Chemical Synthesis and Characterization of an Equinatoxin II(1-85) Analogue.

The chemical synthesis of an 85 residue analogue of the pore-forming protein, Equinatoxin II (EqtII), was achieved. Peptide precursors with over 40 residues were assembled by solid phase synthesis. The EqtII(1-46) fragment was modified to the reactive C-terminal thioester and native chemical ligation was performed with the A47C mutated EqtII(47-85) peptide to form the EqtII(1-85) analogue. Circular dichroism spectroscopy indicated that the N-terminal domain of EqtII(1-46) and EqtII(1-85) maintains predominantly an α-helical structure in solution and also in the presence of lipid micelles. This demonstrates the feasibility of assembling the full 179 residue protein EqtII via chemical means. Site-specific isotopic labels could be incorporated for structural studies in membranes by solid-state NMR spectroscopy.

Native chemical ligation at Asx-Cys, Glx-Cys: chemical synthesis and high-resolution X-ray structure of ShK toxin by racemic protein crystallography.

We have re-examined the utility of native chemical ligation at -Gln/Glu-Cys- [Glx-Cys] and -Asn/Asp-Cys- [Asx-Cys] sites. Using the improved thioaryl catalyst 4-mercaptophenylacetic acid (MPAA), native chemical ligation could be performed at -Gln-Cys- and Asn-Cys- sites without side reactions. After optimization, ligation at a -Glu-Cys- site could also be used as a ligation site, with minimal levels of byproduct formation. However, -Asp-Cys- is not appropriate for use as a site for native chemical ligation because of formation of significant amounts of ß-linked byproduct. The feasibility of native chemical ligation at -Gln-Cys- enabled a convergent total chemical synthesis of the enantiomeric forms of the ShK toxin protein molecule. The D-ShK protein molecule was ~50,000-fold less active in blocking the Kv1.3 channel than the L-ShK protein molecule. Racemic protein crystallography was used to obtain high-resolution X-ray diffraction data for ShK toxin. The structure was solved by direct methods and showed significant differences from the previously reported NMR structures in some regions of the ShK protein molecule.

The membranotropic activity of N-terminal peptides from the pore-forming proteins sticholysin I and II is modulated by hydrophobic and electrostatic interactions as well as lipid composition.

The sea anemone Stichodactyla helianthus produces two pore-forming proteins, sticholysins I and II (St I and St II). Despite their high identity (93%), these toxins exhibit differences in hemolytic activity that can be related to those found in their N-terminal. To clarify the contribution of the N-terminal amino acid residues to the activity of the toxins, we synthesized peptides spanning residues 1-31 of St I (StI1-31) or 1-30 of St II (StII1-30) and demonstrated that StII1-30 promotes erythrocyte lysis to a higher extent than StI1-31. For a better understanding of the molecular mechanism underlying the peptide activity, here we studied their binding to lipid monolayers and pemeabilizing activity in liposomes. For this, we examined the effect on peptide membranotropic activity of including phospatidic acid and cholesterol in a lipid mixture of phosphatidylcholine and sphingomyelin. The results suggest the importance of continuity of the 1-10 hydrophobic sequence in StII1-30 for displaying higher binding and activity, in spite of both peptides' abilities to form pores in giant unilamellar vesicles. Thus, the different peptide membranotropic action is explained in terms of the differences in hydrophobic and electrostatic peptide properties as well as the enhancing role of membrane inhomogeneities.

Bunodosine 391: an analgesic acylamino acid from the venom of the sea anemone Bunodosoma cangicum.

A new acylamino acid, bunodosine 391 (BDS 391), was isolated from the venom of the sea anemone Bunodosoma cangicum. The structure was elucidated by spectroscopic analyses (2D NMR, ESIMS/MS) and verified by its synthesis. Intraplantar injection of BDS 391 into the hind paw of a rat induced a potent analgesic effect. This effect was not altered by naloxone (an opioid receptor antagonist), but was completely reversed by methysergide (a serotonin receptor antagonist), indicating that the effect is mediated by activation of serotonin receptors.

