Katherine J. Nielsen

Novel omega -Conotoxins from Conus catus Discriminate among Neuronal Calcium Channel Subtypes

ω-Conotoxins selective for N-type calcium channels are useful in the management of severe pain. In an attempt to expand the therapeutic potential of this class, four new ω-conotoxins (CVIA–D) have been discovered in the venom of the piscivorous cone snail, Conus catus, using assay-guided fractionation and gene cloning. Compared with other ω-conotoxins, CVID has a novel loop 4 sequence and the highest selectivity for N-type over P/Q-type calcium channels in radioligand binding assays. CVIA−D also inhibited contractions of electrically stimulated rat vas deferens. In electrophysiological studies, ω-conotoxins CVID and MVIIA had similar potencies to inhibit current through central (α1B-d) and peripheral (α1B-b) splice variants of the rat N-type calcium channels when coexpressed with rat β3 in Xenopus oocytes. However, the potency of CVID and MVIIA increased when α1B-d and α1B-b were expressed in the absence of rat β3, an effect most pronounced for CVID at α1B-d (up to 540-fold) and least pronounced for MVIIA at α1B-d (3-fold). The novel selectivity of CVID may have therapeutic implications. 1H NMR studies reveal that CVID possesses a combination of unique structural features, including two hydrogen bonds that stabilize loop 2 and place loop 2 proximal to loop 4, creating a globular surface that is rigid and well defined.

Structure-activity relationships of ?-conotoxins at N-type voltage-sensitive calcium channels

Due to their selectivity towards voltage‐sensitive calcium channels (VSCCs) ω‐conotoxins are being exploited as a new class of therapeutics in pain management and may also have potential application in ischaemic brain injury. Here, the structure–activity relationships (SARs) of several ω‐conotoxins including GVIA, MVIIA, CVID and MVIIC are explored. In addition, the three‐dimensional structures of these ω‐conotoxins and some structurally related peptides that form the cysteine knot are compared, and the effects of the solution environment on structure discussed. The diversity of binding and functional assays used to measure ω‐conotoxin potencies at the N‐type VSCC warranted a revaluation of the relationship between these assays. With one exception, [A22]‐GVIA, this analysis revealed a linear correlation between functional (peripheral N‐type VSCCs) and radioligand binding assays (central N‐type VSCCs) for the ω‐conotoxins and analogues that were tested over three studies. The binding and functional results of several studies are compared in an attempt to identify and distinguish those residues that are important in ω‐conotoxin function as opposed to those that form part of the structural scaffold. Further to determining what ω‐conotoxin residues are important for VSCC binding, the range of possible interactions between the ligand and channel are considered and the factors that influence the selectivity of MVIIA, GVIA and CVID towards N‐type VSCCs examined. Copyright

Determination of the Solution Structures of Conantokin-G and Conantokin-T by CD and NMR Spectroscopy

Conantokin-G and conantokin-T are two paralytic polypeptide toxins originally isolated from the venom of the fish-hunting cone snails of the genus Conus. Conantokin-G and conantokin-T are the only naturally occurring peptidic compounds which possess N-methyl-D-aspartate receptor antagonist activity, produced by a selective non-competitive antagonism of polyamine responses. They are also structurally unusual in that they contain a disproportionately large number of acid labile post-translational γ-carboxyglutamic acid (Gla) residues. Although no precise structural information has previously been published for these peptides, early spectroscopic measurements have indicated that both conantokin-G and conantokin-T form α-helical structures, although there is some debate whether the presence of calcium ions is required for these peptides to adopt this fold. We now report a detailed structural study of synthetic conantokin-G and conantokin-T in a range of solution conditions using CD and 1H NMR spectroscopy. The three-dimensional structures of conantokin-T and conantokin-G were calculated from 1H NMR-derived distance and dihedral restraints. Both conantokins were found to contain a mixture of α- and 310 helix, that give rise to curved and straight helical conformers. Conantokin-G requires the presence of divalent cations (Zn2+, Ca2+, Cu2+, or Mg2+) to form a stable α-helix, while conantokin-T adopts a stable α-helical structure in aqueous conditions, in the presence or absence of divalent cations (Zn2+, Ca2+, Cu2+, or Mg2+).

