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Logo of jbcThe Journal of Biological Chemistry
 
J Biol Chem. 2016 March 25; 291(13): 7205–7220.
Published online 2015 January 27. doi:  10.1074/jbc.M115.697672
PMCID: PMC4807300

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

Abstract

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 an S-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 β-strands stabilized by three disulfide bridges. The loop region linking the β-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.

Keywords: disulfide, high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), peptide chemical synthesis, sodium channel, conotoxin, cysteine, structure-activity relationship studies, two-electrode voltage clamp electrophysiology

Introduction

Marine snails of the genus Conus employ a complex venom mixture to subdue prey and as an effective means of defending against predation. The active venoms contain an array of peptides that bind to and modulate the properties of ion channels, G-protein-coupled receptors, and neurotransmitter receptors (1). Several peptides modulate the activities of voltage-gated sodium channels (VGSCs),4 which are implicated in numerous neurological disorders, as well as neuropathic pain. Four classes of conopeptides have been shown to affect VGSC activity. Peptides belonging to the ι- and δ-families promote activation and inhibit inactivation, respectively, whereas the μ- and μO-conotoxins inhibit VGSCs by either blocking the Na+ conductance pore or preventing channel activation, respectively (2, 3). Recently, the founding member of a fifth class of VGSC inhibitors was identified that blocks the channel through interaction with a previously unidentified neurotoxin binding site, site 8 (Fig. 1A) (4).

FIGURE 1.
Binding site of μO§-GVIIJ on the channel and its peptide sequence. A, binding sites of VGSC-inhibiting conotoxins. Pore-blocking μ-conotoxins bind deep within the Na+ conductance pore and compete with tetrodotoxin for neurotoxin ...

μO§-GVIIJ is a 35-residue peptide isolated from the venom of the piscivorous snail Conus geographus (Fig. 1B). In vitro folding of linear μO§-GVIIJ with thiol-reactive oxidants (i.e. glutathione, l-cystine, or cystamine) resulted in adducts where Cys-24 was disulfide-bonded with glutathione, cysteine, or cysteamine, respectively (abbreviated as GVIIJSSG, GVIIJSSC, and GVIIJSSEA, respectively; see Fig. 1C for structures). Of these, the GVIIJSSC variant most closely resembled the native peptide, which also has a Cys disulfide-bonded to its Cys-24 residue. For largely historical reasons, the glutathione adduct (GVIIJSSG) was tested by two-electrode voltage clamp electrophysiology against rat NaV1.1–1.8 expressed in Xenopus laevis oocytes, and it was found to block all tested NaV1 isoforms with a Kd or IC50 < 0.4 μm except NaV1.5, which was blocked with an IC50 of 207 μm, and NaV1.8, which was not blocked at all (IC50 > 1 mm). When screened against the neuronal VGSC subtype NaV1.2, the three analogs possessed nearly identical off rates (koff values) but different on rates (kon values) (4). An explanation consistent with these results is that the glutathione, cysteine, or cysteamine moiety disulfide-bonded to Cys-24 of the peptide acts as a leaving group when, by disulfide exchange, Cys-24 forms a disulfide bridge with a free cysteine on the α-subunit (specifically Cys-910, in the case of rNaV1.2 (4)). This explanation was supported by the results of experiments that tested four additional adducts with different groups disulfide-bonded to Cys-24 against both rat NaV1.2 and mouse NaV1.6, where it was observed that for a given NaV1 isoform, the adducts had widely varying kon values but the same koff value (5).

Here we have determined the solution structure of μO§-GVIIJ using an analog of this peptide, GVIIJ[C24S], which would not undergo dimerization (5) during NMR studies. The solution structure of GVIIJ[C24S] closely resembled that of the inhibitor cystine knot (ICK) class of peptides in that it possessed two short β-strands and was cross-linked by three disulfide bridges in the “core” of the molecule (6,8). The ICK motif is found in peptides from numerous phyla and is of particular interest for pharmaceutical development because of its inherent stability and amenability to chemical modification for enhanced pharmacological effect (8). Importantly, this structure identified the location and position of residue 24 in a loop linking the two β-strands, where it would be available to interact with the channel.

To complement these structural studies, we performed detailed structure-activity relationship (SAR) studies to identify amino acid residues critical for inhibition of rat NaV1.2 (rNaV1.2), a VGSC isoform found in the central nervous system. SAR studies were performed on rNaV1.2 exogenously expressed in X. laevis oocytes using mutants of a potent analog of μO§-GVIIJ, GVIIJSSEA, in which Cys-24 was modified by cysteamine (Fig. 1C). These studies identified three functionally important residues, Lys-12, Arg-14 and Tyr-16, located on a face of the peptide adjacent to the loop with residue 24. Thus, these results suggest a “functionally bipartite” mechanism of interaction by μO§-GVIIJ, where a disulfide bridge “tethers” the peptide to a channel cysteine at site 8, whereas residues on a different surface of the peptide are also important for interaction with the channel.

