Conorfamide-Sr3, a structurally novel specific inhibitor of the Shaker K+ channel.

Conorfamides (CNFs) are toxins initially characterized from the venom duct of the venomous marine snail Conus spurius from the Gulf of Mexico; at their C-termini, these toxins are amidated and have high sequence similarity with the molluskan cardioexcitatory tetrapeptide Phe-Met-Arg-Phe-NH2 (FMRFamide or FMRFa) and other FMRFa-related peptides (FaRPs) found in the five molluskan classes, and in other invertebrate and vertebrate phyla. These peptides were the first FaRPs found to be present in any venom, and they are biologically active in mice, limpets, and/or freshwater snails. However, the molecular targets of the known CNFs (CNF-Sr1 and CNF-Sr2 from C. spurius, and CNF-Vc1 from C. victoriae) remain unidentified. Very recently, three FaRPs from C. textile have been found to potentiate the currents of acid-sensing ion channels. In this work, we characterized a novel conorfamide, CNF-Sr3 (ATSGPMGWLPVFYRF-NH2), comprised of 15 amino acid residues, and with a specific blocking activity for the Shaker subtype of the voltage-gated potassium channels, without significant effect on the Shab, Shaw, Shal and Eag channels. This peptide is the third type of disulfide-free conotoxins that has been discovered to target K+ channels.

Specific determinants of conantokins that dictate their selectivity for the NR2B subunit of N-methyl-D-aspartate receptors.

Conantokins are naturally-occurring small peptide antagonists of ion flow through NMDA/glycine activated-N-methyl-d-aspartate receptor (NMDAR) ion channels. One member of the conantokin family, conantokin (con)-G, a 17-residue peptide, is selective for NMDARs containing the N-methyl-d-aspartate receptor subunit 2 B (NR2B), whereas the homologous peptides, con-T and con-R, show broader selectivity for NR2 subunits. In this study, con-G, con-R, and con-T variants were chemically synthesized and employed to investigate their subunit selectivities as inhibitors of agonist-evoked ion currents in human embryonic kidney-293 (HEK-293) cells expressing various combinations of NMDAR subunits that contain NR1a or NR1b combined with NR2A or NR2B. Using truncation and point mutants, as well as chimeric conantokins, we determined that the N-terminus of con-G contains all the determinants for NR2B selectivity. With this information, a large number of (con) variants were synthesized and used to establish minimal sequence determinants for selectivity. Tyr at position 5 broadens the NR2 selectivity, and recovery of NR2B selectivity in Tyr5 peptides was achieved by incorporating Ala or Gly at position 8. NR2B selectivity in con-R can be conferred through deletion of the Ala at position 10, thereby shifting the γ-carboxyglutamate (Gla) from position 11 to position 10, where a Gla naturally occurs in con-G and con-T. The nature of the amino acid at position 6 is also linked to subunit selectivity. Our studies suggest that the molecular determinants of conantokins that dictate NMDAR subunit selectivity are housed in specific residues of the N-termini of these peptides. Thus, it is possible to engineer desired NMDAR functional properties into conantokin-based peptides.

Metal ion determinants of conantokin dimerization as revealed in the X-ray crystallographic structure of the Cd(2+)/Mg (2+)-con-T[K7gamma] complex.

Predatory sea snails from the Conus family produce a variety of venomous small helical peptides called conantokins that are rich in gamma-carboxyglutamic acid (Gla) residues. As potent and selective antagonists of the N-methyl-D: -aspartate receptor, these peptides are potential therapeutic agents for a variety of neurological conditions. The two most studied members of this family of peptides are con-G and con-T. Con-G has Gla residues at sequence positions 3, 4, 7, 10, and 14, and requires divalent cation binding to adopt a helical conformation. Although both Ca(2+) and Mg(2+) can fulfill this role, Ca(2+) induces dimerization of con-G, whereas the Mg(2+)-complexed peptide remains monomeric. A variant of con-T, con-T[K7gamma] (gamma is Gla), contains Gla residues at the same five positions as in con-G and behaves very similarly with respect to metal ion binding and dimerization; each peptide binds two Ca(2+) ions and two Mg(2+) ions per helix. To understand the difference in metal ion selectivity, affinity, and the dependence on Ca(2+) for dimer formation, we report here the structure of the monomeric Cd(2+)/Mg(2+)-con-T[K7gamma] complex, and, by comparison with the previously published con-T[K7gamma]/Ca(2+) dimer structure, we suggest explanations for both metal ion binding site specificity and metal-ion-dependent dimerization.

Conotoxin TVIIA, a novel peptide from the venom of Conus tulipa 1. Isolation, characterization and chemical synthesis.