Chemical synthesis and folding of APETx2, a potent and selective inhibitor of acid sensing ion channel 3.

Acid sensing ion channels (ASICs) are pH-sensitive channels that are distributed in the central and peripheral nervous system and which are believed to play a key role in pain perception. APETx2, a 42-residue peptide toxin isolated from the sea anemone Anthopleura elegantissima, is the only known selective inhibitor of ASIC3 channels. Here we describe the total chemical synthesis of APETx2 by solid-phase peptide synthesis and native chemical ligation. The folded synthetic toxin had an IC(50) of 57 nM for inhibition of rat ASIC3 channels expressed in Xenopus oocytes, in agreement with the IC(50) reported for the native toxin (63 nM). The native chemical ligation approach should provide an efficient route for synthesis of other pharmacologically useful disulfide-rich toxins from venomous animals.

Model peptides mimic the structure and function of the N-terminus of the pore-forming toxin sticholysin II.

To investigate the role of the N-terminal region in the lytic mechanism of the pore-forming toxin sticholysin II (St II), we studied the conformational and functional properties of peptides encompassing the first 30 residues of the protein. Peptides containing residues 1-30 (P1-30) and 11-30 (P11-30) were synthesized and their conformational properties were examined in aqueous solution as a function of peptide concentration, pH, ionic strength, and addition of the secondary structure-inducing solvent trifluoroethanol (TFE). CD spectra showed that increasing concentration, pH, and ionic strength led to aggregation of P1-30; as a consequence, the peptide acquired beta-sheet conformation. In contrast, P11-30 exhibited practically no conformational changes under the same conditions, remaining essentially structureless. Moreover, this peptide did not undergo aggregation. These differences clearly point to the modulating effect of the first 10 hydrophobic residues on the peptides aggregation and conformational properties. In TFE both the first ten hydrophobic peptides acquired alpha-helical conformation, albeit to a different extent, P11-30 displayed lower alpha-helical content. P1-30 presented a larger fraction of residues in alpha-helical conformation in TFE than that found in St II's crystal structure for that portion of the protein. Since TFE mimics the membrane environment, such increase in helical content could also occur upon toxin binding to membranes and represent a step in the mechanism of pore formation. The peptides conformational properties correlated well with their functional behavior. Thus, P1-30 exhibited much higher hemolytic activity than P11-30. In addition, P11-30 was able to block the toxin's hemolytic activity. The size of pores formed in red blood cells by P1-30 was estimated by measuring the permeability to PEGs of different molecular mass. The pore radius (0.95 +/- 0.01 nm) was very similar to that of the pore formed by the toxin. The results demonstrate that the synthetic peptide P1-30 is a good model of St II conformation and function and emphasize the contribution of the toxin's N-terminal region, and, in particular, the hydrophobic residues 1-10 to pore formation.

Design and synthesis of type-III mimetics of ShK toxin.

ShK toxin is a structurally defined, 35-residue polypeptide which blocks the voltage-gated Kv1.3 potassium channel in T-lymphocytes and has been identified as a possible immunosuppressant. Our interest lies in the rational design and synthesis of type-III mimetics of protein and polypeptide structure and function. ShK toxin is a challenging target for mimetic design as its binding epitope consists of relatively weakly binding residues, some of which are discontinuous. We discuss here our investigations into the design and synthesis of 1st generation, small molecule mimetics of ShK toxin and highlight any principles relevant to the generic design of type-III mimetics of continuous and discontinuous binding epitopes. We complement our approach with attempted pharmacophore-based database mining.

Synthesis of the C(1)-C(18) segment of lophotoxin and pukalide. Control of 2-alkenylfuran (E/Z)-configuration.

[reaction: see text] The convergent synthesis of the fully functionalized C(1)-C(18) segment 24 of the furanocembranes lophotoxin and pukalide was accomplished in 11 steps and 10% overall yield. The key step was a stereoselective conversion of alkynoate 21 to trimethylsilyl 2-alkenylfuran 22.

Designed peptide analogues of the potassium channel blocker ShK toxin.