To demonstrate that site 8 was physically distinct from site 1, where tetrodotoxin, saxitoxin, and μ-conotoxins bind and plug the Na+-conducting pore (Fig. 1A), we previously employed a “leaky” μ-conotoxin, μ-KIIIA[K7A] (9, 10), and showed that pre-equilibrating rNaV1.2 with this peptide did not interfere with the block by μO§-GVIIJSSG (4). However, as noted (4), μ-KIIIA[K7A] has 16 amino acid residues, so these results did not exclude the possibility that a larger μ-conotoxin, such as μ-GIIIA (with 22 residues), might intrude into the binding space of μO§-GVIIJ. We examined this possibility in the present study with a point mutant of μ-PIIIA (which, like μ-GIIIA, has 22 residues and blocks rNaV1.4 with high affinity), namely μ-PIIIA[R14Q]. We chose this mutant because others have shown that it binds to rNaV1.4 reasonably well with a significant residual current (rINa or “leak”) (11). We show here that pre-equilibrating rNaV1.4 with nearly saturating levels of μ-PIIIA[R14Q] did not interfere with the block by μO§-GVIIJSSG. In contrast, the rate of block by a dimer of μO§-GVIIJ, (μO§-GVIIJ)2, whose monomers were disulfide-bonded to each other via the thiols of their Cys-24 residues (5), was decreased by the presence of μ-PIIIA[R14Q]. These results lead us to conclude that sites 1 and 8 are distinct although not very far apart.

Experimental Procedures

Peptide Synthesis, Oxidative Folding, and Purification

Native Peptide

The native μO§-GVIIJ has three post-translational modifications: bromination of Trp-2, hydroxylation of Pro-6, and S-cysteinylation of Cys-24. All μO§-GVIIJ analogs synthesized for SAR and NMR studies lacked bromination of Trp-2 because it was shown previously that both the brominated and nonbrominated forms of the peptide exhibited comparable functional activity (4), and the cost of the synthesis of the nonbrominated peptide was significantly lower. Structure-activity studies of μO§-GVIIJ were carried out on either the GVIIJSSEA or GVIIJSSC backgrounds, and analogs were designed and synthesized that substituted non-cysteine residues with alanine (i.e. Trp-2, Thr-9, Leu13, Leu-15, Tyr-16, Ser-19, Phe-21, Tyr-25, Thr-26, and Thr-28) or replaced acidic/basic residues with those of the opposite charge (i.e. Asp-5, Lys-12, Arg-14, Asp-23, Lys-27, Lys-30, and Lys-32) (Table 1).

TABLE 1
Characterization of μO§-GVIIJ analogs by HPLC retention time, purity, and molecular mass

Solid Phase Peptide Synthesis

Analogs of μO§-GVIIJ were synthesized on a 50-μmol scale with an AAPPTec Apex 396 synthesizer (AAPPTec, Louisville, KY) using standard solid phase N-(9-fluorenyl)methoxycarbonyl (Fmoc) protocols. Fmoc-protected amino acids, including the pseudoproline dipeptide Fmoc-Tyr(tBu)-Thr(ψMe,Mepro)-OH, were purchased from AAPPTec. N-α-Fmoc-O-t-butyl-l-trans-4-hydroxyproline (Hyp) was purchased from EMD Millipore (Darmstadt, Germany), and Fmoc-γ-carboxy-l-Glu(OtBu)2-OH (Gla or γ) was obtained from Advanced ChemTech (Louisville, KY). Peptides were assembled on preloaded Fmoc-l-Ala-Wang resin (substitution, 0.38 mmol·g−1; Peptides International Inc., Louisville, KY). Side chain protection for amino acids was: Arg, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf); Asp, Gla, and Glu, O-tert-butyl (OtBu); Lys and Trp, tert-butyloxycarbonyl; Hyp, Ser, Thr, and Tyr, tert-butyl (tBu); and Asn and Cys, trityl (Trt). Coupling of each amino acid was achieved using 1 equivalent of 0.4 m benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate and 2 equivalents of 2 m N,N-diisopropylethyl amine in N-methyl-2-pyrrolidone. Amino acids were used in 10-fold excess (60 min of coupling), with the exception of Hyp and the pseudoproline dipeptide Tyr-Thr, which were used in 5-fold excess (90 min of coupling). Double coupling was performed on residues following Cys residues in the μO§-GVIIJ sequence. Fmoc-protecting groups were removed by 20 min of treatment with 20% (v/v) piperidine in dimethylformamide.