A novel conotoxin belonging to the 'four-loop' structural class has been isolated from the venom of the piscivorous cone snail Conus tulipa. It was identified using a chemical-directed strategy based largely on mass spectrometric techniques. The new toxin, conotoxin TVIIA, consists of 30 amino-acid residues and contains three disulfide bonds. The amino-acid sequence was determined by Edman analysis as SCSGRDSRCOOVCCMGLMCSRGKCVSIYGE where O = 4-transL-hydroxyproline. Two under-hydroxylated analogues, [Pro10]TVIIA and [Pro10,11]TVIIA, were also identified in the venom of C. tulipa. The sequences of TVIIA and [Pro10]TVIIA were further verified by chemical synthesis and coelution studies with native material. Conotoxin TVIIA has a six cysteine/four-loop structural framework common to many peptides from Conus venoms including the omega-, delta- and kappa-conotoxins. However, TVIIA displays little sequence homology with these well-characterized pharmacological classes of peptides, but displays striking sequence homology with conotoxin GS, a peptide from Conus geographus that blocks skeletal muscle sodium channels. These new toxins and GS share several biochemical features and represent a distinct subgroup of the four-loop conotoxins.

Design and biological evaluation of non-peptide analogues of omega-conotoxin MVIIA.

Omega-conotoxin MVIIA, a highly potent antagonist of the N-type voltage sensitive calcium channel, has shown utility in several models of pain and ischemia. We report a series of three alkylphenyl ether based analogues which mimic three key amino acids of the toxin. Two of the compounds have been found to exhibit IC50 values of 2.7 and 3.3 microM at the human N-type voltage sensitive calcium channel.

Aromatic substitutions in alpha-conotoxin ImI. Synthesis of iodinated photoactivatable derivative.

Conotoxin ImI is a specific marker of alpha7 nicotinic acetylcholine receptors. To study the role of aromatic indole group of tryptophan 10 in biological activity of ImI, the analogue containing tyrosine at this position was synthesized by solid-phase peptide synthesis. The analogue obtained, as well as its iodinated derivatives, were shown to be active against rat brain alpha7 acetylcholine receptor expressed in Xenopus oocytes. Attachment of bulky aromatic p-benzoylbenzoyl group to N-terminal alpha-amino group of iodinated [Tyr10]ImI only slightly affected the biological activity of the analogue. The data obtained suggest that indole ring of tryptophan 10 is not absolutely necessary for biological activity of conotoxin ImI, and that the N-terminus can accommodate a large aromatic group without loss of biological activity.

Effects of chirality at Tyr13 on the structure-activity relationships of omega-conotoxins from Conus magus.

The effects of chirality inversions of Tyr13 on the structure-activity relationships of omega-conotoxins MVIIA and MVIIC were examined using a combination of 2D 1H NMR spectroscopy and radioligand binding studies specific for N-type ([125I]GVIA) and P/Q-type ([125I]MVIIC) voltage-sensitive calcium channels (VSCCs). A comparison of the Halpha secondary shifts suggests that the structural scaffolds of MVIIA and MVIIC are little altered by the L- to D- inversion of Tyr13; however, the conformations of several residues in loop 2 (residues 9-14) are significantly altered. The experimentally determined 3D structure of [D-Y13]MVIIA indicates that the positions of key residues in this loop which are involved in the binding of MVIIA to the N-type VSCC (Tyr13, Arg10, and Leu11) are so changed as to render the peptide unrecognizable by its cognate ion channel. The large reduction in potency observed for MVIIA and MVIIC at both N-type and P/Q-type VSCCs is likely to stem from the change in conformation and orientation of loop 2.

Accelerated chemical synthesis of peptides and small proteins.

The chemical synthesis of peptides and small proteins is a powerful complementary strategy to recombinant protein overexpression and is widely used in structural biology, immunology, protein engineering, and biomedical research. Despite considerable improvements in the fidelity of peptide chain assembly, side-chain protection, and postsynthesis analysis, a limiting factor in accessing polypeptides containing greater than 50 residues remains the time taken for chain assembly. The ultimate goal of this work is to establish highly efficient chemical procedures that achieve chain-assembly rates of approximately 10-15 residues per hour, thus underpinning the rapid chemical synthesis of long polypeptides and proteins, including cytokines, growth factors, protein domains, and small enzymes. Here we report Boc chemistry that employs O-(7-azabenzotriazol-1-yl)-N,N, N',N'-tetramethyluronium hexafluorophosphate (HATU)/dimethyl sulfoxide in situ neutralization as the coupling agent and incorporates a protected amino acid residue every 5 min to produce peptides of good quality. This rapid coupling chemistry was successfully demonstrated by synthesizing several small to medium peptides, including the "difficult" C-terminal sequence of HIV-1 proteinase (residues 81-99); fragment 65-74 of the acyl carrier protein; conotoxin PnIA(A10L), a potent neuronal nicotinic receptor antagonist; and the pro-inflammatory chemotactic protein CP10, an 88-residue protein, by means of native chemical ligation. The benefits of this approach include enhanced ability to identify and characterize "difficult couplings," rapid access to peptides for biological and structure-activity studies, and accelerated synthesis of tailored large peptide segments (<50 residues) for use in chemoselective ligation methods.

