Structural Basis for the Inhibition of Voltage-gated Sodium Channels by Conotoxin µO§-GVIIJ.

Cone snail toxins are well known blockers of voltage-gated sodium channels, a property that is of broad interest in biology and therapeutically in treating neuropathic pain and neurological disorders. Although most conotoxin channel blockers function by direct binding to a channel and disrupting its normal ion movement, conotoxin µO§-GVIIJ channel blocking is unique, using both favorable binding interactions with the channel and a direct tether via an intermolecular disulfide bond. Disulfide exchange is possible because conotoxin µO§-GVIIJ contains anS-cysteinylated Cys-24 residue that is capable of exchanging with a free cysteine thiol on the channel surface. Here, we present the solution structure of an analog of µO§-GVIIJ (GVIIJ[C24S]) and the results of structure-activity studies with synthetic µO§-GVIIJ variants. GVIIJ[C24S] adopts an inhibitor cystine knot structure, with two antiparallel ß-strands stabilized by three disulfide bridges. The loop region linking the ß-strands (loop 4) presents residue 24 in a configuration where it could bind to the proposed free cysteine of the channel (Cys-910, rat NaV1.2 numbering; at site 8). The structure-activity study shows that three residues (Lys-12, Arg-14, and Tyr-16) located in loop 2 and spatially close to residue 24 were also important for functional activity. We propose that the interaction of µO§-GVIIJ with the channel depends on not only disulfide tethering via Cys-24 to a free cysteine at site 8 on the channel but also the participation of key residues of µO§-GVIIJ on a distinct surface of the peptide.

Interactions of disulfide-deficient selenocysteine analogs of µ-conotoxin BuIIIB with the α-subunit of the voltage-gated sodium channel subtype 1.3.

Inhibitors of the α-subunit of the voltage-gated sodium channel subtype 1.3 (NaV 1.3) are of interest as pharmacological tools for the study of neuropathic pain associated with spinal cord injury and have potential therapeutic applications. The recently described µ-conotoxin BuIIIB (µ-BuIIIB) from Conus bullatus was shown to block NaV 1.3 with submicromolar potency (Kd = 0.2 µm), making it one of the most potent peptidic inhibitors of this subtype described to date. However, oxidative folding of µ-BuIIIB results in numerous folding isoforms, making it difficult to obtain sufficient quantities of the active form of the peptide for detailed structure-activity studies. In the present study, we report the synthesis and characterization of µ-BuIIIB analogs incorporating a disulfide-deficient, diselenide-containing scaffold designed to simplify synthesis and facilitate structure-activity studies directed at identifying amino acid residues involved in NaV 1.3 blockade. Our results indicate that, similar to other µ-conotoxins, the C-terminal residues (Trp16, Arg18 and His20) are most crucial for NaV 1 blockade. At the N-terminus, replacement of Glu3 by Ala resulted in an analog with an increased potency for NaV 1.3 (Kd = 0.07 µm), implicating this position as a potential site for modification for increased potency and/or selectivity. Further examination of this position showed that increased negative charge, through γ-carboxyglutamate replacement, decreased potency (Kd = 0.33 µm), whereas replacement with positively-charged 2,4-diamonobutyric acid increased potency (Kd = 0.036 µm). These results provide a foundation for the design and synthesis of µ-BuIIIB-based analogs with increased potency against NaV 1.3.

A disulfide tether stabilizes the block of sodium channels by the conotoxin µO§-GVIIJ.

A cone snail venom peptide, µO§-conotoxin GVIIJ from Conus geographus, has a unique posttranslational modification, S-cysteinylated cysteine, which makes possible formation of a covalent tether of peptide to its target Na channels at a distinct ligand-binding site. µO§-conotoxin GVIIJ is a 35-aa peptide, with 7 cysteine residues; six of the cysteines form 3 disulfide cross-links, and one (Cys24) is S-cysteinylated. Due to limited availability of native GVIIJ, we primarily used a synthetic analog whose Cys24 was S-glutathionylated (abbreviated GVIIJSSG). The peptide-channel complex is stabilized by a disulfide tether between Cys24 of the peptide and Cys910 of rat (r) NaV1.2. A mutant channel of rNaV1.2 lacking a cysteine near the pore loop of domain II (C910L), was >10(3)-fold less sensitive to GVIIJSSG than was wild-type rNaV1.2. In contrast, although rNaV1.5 was >10(4)-fold less sensitive to GVIIJSSG than NaV1.2, an rNaV1.5 mutant with a cysteine in the homologous location, rNaV1.5[L869C], was >10(3)-fold more sensitive than wild-type rNaV1.5. The susceptibility of rNaV1.2 to GVIIJSSG was significantly altered by treating the channels with thiol-oxidizing or disulfide-reducing agents. Furthermore, coexpression of rNaVß2 or rNaVß4, but not that of rNaVß1 or rNaVß3, protected rNaV1.1 to -1.7 (excluding NaV1.5) against block by GVIIJSSG. Thus, GVIIJ-related peptides may serve as probes for both the redox state of extracellular cysteines and for assessing which NaVß- and NaVα-subunits are present in native neurons.