Peptide Cleavage, Purification, and Folding

Peptides were cleaved from the resin by treatment with reagent K (trifluoroacetic acid (TFA), H2O, phenol, thioanisole, 1,2-ethanedithiol; 82.5/5/5/5/2.5 by volume) for 3 h at room temperature. The crude peptide was separated from resin by vacuum filtration. The cleavage product was precipitated in cold methyl-tert-butyl ether, centrifuged, and washed again with the ether. Crude peptide was purified by reversed phase HPLC using a Vydac C18 semipreparative column (218TP510, 250 × 10 mm, 5-μm particle size) eluted with a linear gradient ranging from 15 to 45% solvent B (90% acetonitrile in 0.1% TFA) in 30 min. Oxidative folding of the linear peptide was performed in a buffered solution containing 20 μm peptide, 0.1 m Tris-HCl, pH 7.5, and 1 mm EDTA. Oxidizing reagents used included a 1:1 mm mixture of reduced:oxidized glutathione (GVIIJ[C24S]), 1 or 2 mm cystamine dihydrochloride (GVIIJSSEA), or 2 ml of an l-cystine-containing solution (6 mg/ml) in 5% (v/v) acetonitrile and 0.1% TFA (GVIIJSSC), as described previously (4, 5). Oxidative folding was carried out overnight at room temperature. The oxidation reaction was quenched by acidification with formic acid (final concentration, 8% (v/v)). Folded analogs were purified by semipreparative reversed phase HPLC. Purities for analogs used in NMR studies were greater than 95%, whereas those used in electrophysiological assays ranged from 80 to 99%. The identities of the oxidized peptides were confirmed by MALDI-TOF mass spectrometry, except GVIIJ[D23γ]SSEA, for which electrospray mass spectrometry was employed.

Other Toxins

Syntheses of μ-GIIIA, and μ-PIIIA[R14Q] were performed essentially as previously described (12, 13). The synthesis of (μO§-GVIIJ)2 (a dimer in which Cys-24 of one monomer was disulfide-bonded to its counterpart on the other monomer) was described recently (5).

Nuclear Magnetic Resonance Spectroscopy

NMR spectra were acquired using either a Varian Inova 600 or Bruker DRX-600 spectrometer. Lyophilized samples were dissolved to final concentrations of 1.4 mm (GVIIJ[C24S]) or 0.68 mm (GVIIJSSEA) in a mixture of 90% H2O, 10% 2H2O at pH 3.2. Chemical shift assignments for backbone and side chain proton resonances were obtained from two-dimensional [1H,1H] total correlation spectroscopy (TOCSY; spin lock time, 30 and 70 ms) and [1H,1H] NOESY (mixing time, 150 ms) spectra acquired at 298 K. For GVIIJ[C24S], 15N chemical shifts were obtained from a two-dimensional [15N,1H] HSQC spectrum, and 13C chemical shifts were obtained using both two-dimensional [13C,1H] HSQC and HMBC spectra. To obtain amide temperature coefficients, a series of one-dimensional 1H spectra and two-dimensional TOCSY(spin lock time, 70 ms) experiments were acquired at 288, 293, and 298 K. Amide (1H → 2H) exchange rates were measured by dissolving lyophilized GVIIJ[C24S] in 2H2O at pH 3.2 (final concentration, 0.71 mm) and then recording a time course of one-dimensional spectra, followed by acquisition of a 70-ms TOCSY spectrum at 298 K. The spectra were processed using TOPSPIN (version 3.2) and were analyzed in CcpNmr-Analysis (version 2.1.5). The spectra were referenced either directly or indirectly to the DSS methyl signal at 0.0 ppm. Chemical shifts were deposited in the BioMagResBank Data Bank (14, 15) with accession numbers 26674 and 26675 for GVIIJ[C24S] and GVIIJSSEA, respectively.