ShK toxin, a potassium channel blocker from the sea anemone Stichodactyla helianthus, is a 35-residue polypeptide cross-linked by 3 disulfide bridges. In an effort to generate truncated peptidic analogues of this potent channel blocker, we have evaluated three analogues, one in which the native sequence was truncated and then stabilized by the introduction of additional covalent links (a non-native disulfide and two lactam bridges), and two in which non-native structural scaffolds stabilized by disulfide and/or lactam bridges were modified to include key amino acid residues from the native toxin. The effect of introducing a lactam bridge in the first helix of ShK toxin (to create cyclo14/18[Lys14,Asp18]ShK) was also examined to confirm that this modification was compatible with activity. All four analogues were tested in vitro for their ability to block Kv1.3 potassium channels in Xenopus oocytes, and their solution structures were determined using 1H NMR spectroscopy. The lactam bridge in full-length ShK is well tolerated, with only a 5-fold reduction in binding to Kv1.3. The truncated and stabilized analogue was inactive, apparently due to a combination of slight deviations from the native structure and alterations to side chains required for binding. One of the peptide scaffolds was also inactive because it failed to adopt the required structure, but the other had a K(d) of 92 microM. This active peptide incorporated mimics of Lys22 and Tyr23, which are essential for activity in ShK, and an Arg residue that could mimic Arg11 or Arg24 in the native toxin. Modification of this peptide should produce a more potent, low molecular weight peptidic analogue which will be useful not only for further in vitro and in vivo studies of the effect of blocking Kv1.3, but also for mapping the interactions with the pore and vestibule of this K(+) channel that are required for potent blockade.

A helical capping motif in ShK toxin and its role in helix stabilization.

ShK toxin, a 35-residue polypeptide cross-linked by three disulfides, is a potent blocker of voltage-gated potassium channels and is of interest as a lead in the development of new immunosuppressant agents. ShK toxin contains two short stretches of alpha-helix, the first of which is preceded by a putative N-capping box encompassing residues Thr13 and Gln16. (1)H and (13)C NMR data support the presence of this structural motif, but the hydrogen bonds involving residues 13 and 16 in the solution structure of ShK toxin do not match the pattern expected for a conventional N-cap motif. They do, however, fit the pattern for the recently described ST-motif, class 4a (Wan and Milner-White (1999) Journal of Molecular Biology, 1999, Vol. 286, pp. 1651-1662). The (1)H NMR chemical shifts, nuclear Overhauser effects, and amide exchange rates of native ShK toxin are compared with those of three synthetic analogues with the substitutions Thr13 to Ala and Gln16 to Glu and Ala in order to determine the contribution of this motif to the structure and stability of ShK toxin. Disruption of the capping interactions destabilizes the helices, with the Thr13 to Ala substitution being much more disruptive than Gln16 to Ala, consistent with the lack of hydrogen bonding to the side chain of residue i + 4 in a class 4a ST-motif. Mutation of residues 13 and 16 has only a minor effect on potassium channel binding, probably because the disulfide bonding network minimizes the effect of loss of the capping motif on the overall structure. The implications of these findings for the design of ShK analogues are discussed.

A potassium-channel toxin from the sea anemone Bunodosoma granulifera, an inhibitor for Kv1 channels. Revision of the amino acid sequence, disulfide-bridge assignment, chemical synthesis, and biological activity.

