of phage SPP1 self-assembles into tubes exhibiting a phage tail-like helical architecture. Conclusion: A three-dimensional model is proposed for the self-assembled tubes. Significance: This work opens the way for the generation of artificial tubular structures. The majority of known bacteriophages have long tails that serve for bacterial target recognition and viral DNA delivery into the host. These structures form a tube from the viral capsid to the bacterial cell. The tube is formed primarily by a helical array of tail tube protein (TTP) subunits. In phages with a contractile tail, the TTP tube is surrounded by a sheath structure. Here, we report the first evidence that a phage TTP, gp17.1 of siphophage SPP1, self-assembles into long tubes in the absence of other viral proteins. gp17.1 does not exhibit a stable globular structure when monomeric in solution, even if it was confidently predicted to adopt the β-sandwich fold of phage λ TTP. However, Fourier transform infrared and nuclear magnetic resonance spectroscopy analyses showed that its β-sheet content increases significantly during tube assembly, suggesting that gp17.1 acquires a stable β-sandwich fold only after self-assembly. EM analyses revealed that the tube is formed by hexameric rings stacked helicoidally with the same organization and helical parameters found for the tail of SPP1 virions. These parameters were used to build a pseudo-atomic model of the TTP tube. The large loop spanning residues 40–56 is located on the inner surface of the tube, at the interface between adjacent monomers and hexamers. In line with our structural predictions, deletion of this loop hinders gp17.1 tube assembly in vitro and interferes with SPP1 tail assembly during phage particle morphogenesis in bacteria.