Distinct disulfide isomers of µ-conotoxins KIIIA and KIIIB block voltage-gated sodium channels.

In the preparation of synthetic conotoxins containing multiple disulfide bonds, oxidative folding can produce numerous permutations of disulfide bond connectivities. Establishing the native disulfide connectivities thus presents a significant challenge when the venom-derived peptide is not available, as is increasingly the case when conotoxins are identified from cDNA sequences. Here, we investigate the disulfide connectivity of µ-conotoxin KIIIA, which was predicted originally to have a [C1-C9,C2-C15,C4-C16] disulfide pattern based on homology with closely related µ-conotoxins. The two major isomers of synthetic µ-KIIIA formed during oxidative folding were purified and their disulfide connectivities mapped by direct mass spectrometric collision-induced dissociation fragmentation of the disulfide-bonded polypeptides. Our results show that the major oxidative folding product adopts a [C1-C15,C2-C9,C4-C16] disulfide connectivity, while the minor product adopts a [C1-C16,C2-C9,C4-C15] connectivity. Both of these peptides were potent blockers of Na(V)1.2 (K(d) values of 5 and 230 nM, respectively). The solution structure for µ-KIIIA based on nuclear magnetic resonance data was recalculated with the [C1-C15,C2-C9,C4-C16] disulfide pattern; its structure was very similar to the µ-KIIIA structure calculated with the incorrect [C1-C9,C2-C15,C4-C16] disulfide pattern, with an α-helix spanning residues 7-12. In addition, the major folding isomers of µ-KIIIB, an N-terminally extended isoform of µ-KIIIA identified from its cDNA sequence, were isolated. These folding products had the same disulfide connectivities as µ-KIIIA, and both blocked Na(V)1.2 (K(d) values of 470 and 26 nM, respectively). Our results establish that the preferred disulfide pattern of synthetic µ-KIIIA and µ-KIIIB folded in vitro is 1-5/2-4/3-6 but that other disulfide isomers are also potent sodium channel blockers. These findings raise questions about the disulfide pattern(s) of µ-KIIIA in the venom of Conus kinoshitai; indeed, the presence of multiple disulfide isomers in the venom could provide a means of further expanding the snail's repertoire of active peptides.

Structure/function characterization of micro-conotoxin KIIIA, an analgesic, nearly irreversible blocker of mammalian neuronal sodium channels.

Peptide neurotoxins from cone snails continue to supply compounds with therapeutic potential. Although several analgesic conotoxins have already reached human clinical trials, a continuing need exists for the discovery and development of novel non-opioid analgesics, such as subtype-selective sodium channel blockers. Micro-conotoxin KIIIA is representative of micro-conopeptides previously characterized as inhibitors of tetrodotoxin (TTX)-resistant sodium channels in amphibian dorsal root ganglion neurons. Here, we show that KIIIA has potent analgesic activity in the mouse pain model. Surprisingly, KIIIA was found to block most (>80%) of the TTX-sensitive, but only approximately 20% of the TTX-resistant, sodium current in mouse dorsal root ganglion neurons. KIIIA was tested on cloned mammalian channels expressed in Xenopus oocytes. Both Na(V)1.2 and Na(V)1.6 were strongly blocked; within experimental wash times of 40-60 min, block was reversed very little for Na(V)1.2 and only partially for Na(V)1.6. Other isoforms were blocked reversibly: Na(V)1.3 (IC50 8 microM), Na(V)1.5 (IC50 284 microM), and Na(V)1.4 (IC50 80 nM). "Alanine-walk" and related analogs were synthesized and tested against both Na(V)1.2 and Na(V)1.4; replacement of Trp-8 resulted in reversible block of Na(V)1.2, whereas replacement of Lys-7, Trp-8, or Asp-11 yielded a more profound effect on the block of Na(V)1.4 than of Na(V)1.2. Taken together, these data suggest that KIIIA is an effective tool to study structure and function of Na(V)1.2 and that further engineering of micro-conopeptides belonging to the KIIIA group may provide subtype-selective pharmacological compounds for mammalian neuronal sodium channels and potential therapeutics for the treatment of pain.