Structural Constraints and Calculations

Distance constraints for structural calculations were generated from assigned cross-peaks in NOESY spectra (150-ms mixing time) acquired at 298 K, pH 3.2. Dihedral angle constraints ([var phi] and ψ) were generated from TALOS-N predictions (16) and from 3JHN-Hα coupling constants according to the following criteria: 3JHN-Hα > 8 Hz, [var phi] = −120 ± 40°; 3JHN-Hα < 6 Hz, [var phi] = −60 ± 40°. Disulfide bond constraints were added according to the previous experimentally determined connectivity illustrated in Fig. 1C (4). Deuterium exchange rates and temperature coefficients were used to identify H-bonded backbone amides, where H-bond acceptors could be identified from initial rounds of structural calculation (present and consistent in ≥80% of structures) and were included in subsequent structural calculations. Initial structure calculations were optimized for a low target function using the noeassign macro in CYANA 3.0 (17). Structures generated by CYANA were then used to resolve the assignment of any remaining ambiguous inter-residue NOEs. Following optimization, the final set of constraints was entered into CNS version 1.3 (18), and an ensemble of 100 structures was generated. Of these, the 20 lowest energy structures without violations were selected to represent the solution structure of GVIIJ[C24S]. Validation of the final calculated structures was accomplished using PROCHECK-NMR (19). Secondary structure prediction was performed using DSSP (20) and PROMOTIF (21) based on the closest to average structure of GVIIJ[C24S]. Classification of β-turns was based on the criteria reported in Ref. 22. Structures were analyzed using MOLMOL (23), and structural representations were constructed using UCSF Chimera.

Structure-Activity Studies on GVIIJSSEA and GVIIJSSC with rNaV1.2 and Competition Experiments of μ- versus μO§-Conotoxins with rNaV1.4

Two-electrode Voltage Clamp Electrophysiology

All μO§-GVIIJ analogs were screened against rat NaV1.2 exogenously expressed in X. laevis oocytes. Oocytes were also used to express rat NaV1.4 in experiments to assess competition between μ-PIIIA[R14Q] and μO§-GVIIJSSG or μO§-GVIIJ dimer and, in positive control tests, between μ-PIIIA[R14Q] and μ-GIIIA. The NaV1.2 (NM_012647) and NaV1.4 (NM_013178) clones were obtained from Alan Golden (University of California, Irvine). The oocytes were injected with 30–50 nl of NaV1.2 or NaV1.4 cRNA (1.5 or 0.6 ng, respectively) in RNase-free water and incubated overnight at 16 °C in ND96 (96 nm NaCl, 2 mm KCl, 1.8 mm CaCl2, and 5 mm HEPES, pH 7.5), supplemented with antibiotics (100 units/ml penicillin, 0.1 mg/ml streptomycin). Voltage clamping was performed with a Warner OC-725C amplifier (Warner Instruments) using 3 m KCl-filled microelectrodes (resistance < 0.5 mΩ) (24). An oocyte was placed in a 4-mm diameter well (30 μl volume) filled with ND96 and clamped at a holding potential of −80 mV. Voltage-gated sodium currents (INa) were induced at 20-s intervals with a 50-ms depolarizing pulse to −10 mV. The peptides to be tested were dissolved in ND96 at 10 times the desired final concentration (0.1–333 μm) and applied in 3-μl volumes to the static bath containing the voltage clamped oocyte. A static bath was used to conserve the limited quantities of analogs. Immediately following peptide application, the solution in the well was gently mixed through repeated pipetting of the bath solution. All experiments were performed at room temperature. Washout of peptide was performed by perfusing the chamber initially at a rate of 1 ml/min for 1 min followed by 0.2 ml/min for 19 min. Use of X. laevis frogs, which provided oocytes for this study, followed protocols approved by the University of Utah Institutional Animal Care and Use Committee, which conform to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Analysis of Electrophysiological Data

The time course of peak INa was plotted before and during toxin exposure, as well as following toxin washout. The observed rate constants (kobs values) were determined by fitting of the onset of block to a single-exponential function for ≥3 oocytes at each of three different peptide concentrations. Because all μO§-GVIIJ analogs tested were very slowly reversible (i.e. < 50% recovery after 20 min washout or koff < 0.035 min−1), off rates (koff values) were estimated from the level of recovery after 20 min washout for ≥ 9 oocytes, assuming a single-exponential time course (25). To minimize effects of baseline drift, times longer than 20 min were not used. kobs values were plotted against [peptide], and kon values were obtained from the slope of the linear regression line, assuming the equation, kobs = kon[peptide] + koff (26). Electrophysiology data are presented as the means ± S.D., unless specified otherwise, and statistical significance was determined using the two-tailed unpaired t test in GraphPad Prism or Excel. Percentage block of the peak sodium current (INa) by conotoxins or their analogs was calculated as defined in Ref. 25 by the following equation: % block (peak INa) = [(average ≥ 3 traces at steady state)/(average ≥ 3 baseline traces)] × 100.