Functional determinants by which snake and cone snail toxins block the alpha 7 neuronal nicotinic acetylcholine receptors.

Snakes and cone snails produce toxins which block muscular and/or neuronal nicotinic acetylcholine receptors (AChRs). This paper mostly focuses on the determinants by which a snake long chain curaremimetic toxin and the cone snail toxin ImI bind specifically to the alpha 7 neuronal receptor. In both cases, the site involves a small turn-like structure constrained by two half-cystines.

Two distinct structures of alpha-conotoxin GI in aqueous solution.

The detailed analysis of conformational space of alpha-conotoxin GI in aqueous solution has been performed on the basis of two-dimensional NMR spectroscopy data using multiconformational approach. As the result, two topologically distinct interconvertible sets of GI conformations (populations of 78% and 22%) have been found. A common feature of the two sets is the Asn4-Cys7 beta-turn. The Gly8 to Tyrll region has a structure of right-handed helical turn in the major set and two sequential bends in the minor one. N-terminus and C-terminus also have different orientations, anti-parallel in the major conformational set and parallel in the minor one. An average pairwise rmsd for backbone heavy atoms is 0.56 A in the major set, 0.23 A in the minor, and 1.85 A between the structures of the two sets. The X-ray structure of GI [Guddat, L. W., Martin, J. A., Shan, L., Edmundson, A. B. & Gray, W. R. (1996) Biochemistry 35, 11329 - 11335] has the same folding pattern as the major NMR set, the average backbone rmsd between the two structures being 0.77 A.

Three-dimensional structure of kappa-conotoxin PVIIA, a novel potassium channel-blocking toxin from cone snails.

kappa-Conotoxin PVIIA from the venom of Conus purpurascens is the first cone snail toxin that was described to block potassium channels. We synthesized chemically this toxin and showed that its disulfide bridge pattern is similar to those of omega- and delta-conotoxins. kappa-conotoxin competes with radioactive alpha-dendrotoxin for binding to rat brain synaptosomes, confirming its capacity to bind to potassium channels; however, it behaves as a weak competitor. The three-dimensional structure of kappa-conotoxin PVIIA, as elucidated by NMR spectroscopy and molecular modeling, comprises two large parallel loops stabilized by a triple-stranded antiparallel beta-sheet and three disulfide bridges. The overall fold of kappa-conotoxin is similar to that of calcium channel-blocking omega-conotoxins but differs from those of potassium channel-blocking toxins from sea anemones, scorpions, and snakes. Local topographies of kappa-conotoxin PVIIA that might account for its capacity to recognize Kv1-type potassium channels are discussed.

Conantokin-T selectively antagonizes N-methyl-D-aspartate-evoked responses in rat hippocampal slice.

This study investigated the mode of action of conantokin-T, a 21 amino acid peptide toxin isolated from the venom of the fish-hunting cone snail Conus tulipa, on excitatory synaptic transmission in rat hippocampal slices using intracellular recording techniques. Superfusion of conantokin-T (1-500 nM) specifically and irreversibly decreased the pharmacologically isolated N-methyl-D-aspartate receptor (NMDA)-mediated excitatory postsynaptic potential (EPSPNMDA) in a concentration-dependent manner but had no effect on normal excitatory synaptic transmission (EPSP). The sensitivity of postsynaptic neurons to NMDA but not to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid was also antagonized by conantokin-T pretreatment. In addition, the conantokin-T-induced depression of EPSPNMDA could be antagonized by prior treatment of hippocampal slices with either DL-2-amino-5-phosphonovaleate (10 microM) or ifenprodil (20 microM). However, 7-chlorokynurenic acid (1 microM) had no effect on the action of conantokin-T. These findings indicated that conantokin-T modulates the NMDA receptor by an interaction with its glutamate binding site and polyamine recognition site.

Solution structure and proposed binding mechanism of a novel potassium channel toxin kappa-conotoxin PVIIA.