Conotoxins containing nonnatural backbone spacers: cladistic-based design, chemical synthesis, and improved analgesic activity.

Disulfide-rich neurotoxins from venomous animals continue to provide compounds with therapeutic potential. Minimizing neurotoxins often results in removal of disulfide bridges or critical amino acids. To address this drug-design challenge, we explored the concept of disulfide-rich scaffolds consisting of isostere polymers and peptidic pharmacophores. Flexible spacers, such as amino-3-oxapentanoic or 6-aminohexanoic acids, were used to replace conformationally constrained parts of a three-disulfide-bridged conotoxin, SIIIA. The peptide-polymer hybrids, polytides, were designed based on cladistic identification of nonconserved loci in related peptides. After oxidative folding, the polytides appeared to be better inhibitors of sodium currents in dorsal root ganglia and sciatic nerves in mice. Moreover, the polytides appeared to be significantly more potent and longer-lasting analgesics in the inflammatory pain model in mice, when compared to SIIIA. The resulting polytides provide a promising strategy for transforming disulfide-rich peptides into therapeutics.

Albumin is a redox-active crowding agent that promotes oxidative folding of cysteine-rich peptides.

Oxidative folding that occurs in a crowded cellular milieu is characterized by multifaceted interactions that occur among nascent polypeptides and resident components of the endoplasmic reticulum (ER) lumen. Macromolecular crowding has been considered an essential factor in the folding of polypeptides, but the excluded volume effect has not been evaluated for small, disulfide-rich peptides. In the research presented, we examined how macromolecular crowding agents, such as albumin, ovalbumin, and polysaccharides, influenced the kinetics and thermodynamics of forming disulfide bonds in four model peptides of varying molecular size from 13 residues (1.4 kDa) to 58-residues (6.5 kDa): conotoxins: GI, PVIIA, r11a, and bovine pancreatic trypsin inhibitor. Our results indicate that the excluded volume effect does not significantly alter the folding rates nor equilibria for these peptides. In stark contrast, folding reactions were dramatically accelerated, when protein-based crowding agents were present at concentrations lower than those predicted to provide the excluded volume effect. Submillimolar albumin alone was as effective as glutathione in promoting the oxidative folding of GI conotoxin at concentrations typically found in the ER. To the best of our knowledge, this is the first report and quantitative characterization of oxidative folding of peptides mediated by other than thioredoxin-based protein disulfide bonds. Our work raises a possibility that concurrent secretory and ER-resident proteins may influence the oxidative folding of small, cysteine-rich peptides not as crowding agents, but as redox-active factors.

Oxidative folding of conotoxins sharing an identical disulfide bridging framework.

Conotoxins are short, disulfide-rich peptide neurotoxins produced in the venom of predatory marine cone snails. It is generally accepted that an estimated 100,000 unique conotoxins fall into only a handful of structural groups, based on their disulfide bridging frameworks. This unique molecular diversity poses a protein folding problem of relationships between hypervariability of amino acid sequences and mechanism(s) of oxidative folding. In this study, we present a comparative analysis of the folding properties of four conotoxins sharing an identical pattern of cysteine residues forming three disulfide bridges, but otherwise differing significantly in their primary amino acid sequence. Oxidative folding properties of M-superfamily conotoxins GIIIA, PIIIA, SmIIIA and RIIIK varied with respect to kinetics and thermodynamics. Based on rates for establishing the steady-state distribution of the folding species, two distinct folding mechanisms could be distinguished: first, rapid-collapse folding characterized by very fast, but low-yield accumulation of the correctly folded form; and second, slow-rearrangement folding resulting in higher accumulation of the properly folded form via the reshuffling of disulfide bonds within folding intermediates. Effects of changing the folding conditions indicated that the rapid-collapse and the slow-rearrangement mechanisms were mainly determined by either repulsive electrostatic or productive noncovalent interactions, respectively. The differences in folding kinetics for these two mechanisms were minimized in the presence of protein disulfide isomerase. Taken together, folding properties of conotoxins from the M-superfamily presented in this work and from the O-superfamily published previously suggest that conotoxin sequence diversity is also reflected in their folding properties, and that sequence information rather than a cysteine pattern determines the in vitro folding mechanisms of conotoxins.