Ziad Fajloun

Design and Characterization of a Highly Selective Peptide Inhibitor of the Small Conductance Calcium-activated K+Channel, SkCa2

Apamin-sensitive small conductance calcium-activated potassium channels (SKCa1–3) mediate the slow afterhyperpolarization in neurons, but the molecular identity of the channel has not been defined because of the lack of specific inhibitors. Here we describe the structure-based design of a selective inhibitor of SKCa2. Leiurotoxin I (Lei) and PO5, peptide toxins that share the RXCQ motif, potently blocked human SKCa2 and SKCa3 but not SKCa1, whereas maurotoxin, Pi1, Tsκ, and PO1 were ineffective. Lei blocked these channels more potently than PO5 because of the presence of Ala1, Phe2, and Met7. By replacing Met7 in the RXCQ motif of Lei with the shorter, unnatural, positively charged diaminobutanoic acid (Dab), we generated Lei-Dab7, a selective SKCa2 inhibitor (K d = 3.8 nm) that interacts with residues in the external vestibule of the channel. SKCa3 was rendered sensitive to Lei-Dab7 by replacing His521 with the corresponding SKCa2 residue (Asn367). Intracerebroventricular injection of Lei-Dab7 into mice resulted in no gross central nervous system toxicity at concentrations that specifically blocked SKCa2 homotetramers. Lei-Dab7 will be a useful tool to investigate the functional role of SKCa2 in mammalian tissues.

Chemical synthesis and characterization of maurocalcine, a scorpion toxin that activates Ca2+ release channel/ryanodine receptors.

Maurocalcine is a novel toxin isolated from the venom of the chactid scorpion Scorpio maurus palmatus. It is a 33‐mer basic peptide cross‐linked by three disulfide bridges, which shares 82% sequence identity with imperatoxin A, a scorpion toxin from the venom of Pandinus imperator. Maurocalcine is peculiar in terms of structural properties since it does not possess any consensus motif reported so far in other scorpion toxins. Due to its low concentration in venom (0.5% of the proteins), maurocalcine was chemically synthesized by means of an optimized solid‐phase method, and purified after folding/oxidation by using both C18 reversed‐phase and ion exchange high‐pressure liquid chromatographies. The synthetic product (sMCa) was characterized. The half‐cystine pairing pattern of sMCa was identified by enzyme‐based cleavage and Edman sequencing. The pairings were Cys3‐Cys17, Cys10‐Cys21, and Cys16‐Cys32. In vivo, the sMCa was lethal to mice following intracerebroventricular inoculation (LD50, 20 μg/mouse). In vitro, electrophysiological experiments based on recordings of single channels incorporated into planar lipid bilayers showed that sMCa potently and reversibly modifies channel gating behavior of the type 1 ryanodine receptor by inducing prominent subconductance behavior.

A new fold in the scorpion toxin family, associated with an activity on a ryanodine-sensitive calcium channel.

We determined the structure in solution by 1H two‐dimensional NMR of Maurocalcine from the venom of Scorpio maurus. This toxin has been demonstrated to be a potent effector of ryanodyne‐sensitive calcium channel from skeletal muscles. This is the first description of a scorpion toxin which folds following the Inhibitor Cystine Knot fold (ICK) already described for numerous toxic and inhibitory peptides, as well as for various protease inhibitors. Its three dimensional structure consists of a compact disulfide‐bonded core from which emerge loops and the N‐terminus. A double‐stranded antiparallel β‐sheet comprises residues 20–23 and 30–33. A third extended strand (residues 9–11) is perpendicular to the β‐sheet. Maurocalcine structure mimics the activating segment of the dihydropyridine receptor II‐III loop and is therefore potentially useful for dihydropyridine receptor/ryanodine receptor interaction studies. Proteins 2000;40:436–442.

Maurotoxin Versus Pi1/HsTx1 Scorpion Toxins TOWARD NEW INSIGHTS IN THE UNDERSTANDING OF THEIR DISTINCT DISULFIDE BRIDGE PATTERNS

Maurotoxin (MTX) is a scorpion toxin acting on several K+ channel subtypes. It is a 34-residue peptide cross-linked by four disulfide bridges that are in an “uncommon” arrangement of the type C1-C5, C2-C6, C3-C4, and C7-C8 (versus C1-C5, C2-C6, C3-C7, and C4-C8 for Pi1 or HsTx1, two MTX-related scorpion toxins). We report here that a single mutation in MTX, in either position 15 or 33, resulted in a shift from the MTX toward the Pi1/HsTx1 disulfide bridge pattern. This shift is accompanied by structural and pharmacological changes of the peptide without altering the general α/β scaffold of scorpion toxins.