Results

Peptide Synthesis and Analysis

GVIIJ[C24S] and GVIIJSSEA, which were used for NMR studies, were synthesized as described under “Experimental Procedures.” All peptide analogs used in SAR studies were synthesized following folding and purification protocols described for GVIIJSSEA. These syntheses generated, on average, between 500 and 900 nmol of purified linear peptide per 100 mg of resin cleaved. Oxidative folding of most of the linear analogs produced a single major product comprising between 40 and 60% of the total folding mixture. The exceptions were the [O6A], [D23N], and [D23γ] analogs, for which the major product comprised only 9, 17, and 28% of the mixture, respectively. Folding of the [F21A], [T26A], and [T28A] analogs in the presence of cystamine dihydrochloride did not lead to a single major product as monitored by reversed phase HPLC. However, oxidation of these analogs in the presence of l-cystine showed modest improvement in producing a major folding product. The [S19A] analog exhibited poor folding properties independent of the oxidant used, with the major peak being just 8% of the total mixture. Broadening and “tailing” of the purified HPLC peaks characterized all “poorly” folding analogs, resulting in final purities of 80–93%.

NMR Spectroscopy

Good quality 1H NMR spectra were obtained for GVIIJ[C24S] and GVIIJSSEA at pH 3.2. Comparison of NOESY spectra of GVIIJ[C24S] at pH 3.2 with spectra at pH 7.4 showed that several key long-range NOEs involving pairs of nonexchangeable protons (e.g. Lys-30 Hβ-Asp-23 Hβ; Cys-29 Hβ-Hyp-6 Hα; Ala-8 Hβ-Cys-29 Hα; Thr-9 Hα-Thr-28 Hγ2; Gly-7 Hα-Thr-28 Hα) were present at both pH values, implying that the structure determined at pH 3.2 was representative of that at physiological pH. Sequential assignments were derived from two-dimensional TOCSY and NOESY spectra. The temperature dependence of amide proton chemical shifts is shown in Fig. 2A. Deviations from random coil values (27, 28) for backbone HN and Hα chemical shifts of GVIIJ[C24S] and GVIIJSSEA are plotted in Fig. 3. The near identity of these plots confirms adoption of similar backbone structures for these two analogs. Both structures have chemical shifts close to random coil values at the N and C termini. Small differences between the two peptides were observed for the residues of loop 2 (Gly-11–Tyr-16) and loop 4 (Asp-23–Thr-28).

FIGURE 2.
Characterization of GVIIJ[C24S] by NMR spectroscopy. A, temperature dependence experiments identified several residues that exhibited amide proton chemical shifts of magnitude < 4 ppb/K, including Trp-2, Arg-14, Cys-18, Gly-20, Phe-21, Asp-23, ...
FIGURE 3.
Deviation of backbone amide (HN) and Cα proton (Hα) chemical shifts from random coil values at 25 °C (28). A, HN chemical shift deviations for GVIIJ[C24S] (red) and GVIIJSSEA (blue). B, Hα chemical shift deviations for ...

Solution Structures

Structures were generated in CYANA and then refined in CNS (18) using a total of 401 NOE-derived distance constraints, 39 dihedral, 9 χ1 angle constraints (from 3JHN-Hα J-coupling measurements and TALOS-N predictions), and 5 hydrogen bond restraints (from amide temperature coefficients and 2H exchange experiments). A summary of experimental constraints and structural statistics for GVIIJ[C24S] is provided in Table 2.

TABLE 2
Structural statistics for GVIIJ[C24S]

GVIIJ[C24S] is structurally well defined, with a mean global RMSD of 0.76 Å (over the backbone heavy atoms N, CA, and C of residues 2–32) (Table 2 and Fig. 4A). The structure is characterized by two short antiparallel β-strands encompassing Cys-22–Asp-23 (β1) and Thr-28–Cys-29 (β2), linked by a distorted type I β-turn (22) spanning residues Asp-23 (i)–Thr-26 (i + 3) (backbone heavy atom RMSD of 0.07 Å) (Fig. 4B). The β-strands, together with the β-turn, form a 4:4 β-hairpin (29), near the tip of which is the side chain of residue 24. These secondary structure features were supported by weak or absent sequential HN-HN NOEs and stronger HN-Hα sequential NOEs, as well as a number of 3JHNHα coupling constants ≥ 8 Hz (Fig. 5, A and B). Hydrogen bonds were observed between Asp-23 HN–Thr-28 O, Thr-28 HN–Asp-23 O and Lys-30 HN–Phe-21 O (20 of 20 structures), as well as between Lys-27 HN–Ser-24 O (7 of 20 structures) and Thr-26 HN–Asp-23 O (14 of 20 structures). Within this region, backbone amides of Phe-21, Asp-23, Ser-24, Thr-26, Lys-27, Thr-28, and Cys-29 were slowly exchanging (Figs. 2, B and C, and and44A), and many (e.g. Phe-21, Asp-23, Thr-26, Thr-28, and Cys-29) exhibited temperature coefficients with magnitudes of <4 ppb/K (Fig. 2A).