The potassium channel toxin secreted by the sea anemone Bunodosoma granulifera (BgK) is a 37-amino-acid peptide containing three disulfide bridges. Because a synthetic peptide corresponding to the reported sequence of BgK was found not to fold properly, the sequence was determined again. The new sequence differed from the previous one in the C-terminal tetrapeptide, which contains two cysteines involved in disulfide bridging. The revised sequence is: V C R D W F K E T A C R H A K S L G N C R T S Q K Y R A N C A K T C E L C. The toxin BgK was synthesized according to the new sequence and folded successfully. Disulfide bridges were assigned by peptide mapping on both natural and synthetic forms to be between Cys2-Cys37, Cys11-Cys30 and Cys20-Cys34. The toxin contains a C-terminal free carboxylate as shown by comparing the native toxin with two synthetic peptides containing the C-terminus in either the carboxylate or carboxamido form. Synthetic BgK inhibits binding of 125I-alpha-dendrotoxin to rat brain synaptosomal membranes, similarly to natural BgK (nanomolar range). No activity was observed on maxi-K+ channels incorporated into planar lipid bilayers. The ability of BgK to block voltage-dependent K+ channels was determined from recordings of whole cell currents in Xenopus oocytes injected with cRNA encoding three cloned Kv1 channels (Kv1.1, Kv1.2, Kv1.3) and one Kv3 (Kv3.1) channel. The Shaker-related Kv1 channels are equally affected by BgK, while the Shaw-related channel Kv3.1 is insensitive up to 0.125 microM toxin. Indeed, half blockage of the current through the three Kv1 channels tested occurred in the same concentration range (Kd = 6 nM for Kv1.1, 15 nM for Kv1.2, 10 nM for Kv1.3). The specificity of BgK for the Shaker-related K+ channels indicates that BgK is able to discriminate a large group of neuronal Kv1 channels in situ. The sequence, the disulfide bridge pattern, the secondary structure and the biological activity of BgK demonstrated that the sea anemone toxins, i.e. BgK, ShK and Kaliseptine, constitute novel molecular probes useful for investigating K+ channel properties.

An essential binding surface for ShK toxin interaction with rat brain potassium channels.

An "Ala scan" analysis of ShK toxin, a 35-residue basic peptide possessing three disulfide bonds, identifies seven side chains which influence binding to brain delayed rectifier potassium channels. Additional analogs were synthesized and tested to further decipher the roles of these residues, particularly Tyr23. The inhibitory effects of these analogs on 125I-labeled dendrotoxin binding to rat brain membranes showed that replacement of Tyr23 with Ala drastically lowered the affinity of the toxin for the Kv1.2 channels. Ala substitution of Phe27 reduced potency more than 15-fold. Monosubstituted Ala analogs for Ile7, Ser20, or Lys30 each displayed 5-fold reductions in potency. Thus, aromaticity at position 23 is important for effective delayed rectifier brain K channel binding. In contrast, the aromatic residue at position 27 was not critical, since cyclohexylalanine substitution increased affinity. The solution structure of ShK toxin clusters Ile7, Arg11, Ser20, Lys22, Tyr23, and Phe27 in close proximity, forming the potassium channel binding surface of the toxin. We propose an essential binding surface on the toxin in which Lys22 and Tyr23 are major contributors, through ionic and aromatic (hydrophobic) interactions, with the potassium channel.

Identification of three separate binding sites on SHK toxin, a potent inhibitor of voltage-dependent potassium channels in human T-lymphocytes and rat brain.

Eighteen synthetic analogs of ShK toxin, a thirty-five residue K channel blocker derived from the sea anemone Stichodactyla helianthus, were prepared in order to identify functionally important residues. CD spectra of sixteen of the analogs were virtually identical with the spectrum of wild-type toxin, indicating that the conformations were not affected by the substitutions. A conserved residue, Lys22, is essential for ShK binding to rat brain K channels which are primarily of the Kv1.2 type. However, a cationic side chain at position 22 is not essential for binding to the human Jurkat T-lymphocyte Kv1.3 channel. While decreasing bulkiness at this position affected toxin affinity for the brain K channels, increasing bulkiness decreased toxin affinity for both brain and lymphocyte K channels. In contrast to the rat brain channels, ShK binding to Kv1.3 was sensitive to substitution at Lys9 and Arg11.

Chemical synthesis and characterization of ShK toxin: a potent potassium channel inhibitor from a sea anemone.