BACKGROUND: kappa-PVIIA is a 27-residue polypeptide isolated from the venom of Conus purpurascens and is the first member of a new class of conotoxins that block potassium channels. By comparison to other ion channels of eukaryotic cell membranes, voltage-sensitive potassium channels are relatively simple and methodology has been developed for mapping their interactions with small-peptide toxins. PVIIA, therefore, is a valuable new probe of potassium channel structure. This study of the solution structure and mode of channel binding of PVIIA forms the basis for mapping the interacting residues at the conotoxin-ion channel interface. RESULTS: The three-dimensional structure of PVIIA resembles the triple-stranded beta sheet/cystine-knot motif formed by a number of toxic and inhibitory peptides. Subtle structural differences, predominantly in loops 2 and 4, are observed between PVIIA and other conotoxins with similar structural frameworks, however. Electrophysiological binding data suggest that PVIIA blocks channel currents by binding in a voltage-sensitive manner to the external vestibule and occluding the pore. Comparison of the electrostatic surface of PVIIA with that of the well-characterised potassium channel blocker charybdotoxin suggests a likely binding orientation for PVIIA. CONCLUSIONS: Although the structure of PVIIA is considerably different to that of the alphaK scorpion toxins, it has a similar mechanism of channel blockade. On the basis of a comparison of the structures of PVIIA and charybdotoxin, we suggest that Lys19 of PVIIA is the residue which is responsible for physically occluding the pore of the potassium channel.

Calcium binding properties of synthetic gamma-carboxyglutamic acid-containing marine cone snail "sleeper" peptides, conantokin-G and conantokin-T.

Total chemical synthesis of two Conus-derived peptides, conantokin-G (con-G), a 17-residue polypeptide containing five residues of gamma-carboxyglutamic acid (Gla), and conantokin-T (con-T), a 21-residue polypeptide possessing four residues of Gla, was accomplished. Calcium binding isotherms were obtained for each peptide, and these differed considerably from each other. The binding isotherm for con-G was complex and could only be fit to degenerate models involving multiple Ca2+ binding sites. The data for Ca2+ binding to con-T was uniquely fit to a simple one-site model. In the case of con-G, circular dichroism (CD) studies revealed a polypeptide without observable alpha-helicity in the absence of Ca2+ and a dramatic shift to a high degree of alpha-helix at saturating Ca2+ concentrations. In contrast, apo-con-T possessed significant alpha-helical structure, and saturation with Ca2+ produced a less substantial change in its alpha-helical content. Titrations with Ca2+ of the change in alpha-helical content of con-T produced a C50 value for Ca2+ that was essentially the same as its Kd from direct binding studies, demonstrating that occupancy of the single macroscopic binding site resulted in the conformational change. Similar titrations with con-G provided a C50 value in concert with the Kd for binding of Ca2+ to this peptide. Moreover, in agreement with these particular Ca(2+)-induced structural changes, gel filtration analyses demonstrated significantly reduced hydrodynamic volumes of both of these polypeptides after saturation of their apo forms with Ca2+, with con-G showing a more pronounced change than con-T. One-dimensional H-NMR spectra showed both line broadening and changes in chemical shifts of several peptide amide proton resonances after addition of Ca2+ to con-G, again suggestive of a large Ca(2+)-induced conformational change in this polypeptide. A derivative of con-G was synthesized with all amino acids present in the D-configuration (D-con-G). This variant peptide displayed Ca2+ binding isotherms nearly identical to those of con-G and underwent a Ca(2+)-induced conformational change very similar to that of con-G. Intracranial injections of con-G and con-T in young (< 2 weeks) and older (3-4 weeks) mice produced the expected "sleep-like" and hyperactive effects, respectively. The variant, D-con-G, was inactive in these assays. These studies demonstrate that synthetic con-G and con-T possess their expected bioactivities and undergo large and defined conformational alterations in the presence of Ca2+. We propose that binding of Ca2+ to these polypeptides contributes to their ability to adopt a defined conformation, and this divalent cation-dependent conformation is necessary for their neuroactivities.

Synthesis and biological characterization of a series of analogues of omega-conotoxin GVIA.

The 27-residue polypeptide omega-conotoxin GVIA (omega-CgTx), from the venom of the cone shell Conus geographus, blocks N-type neuronal calcium channels. It contains three disulphide bridges. We report here the synthesis and biological characterization of a series of analogues in which one disulphide has been replaced by substitution of appropriate Cys residues with Ser, viz. [Ser1,16]-omega -CgTx, [Ser8,19]-omega-CgTx, [Ser15,26)-omega-CgTx, [Ser16]-omega-CgTx8-27 and [Ser15]-omega-CgTx1-19. All syntheses were conducted manually using either Boc or Fmoc methodology. Deprotected peptides were oxidized to their bridged forms using either aerial oxidation or aqueous dimethyl sulphoxide. Peptides were purified using RP-HPLC, and their purity and identity were checked by RP-HPLC, capillary electrophoresis and mass spectrometry. Inhibition of neuronal N-type calcium channels was assessed as the inhibition of the twitch responses of rat vas deferens stimulated with single electrical pulses at 20 second intervals. None of these analogues was biologically active, suggesting that the disulphides play an important role in maintaining biological activity.