FIGURE 4.
Structure of the μO§-GVIIJ analog GVIIJ[C24S]. A, family of 20 final structures for GVIIJ[C24S], superimposed on backbone heavy atoms of residues 2–32. Disulfide bonds are shown in orange according to the previously established ...
FIGURE 5.
Characterization of the final 20 structures of GVIIJ[C24S]. A, sequential and medium-range NOE connectivities for GVIIJ[C24S] (298 K, pH 3.2) according to CYANA structure calculation. Intensities of dNN, dαN, and dβN are proportional to ...

Within loop 1 (Gly-4–Thr-9), adjacent to the β2-strand, is a small segment of the peptide that exhibits modest levels of extended structure (Fig. 4B). Indeed, hydrogen bonds were observed between Cys-29 HN and Ala-8 O (14 of 20 structures) and Cys-10 HN-Lys-27 O (20 of 20 structures). In ICK peptides that display intercysteine loop lengths similar to those of μO§-GVIIJ, this region of extended structure is often present, sometimes as a third β-strand (6, 7). However, in GVIIJ[C24S], this region was too short to be designated as such by DSSP or PROMOTIF secondary structure prediction programs.

Other secondary structure features include a type IV β-turn encompassing Cys-10–Leu-13 within intercysteine loop 2 (Gly-11–Tyr-16) (Fig. 4C). This loop was less well defined than the remainder of the peptide, with angular order parameters for [var phi] and ψ < 0.8 and RMSD of 1.2 Å over the backbone heavy atoms N, CA, and C of residues Gly-11–Tyr-16 (Fig. 5, D–F). Several rapidly exchanging backbone amide protons were identified in this region by hydrogen-deuterium exchange experiments (Figs. 2, B and C, and and55A), indicating that this loop was solvent-exposed. Backbone angular order parameters for residues at the N and C termini were also ≤0.8 (Fig. 5, D–F).

Oxidative Folding of μO§-GVIIJ Analogs

Residue 24, which in the native peptide presents the “extra” cysteine responsible for tethering to the channel, is located within the β-turn of loop 4 (Fig. 6A). Asp-23, at the start of the turn, was critical for folding of the GVIIJSSEA analogs, with substitution by either lysine or alanine resulting in inefficient formation of a major folding product (Fig. 6B). A major product was observed in the [D23N] and [D23γ] analogs, albeit in lower overall yields compared with efficiently folding analogs. This restoration in folding was presumably due to interactions between Oγ in the side chain of Asp-23 and the backbone HN of Tyr-25 (i + 2) and HN of Thr-26 (i + 3) (22). Hydrogen bonds between the side chain of Asp-23 and the HN of Tyr-25 were observed in half the structures and between Asp-23 and Thr-26 in ~25% of structures. Replacement of Tyr-25 by Asp made the peptide more prone to dimerization, decreasing the yield for the desired product by ~10%. In many type I β-turns, Asp is one of three preferred residues for electrostatic interactions with the backbone HN of residue 25 (22); it may be that this substitution further exposed Cys-24, where it could conceivably undergo dimerization. Restoration of the folding yield was observed for the Arg-25 (i + 2) mutation, which may have resulted from stabilization of loop 4 through a weak interaction between Nϵ of Arg-25 and the main chain oxygen of Asp-23.

FIGURE 6.
Structure-activity relationship studies of residues in the Cys-24-containing loop 4. A, the closest to average backbone structure of GVIIJ[C24S] is stabilized by numerous H-bonds between backbone atoms, as well as by side chain to backbone interactions ...

Several residues in close proximity to this β-turn also appeared to be important for stabilization of loop 4 (Asp-23–Thr-28). In all 20 structures, the side chain of Lys-30 points back into the loop where it could participate in electrostatic interactions with either Asp-23 or Thr-26. In the closest to average structure of GVIIJ[C24S], the Hη of Lys-30 and the side chain oxygen atoms of Asp-23 or Thr-26 were separated by just 3.4 and 4.4 Å, respectively (Fig. 6A). The importance of such interactions was illustrated by the inability of the [K30D] analog to produce a single, major folding product. Replacement of Lys-30 with an acidic residue such as aspartate presumably had a destabilizing effect on loop 4 (Asp-23–Thr-28), but substitution with a small, uncharged residue in the [K30A] analog restored efficient folding. Other residues found to be important for the folding of μO§-GVIIJ included Thr-26 and Thr-28. Neither [T26A] nor [T28A] yielded a single major product following cystamine oxidation. Again, based on the calculated distances between the side chains in the closest to average structure, this was probably caused by removal of stabilizing interactions between the side chains of these residues and Hη of Lys-30. The use of a different oxidant (i.e. l-cystine) resulted in formation of a major product in each case, albeit in significantly lower yields compared with other analogs used in this study (Fig. 6B).