Isolation and Structure-Activity of μ-Conotoxin TIIIA, A Potent Inhibitor of Tetrodotoxin-Sensitive Voltage-Gated Sodium Channels

μ-Conotoxins are three-loop peptides produced by cone snails to inhibit voltage-gated sodium channels during prey capture. Using polymerase chain reaction techniques, we identified a gene sequence from the venom duct of Conus tulipa encoding a new μ-conotoxin-TIIIA (TIIIA). A 125I-TIIIA binding assay was established to isolate native TIIIA from the crude venom of Conus striatus. The isolated peptide had three post-translational modifications, including two hydroxyproline residues and C-terminal amidation, and <35% homology to other μ-conotoxins. TIIIA potently displaced [3H]saxitoxin and 125I-TIIIA from rat brain (Nav1.2) and skeletal muscle (Nav1.4) membranes. Alanine and glutamine scans of TIIIA revealed several residues, including Arg14, that were critical for high-affinity binding to tetrodotoxin (TTX)-sensitive Na+ channels. We were surprised to find that [E15A]TIIIA had a 10-fold higher affinity than TIIIA for TTX-sensitive sodium channels (IC50, 15 vs. 148 pM at rat brain membrane). TIIIA was selective for Nav1.2 and -1.4 over Nav1.3, -1.5, -1.7, and -1.8 expressed in Xenopus laevis oocytes and had no effect on rat dorsal root ganglion neuron Na+ current. 1H NMR studies revealed that TIIIA adopted a single conformation in solution that was similar to the major conformation described previously for μ-conotoxin PIIIA. TIIIA and analogs provide new biochemical probes as well as insights into the structure-activity of μ-conotoxins.

THE INTERACTIONS OF THE N-TERMINAL FUSOGENIC PEPTIDE OF HIV-1 GP41 WITH NEUTRAL PHOSPHOLIPIDS

Abstract We have studied the interactions with neutral phospholipid bilayers of FPI, the 23-residue fusogenic N-terminal peptide of the HIV-1LAI transmembrane glycoprotein gp41, by CD, EPR, NMR, and solid state NMR (SSNMR) with the objective of understanding how it lyses and fuses cells. Using small unilamellar vesicles made from egg yolk phoshatidylcholine which were not fused or permeabilised by the peptide we obtained results suggesting that it was capable of inserting as an α-helix into neutral phospholipid bilayers but was only completely monomeric at peptide/lipid (P/L) ratios of 1/2000 or lower. Above this value, mixed populations of monomeric and multimeric forms were found with the proportion of multimer increasing proportionally to P/L, as calculated from studies on the interaction between the peptide and spin-labelled phospholipid. The CD data indicated that, at P/L between 1/200 and 1/100, approximately 68% of the peptide appeared to be in α-helical form. When P/L=1/25 the α-helical content had decreased to 41%. Measurement at a P/L of 1/100 of the spin lattice relaxation effect on the 13C nuclei of the phospholipid acyl chains of an N-terminal spin label attached to the peptide showed that most of the peptide N-termini were located in the interior hydrocarbon region of the membrane. SSNMR on multilayers of ditetradecylphosphatidyl choline at P/Ls of 1/10, 1/20 and 1/30 showed that the peptide formed multimers that affected the motion of the lipid chains and disrupted the lipid alignment. We suggest that these aggregates may be relevant to the membrane-fusing and lytic activities of FPI and that they are worthy of further study.

Neuronally Selective μ-Conotoxins from Conus striatus Utilize an α-Helical Motif to Target Mammalian Sodium Channels

μ-Conotoxins are small peptide inhibitors of muscle and neuronal tetrodotoxin (TTX)-sensitive voltage-gated sodium channels (VGSCs). Here we report the isolation of μ-conotoxins SIIIA and SIIIB by 125I-TIIIA-guided fractionation of milked Conus striatus venom. SIIIA and SIIIB potently displaced 125I-TIIIA from native rat brain Nav1.2 (IC50 values 10 and 5 nm, respectively) and muscle Nav1.4 (IC50 values 60 and 3 nm, respectively) VGSCs, and both inhibited current through Xenopus oocyte-expressed Nav1.2 and Nav1.4. An alanine scan of SIIIA-(2–20), a pyroglutamate-truncated analogue with enhanced neuronal activity, revealed residues important for affinity and selectivity. Alanine replacement of the solvent-exposed Trp-12, Arg-14, His-16, Arg-18 resulted in large reductions in SIIIA-(2–20) affinity, with His-16 replacement affecting structure. In contrast, [D15A]SIIIA-(2–20) had significantly enhanced neuronal affinity (IC50 0.65 nm), while the double mutant [D15A/H16R]SIIIA-(2–20) showed greatest Nav1.2 versus 1.4 selectivity (136-fold). 1H NMR studies revealed that SIIIA adopted a single conformation in solution comprising a series of turns and anα-helical motif across residues 11–16 that is not found in larger μ-conotoxins. The structure of SIIIA provides a new structural template for the development of neuronally selective inhibitors of TTX-sensitive VGSCs based on the smaller μ-conotoxin pharmacophore.

A peptide corresponding to the N-terminal 13 residues of T4 lysozyme forms an α-helix

Solid‐phase methods have been used to synthesize LYS(1–13), a peptide corresponding to the first 13 residues of T4 lysozyme. 2D 1H NMR techniques were used to investigate its solution structure in the presence of SDS micelles. The identification of numerous medium‐range NOESY crosspeaks and several slowly exchanging NH protons indicated the presence of an α‐helical structure. This was confirmed by simulated annealing calculations performed using XPLOR.