Cobatoxin 1 from Centruroides noxius scorpion venom: chemical synthesis, three-dimensional structure in solution, pharmacology and docking on K+ channels

CoTX1 (cobatoxin 1) is a 32-residue toxin with three disulphide bridges that has been isolated from the venom of the Mexican scorpion Centruroides noxius Hoffmann. Here we report the chemical synthesis, disulphide bridge organization, 3-D (three-dimensional) solution structure determination, pharmacology on K+ channel subtypes (voltage-gated and Ca2+-activated) and docking-simulation experiments. An enzyme-based cleavage of the synthetic folded/oxidized CoTX1 indicated half-cystine pairs between Cys3-Cys22, Cys8-Cys27 and Cys12-Cys29. The 3-D structure of CoTX1 (solved by 1H-NMR) showed that it folds according to the common alpha/beta scaffold of scorpion toxins. In vivo, CoTX1 was lethal after intracerebroventricular injection to mice (LD50 value of 0.5 microg/mouse). In vitro, CoTX1 tested on cells expressing various voltage-gated or Ca2+-activated (IKCa1) K+ channels showed potent inhibition of currents from rat K(v)1.2 ( K(d) value of 27 nM). CoTX1 also weakly competed with 125I-labelled apamin for binding to SKCa channels (small-conductance Ca2+-activated K+ channels) on rat brain synaptosomes (IC50 value of 7.2 microM). The 3-D structure of CoTX1 was used in docking experiments which suggests a key role of Arg6 or Lys10, Arg14, Arg18, Lys21 (dyad), Ile23, Asn24, Lys28 and Tyr30 (dyad) residues of CoTX1 in its interaction with the rat K(v)1.2 channel. In addition, a [Pro7,Gln9]-CoTX1 analogue (ACoTX1) was synthesized. The two residue replacements were selected aiming to restore the RPCQ motif in order to increase peptide affinity towards SKCa channels, and to alter the CoTX1 dipole moment such that it is expected to decrease peptide activity on K(v) channels. Unexpectedly, ACoTX1 exhibited an activity similar to that of CoTX1 towards SKCa channels, while it was markedly more potent on IKCa1 and several voltage-gated K+ channels.

CD26 modulates nociception in mice via its dipeptidyl-peptidase IV activity.

BACKGROUND CD26 is a multifunctional cell surface glycoprotein expressed by T and B cells. It exhibits a dipeptidyl-peptidase activity (DPP-IV) that cleaves the penultimate proline from the N-terminus of polypeptides, thereby regulating their activity and concentration. METHODS Using CD26-/- mice resulting from targeted inactivation of the gene, we examined the consequences of a DPP-IV defect on behavioural response to nociceptive stimuli and concentration of the pain modulator peptides substance P (SP) and endomorphin 2, two DPP-IV substrates. RESULTS CD26 inactivation induced a three-fold decrease in circulating endopeptidase activity while that found in brain extracts was normal, albeit very weak. CD26-/- mice had high SP concentrations in plasma (3.4+/-1 pg/ml versus 1.5+/-0.3 pg/ml, P<10(-3)) but not in brain extracts (35+/-12 pg/ml versus 32+/-9 pg/ml, P>0.05). Endomorphin-2 levels in the two groups were in the same range for plasma and brain extracts. CD26-/- mice displayed short latencies to nociceptive stimuli (hot plate test: 6.6+/-1.2 s versus 8.6+/-1.5 s, P<10(-4); tail pinch test: 3.1+/-0.6 s versus 4.2+/-0.8 s, P<10(-3)). Administration of an SP (NK1) receptor antagonist or DPP-IV to CD26-/- mice normalised latencies. DPP-IV inhibitors decreased latencies only in CD26+/+ mice. CONCLUSIONS Our observations represent the first fundamental evidence showing that DPP-IV influences pain perception via modulation of the peripheral SP concentration. Our work also highlights the role of peripheral NK1 receptors in nociception.