Structure-Activity Relationship Studies of the Block of NaV1.2 by μO§-GVIIJ Analogs

The ability of peptides to block NaV1.2 expressed in Xenopus oocytes was assessed electrophysiologically as described under “Experimental Procedures.” We used rNaV1.2 as the target channel to maintain consistency with the previous studies of μO§-GVIIJ analogs (4, 5). The GVIIJ[C24S] analog, which was used in NMR studies, blocked NaV1.2 rapidly and very reversibly (Fig. 7A). The rapidity precluded accurate determination of kon and koff values. The IC50 for GVIIJ[C24S] was 4.8 μm (with 95% confidence interval (CI) of 3.7–6.2 μm) (Fig. 7B).

FIGURE 7.
NaV1.2 block by μO§-GVIIJ analogs. A, comparison of NaV1.2 blockade by 3.3 μm (filled circles) and 33 μm (open circles) GVIIJ[C24S]. Block was rapidly reversible at all concentrations tested. Responses did not completely ...

All SAR studies were performed with analogs of GVIIJSSEA, which itself is slightly more potent than GVIIJSSC, against NaV1.2 (Kd = 2.3 versus 3.4 nm) (5). An initial series of analogs was designed and synthesized to investigate the importance of the residues of loop 4 (Asp-23–Thr-28) in the immediate vicinity of Cys-24. Amino acid substitutions in loop 4 appeared to have significant effects on the structural stability of the peptide, as evidenced by the inability of some of the variants to form a single, major folding product (Fig. 6B). However, most of these substitutions had little effect on the ability of the peptide to block rNaV1.2 (Fig. 6C). The on rate constants varied considerably across all analogs, whereas recovery from block (reflected in koff) was very slow and showed little variation (Table 3 and Fig. 7). The largest kon values were observed in mutants of Thr-9 ([T9A]), Asp-23 ([D23N]), and Lys-30 ([K30A]), with values in the range of 4–5 μm−1·min−1. Conversely, the analogs with the smallest kon values were located in loop 2 (Gly-11–Tyr-16), namely Lys-12 ([K12D]), Arg-14 ([R14D]), and Tyr-16 ([Y16A]) (kon = 0.01, 0.03 and 0.06 μm−1·min−1, respectively). The lower kon values observed with these analogs, in turn, resulted in increased Kd values ranging from ~50- to 280-fold higher than GVIIJSSEA (Fig. 7, C–F, and Table 3).

TABLE 3
Kinetics of block of rNaV1.2 by μO§-GVIIJ analogs

Block by μ-GIIIA, μO§-GVIIJSSG, or (μO§-GVIIJ)2 of Naïve rNaV1.4 versus rNaV1.4 Pre-equilibrated with μ-PIIIA[R14Q]

μ-PIIIA[R14Q] blocked NaV1.4 with an IC50 of 0.25 μm (95% CI of 0.19–0.33 μm) (Fig. 8A). At saturating concentrations, μ-PIIIA[R14Q] blocked incompletely and left a residual current (rINa) of 27 ± 3.5% (Fig. 8A). As a positive control for competition between μ-PIIIA[R14Q] and another site 1 antagonist, we used μ-GIIIA, the first μ-conotoxin used to help define site 1 (12, 30). μ-GIIIA (10 μm) rapidly blocked the INa of rNaV1.4 (Fig. 8B, inset). However, when rNaV1.4 was pre-equilibrated with 33 μm μ-PIIIA[R14Q], the block of the resulting rINa by μ-GIIIA was significantly slower (Fig. 8B and Table 4), as would be expected if the two μ-conotoxins competed for the same site on the channel. In contrast, the rate of block by 3.3 μm μO§-GVIIJSSG was unaffected by the presence of 33 μm μ-PIIIA[R14Q] (Fig. 8C and Table 4), which indicates that the two peptides (μ- and μO§-conotoxins) do not compete for the same site to block the channel. On the other hand, the rate of block by 10 μm (μO§-GVIIJ)2 (i.e. the μO§-GVIIJ dimer) was slightly but significantly, decreased by the presence of μ-PIIIA[R14Q] (Fig. 8D and Table 4).