Synthesis of gamma-carboxyglutamic acid-containing peptides by the Boc strategy.

gamma-Carboxyglutamic acid (Gla) derivatives having several protecting groups at the gamma-carboxyl function were synthesized and examined for their stabilities and removabilities under the conditions used in peptide synthesis by the Boc strategy. Among them, the cyclohexyl (cHx) group of the Gla residue was found to be stable during the synthesis of the protected peptides, but was quantitatively cleaved by the final HF treatment without decarboxylation. Using Boc-Gla(OcHx)2-OH as a starting material, the synthesis of Gla-containing peptides was achieved by the Boc strategy using a standard HF procedure for the final deprotection.

alpha-Conotoxins, small peptide probes of nicotinic acetylcholine receptors.

alpha-Conotoxins, a family of small peptides from the venoms of the Conus marine moluscs, are selective, snake alpha-neurotoxin-competitive antagonists of the nicotinic acetylcholine receptor. A new alpha-conotoxin, SIA, has been purified, sequenced, and synthesized. Cross-linking with bivalent reagents and photoaffinity labeling of the acetylcholine receptor with alpha-conotoxin yield covalent adducts. Surprisingly, cross-linking to other subunits is considerably more efficient than to the alpha subunit. The relative efficiency of photoactivatable cross-linking to different subunits of the receptor is a function of placement of the photoactivatable group on the toxin. Since the structures of alpha-conotoxins can be solved by 2D NMR [see Pardi et al. (1989) Biochemistry 28, 5494-5508; Kobayashi et al. (1989) Biochemistry 28, 4853-4860], this family of toxins should provide a set of new ligands for probing the acetylcholine receptor with considerable precision.

Conopressins and their analogs: synthesis, antidiuretic and behavioral effects.

[Lys8]-Conopressin G (L1), and [Arg8]-Conopressin S (A1) and their four analogs were synthesized using solid phase procedure. These analogs are [2-thiopropionic acid1, lys8]-conopressin (L2), [2-thiopropionic acid1, Arg8]-conopressin (A2), [cis-4-methyl-1-thiocyclohexaneacetic acid1, Lys8], conopressin (L3), and [cis-4-methyl-1-thiocyclohexaneacetic acid1, ARg8]-conopressin (A3). Behavioral and diuretic effects of all six peptides were compared with these of [Arg8]-vasopressin (AVP). Conopressin A1, and L1 and their analogs A2, A3, L2, L3, induced antidiuretic effects. After icv injection of some conopressins, barrel rotatory behavior of rats was observed.

Conantokin-T. A gamma-carboxyglutamate containing peptide with N-methyl-d-aspartate antagonist activity.

Conantokin-T, a 21-amino acid peptide which induces sleep-like symptoms in young mice was purified from the venom of the fish-hunting cone snail, Conus tulipa. The amino acid sequence of the peptide was determined and verified by chemical synthesis. The peptide has 4 residues of the modified amino acid, gamma-carboxyglutamate (Gla). The sequence of the peptide is: Gly-Glu-Gla-Gla-Tyr-Gln-Lys-Met-Leu-Gla-Asn-Leu-Arg-Gla-Ala-Glu-Val-Lys- Lys-Asn-Ala-NH2. Conantokin-T inhibits N-methyl-D-aspartate (NMDA) receptor-mediated calcium influx in central nervous system neurons. This observation suggests that like conantokin-G (a homologous Conus peptide with recently identified NMDA antagonist activity) conantokin-T has NMDA antagonist activity. A sequence comparison of conantokins-T and -G identifies the 4 Gla residues and the N-terminal dipeptide sequence as potential key elements for the biological activity of this peptide.

Synthesis of mu-conotoxin GIIIA: a chemical probe for sodium channels.

mu-Conotoxin GIIIA, a 22 amino acid peptide paralytic toxin which inhibits the muscle voltage-activated sodium channels, was synthesized by a solid phase method. No purification of intermediates was necessary for the synthesis, and a simple air oxidation of the deprotected crude peptide gave the desired toxin. By all criteria applied, the synthetic material was indistinguishable from the authentic natural toxin.