ShK-toxin, a 35 residue peptide isolated from the sea anemone Stichodactyla helianthus, was synthesized using an Fmoc strategy and successfully folded to the biologically active form containing three intramolecular disulfide bonds. The ability of synthetic ShK toxin to inhibit specific [125I]-dendrotoxin I binding to rat brain membranes slightly exceeded (was more potent than) that of the natural ShK toxin sample, but was comparable with previously reported data for ShK toxin. The peptide toxin inhibited [125I]-charybdotoxin binding to Jurkat T lymphocytes with an IC50 value of 32 pM. In addition, Jurkat T lymphocytes Kv1.3 potassium channels were inhibited with an IC50 value of 133 pM. Owing to their unique structure and high affinity for at least some potassium channels, ShK toxin and related sea anemone potassium channel toxins may become useful molecular probes for investigating potassium channels.

Chemical synthesis of a neurotoxic polypeptide from the sea anemone Stichodactyla helianthus.

The sea anemone Stichodactyla helianthus neurotoxin I, a 48-residue polypeptide, was synthesized by automated solid phase methodology. The fully reduced polypeptide was subsequently refolded in the presence of a glutathione oxidoreduction buffer to the biologically active species containing three disulfide bonds. The overall yield after rigorous purification was 12.5%. The circular dichroism (CD), and proton nuclear magnetic resonance (1H NMR) spectra of the HPLC-purified synthetic toxin were indistinguishable from those obtained concurrently with the natural toxin. A subtilisin digest of the synthetic neurotoxin generated peptide fragments identical to that of a sample of the natural toxin subjected to the same treatment. The toxicity of the synthetic polypeptide was identical to that of the natural toxin (crab LD50, 3.1 micrograms/kg). The equilibrium dissociation constant (28 nM) for interaction of the synthetic toxin with crab axolemma vesicles was nearly identical to that of the natural toxin (25 nM).

An analog of lophotoxin reacts covalently with Tyr190 in the alpha-subunit of the nicotinic acetylcholine receptor.

Lophotoxin and lophotoxin analog-1 are natural diterpenes from coral that inhibit nicotinic acetylcholine receptors by covalent reaction with the acetylcholine recognition sites on the alpha-subunits. Although both toxins contain potentially reactive epoxides and alpha,beta-unsaturated aldehydes, the mechanism of their covalent reaction with the receptor is not known. The role of the alpha,beta-unsaturated aldehyde in analog-1 was investigated by reduction of the aldehyde to an alcohol with [3H]NaBH4. The reduced [3H]analog-1 bound selectively and covalently to the alpha-subunit of the receptor. Covalent binding was inhibited by agonists and antagonists, but not by noncompetitive allosteric inhibitors. The apparent dissociation constant of the reduced [3H]analog-1 was approximately 1.5 x 10(-6) M. These results demonstrate that the alpha,beta-unsaturated aldehyde in analog-1 is not an absolute requirement for covalent reaction with the receptor. Receptors were treated with the reduced-[3H]analog-1, and the labeled alpha-subunits were isolated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and digested with staphylococcal V8 protease. A labeled 20-kDa V8 protease fragment was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase high performance liquid chromatography and subjected to sequence analysis. A peptide beginning at Ser173 was identified, and the label appeared in the 18th step corresponding to Tyr190. This assignment was confirmed by digestion of the labeled 20-kDa V8 protease fragment with cyanogen bromide, followed by purification of the labeled cyanogen bromide peptide on reverse-phase high performance liquid chromatography. A peptide beginning at Lys179 was identified, and the label appeared in the 12th step, again corresponding to Tyr190. Tyr190 may react with the coral toxin by nucleophilic addition at one of the carbons associated with an epoxide, and may form part of the alkylammonium-binding subsite of the acetylcholine recognition site.

Synthesis of sequences 1-16 and 63-95 of Cerebratulus lacteus toxin AIII. Hemolytic activity in a toxin fragment.

Sequences 1-16 and 63-95 of C. lacteus toxin AIII were synthesized on benzhydrylamine and PAM resins respectively; the crude peptide products released by HF contained 90 and 33% of the target peptides. Fragment 63-95 was nearly a full agonist, producing greater than 90% of the hemolytic activity of the intact toxin molecule at 10(3)-fold higher concentrations. Fragment 1-16 had no activity.