Parameters affecting in vitro oxidation/folding of maurotoxin, a four-disulphide-bridged scorpion toxin

Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four disulphide bridges that acts on various K(+) channel subtypes. MTX adopts a disulphide bridge organization of the type C1-C5, C2-C6, C3-C4 and C7-C8, and folds according to the common alpha/beta scaffold reported for other known scorpion toxins. Here we have investigated the process and kinetics of the in vitro oxidation/folding of reduced synthetic L-MTX (L-sMTX, where L-MTX contains only L-amino acid residues). During the oxidation/folding of reduced L-sMTX, the oxidation intermediates were blocked by iodoacetamide alkylation of free cysteine residues, and analysed by MS. The L-sMTX intermediates appeared sequentially over time from the least (intermediates with one disulphide bridge) to the most oxidized species (native-like, four-disulphide-bridged L-sMTX). The mathematical formulation of the diffusion-collision model being inadequate to accurately describe the kinetics of oxidation/folding of L-sMTX, we have formulated a derived mathematical description that better fits the experimental data. Using this mathematical description, we have compared for the first time the oxidation/folding of L-sMTX with that of D-sMTX, its stereoisomer that contains only D-amino acid residues. Several experimental parameters, likely to affect the oxidation/folding process, were studied further; these included temperature, pH, ionic strength, redox potential and concentration of reduced toxin. We also assessed the effects of some cellular enzymes, peptidylprolyl cis-trans isomerase (PPIase) and protein disulphide isomerase (PDI), on the folding pathways of reduced L-sMTX and D-sMTX. All the parameters tested affect the oxidative folding of sMTX, and the kinetics of this process were indistinguishable for L-sMTX and D-sMTX, except when stereospecific enzymes were used. The most efficient conditions were found to be: 50 mM Tris/HCl/1.4 mM EDTA, pH 7.5, supplemented by 0.5 mM PPIase and 50 units/ml PDI for 0.1 mM reduced compound. These data represent the first report of potent stereoselective effects of cellular enzymes on the oxidation/folding of a scorpion toxin.

Disulfide bridge reorganization induced by proline mutations in maurotoxin

Maurotoxin (MTX) is a 34‐residue toxin that has been isolated from the venom of the chactidae scorpion Scorpio maurus palmatus, and characterized. Together with Pi1 and HsTx1, MTX belongs to a family of short‐chain four‐disulfide‐bridged scorpion toxins acting on potassium channels. However, contrary to other members of this family, MTX exhibits an uncommon disulfide bridge organization of the type C1–C5, C2–C6, C3–C4 and C7–C8, versus C1–C5, C2–C6, C3–C7 and C4–C8 for both Pi1 and HsTx1. Here, we report that the substitution of MTX proline residues located at positions 12 and/or 20, adjacent to C3 (Cys13) and C4 (Cys19), results in conventional Pi1‐ and HsTx1‐like arrangement of the half‐cystine pairings. In this case, this novel disulfide bridge arrangement is without obvious incidence on the overall three‐dimensional structure of the toxin. Pharmacological assays of this structural analog, [A12,A20]MTX, reveal that the blocking activities on Shaker B and rat Kv1.2 channels remain potent whereas the peptide becomes inactive on rat Kv1.3. These data indicate, for the first time, that discrete point mutations in MTX can result in a marked reorganization of the half‐cystine pairings, accompanied with a novel pharmacological profile for the analog.

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.

Chemical synthesis and 1H-NMR 3D structure determination of AgTx2-MTX chimera, a new potential blocker for Kv1.2 channel, derived from MTX and AgTx2 scorpion toxins

Agitoxin 2 (AgTx2) is a 38‐residue scorpion toxin, cross‐linked by three disulfide bridges, which acts on voltage‐gated K+ (Kv) channels. Maurotoxin (MTX) is a 34‐residue scorpion toxin with an uncommon four‐disulfide bridge reticulation, acting on both Ca2+‐activated and Kv channels. A 39‐mer chimeric peptide, named AgTx2‐MTX, was designed from the sequence of the two toxins and chemically synthesized. It encompasses residues 1–5 of AgTx2, followed by the complete sequence of MTX. As established by enzyme cleavage, the new AgTx2‐MTX molecule displays half‐cystine pairings of the type C1–C5, C2–C6, C3–C7, and C4–C8, which is different from that of MTX. The 3D structure of AgTx2‐MTX solved by 1H‐NMR, revealed both α‐helical and β‐sheet structures, consistent with a common α/β scaffold of scorpion toxins. Pharmacological assays of AgTx2‐MTX revealed that this new molecule is more potent than both original toxins in blocking rat Kv1.2 channel. Docking simulations, performed with the 3D structure of AgTx2‐MTX, confirmed this result and demonstrated the participation of the N‐terminal domain of AgTx2 in its increased affinity for Kv1.2 through additional molecular contacts. Altogether, the data indicated that replacement of the N‐terminal domain of MTX by the one of AgTx2 in the AgTx2‐MTX chimera results in a reorganization of the disulfide bridge arrangement and an increase of affinity to the Kv1.2 channel.