FIGURE 8.
Block of NaV1. 4 by μ-PIIIA[R14Q], μ-GIIIA, μO§-GVIIJ, or (μO§-GVIIJ)2, alone or in binary combinations. Peak INa of voltage clamped oocytes expressing rat NaV1.4 were monitored as described under “Experimental ...
TABLE 4
Values of kobs for the block by μ-GIIIA, μO§-GVIIJ, or (μO§-GVIIJ)2 of control INa and of rINa in presence of 33 μm μ-PIIIA[R14Q]

Discussion

The structural and functional studies of μO§-GVIIJ described here have revealed several important features of the peptide that contribute to its functional activity. The solution structure of GVIIJ[C24S] is a classical ICK motif (6, 7), exhibiting two antiparallel β-strands cross-linked by three disulfide bridges that formed a knot-like structure in which the Cys-17–Cys-29 disulfide crossed the macrocycle formed by the remaining two disulfide bridges and the interconnecting backbone. ICK peptides are abundant in nature, with many being observed in toxin or toxin-like peptides (Fig. 9), and are of significant interest as therapeutic scaffolds because of their relative ease of synthesis, stability and amenability to sequence mutations (8). Important in the structure of GVIIJ[C24S] was the presentation of residue 24, within the β-turn of loop 4 (Asp-23–Thr-26), such that it could readily interact with the channel. It was shown recently that the cysteine residue at this position is a key determinant of the off rate of the peptide (4, 5). Our results lend support to this because all peptide analogs investigated here exhibited koff values that were close to that of unmodified GVIIJSSEA (Table 3). Presumably this is because, once bound to the channel, the energetics of dissociation are resilient to changes in individual amino acid side chains. These results suggested that the structural features that had the greatest influence on the biological activity of μO§-GVIIJ are those that affect kon.

FIGURE 9.
Structural comparison of the GVIIJ[C24S] backbone with those of other ICK motif-containing peptides. The backbone of the closest to average structure of GVIIJ[C24S] (blue) was aligned with the backbones of δ-conotoxin EVIA from Conus ermenius ...

The solution structure of μO§-GVIIJ was solved using an analog in which Cys-24 was replaced with serine to avoid the risk of dimerization during the course of NMR studies. The block by GVIIJ[C24S] was rapidly reversible, in contrast to that of GVIIJSSC or GVIIJSSEA, which blocked nearly irreversibly. The differences in kinetics between GVIIJ[C24S] and GVIIJSSC or GVIIJSSEA can be attributed to the inability of GVIIJ[C24S] to tether to its binding site on the channel. Given the low nanomolar potency of GVIIJSSEA for NaV1.2, this analog was used as the basis for SAR studies to assess the importance of individual non-cysteine residues. Deviations from random coil chemical shift plots were nearly identical for GVIIJ[C24S] and GVIIJSSEA (Fig. 3), which validated their use for structural and functional studies, respectively.

SAR studies focused initially on mutations of residues in loop 4 (Asp-23–Thr-28) in the vicinity of Cys-24. Alignment of this loop with the free cysteine-containing loops of NaVβ2 (Phe-23–Thr-28) or β4 (Phe-55–Gly-60) revealed modest levels of sequence homology (Fig. 6C) (31, 32). This is of interest in light of previous work showing that coexpression of NaV1 subtypes with NaVβ2 or β4 inhibited the ability of μO§-GVIIJ analogs to block (4), suggesting that μO§-GVIIJ and the β-subunits may interact at the same site on the ectodomain of the α-subunit. Numerous residues in this region of the peptide had significant effects on the ability of the analog to fold into a major product, implying that interactions among residues within the β-turn of this loop (Asp-23–Thr-26) were important for stabilization of the peptide, even though they had much less bearing on the functional activity of the peptide. Thus, little difference was observed between Kd values of loop 4 mutants and that of GVIIJSSEA (Table 3).

Our SAR results nonetheless identified a number of functionally important residues in GVIIJSSEA, most of which are located in the structurally less well defined loop 2 (Gly-11–Tyr-16). The [K12D], [R14D], and [Y16A] analogs in this loop exhibited on rate constants that were significantly lower than that of the unmodified peptide, giving rise to higher Kd values. There is a dearth of medium- and long-range NOEs in this region, and the [var phi]- and ψ-angular order parameters for residues in this region fall below 0.8 (Fig. 5). This loop may adopt a more rigid structure upon interaction with the channel, but further studies will be required to confirm this.

As described in the introduction, the lack of overlap of sites 1 and 8 was observed initially with μ-KIIIA[K7A] and rNaV1.2 (4). We had shown previously that μ-KIIIA and its analogs block rNaV1.2 and compete for, and can even co-occupy, site 1 with the guanidinium alkaloids tetrodotoxin, saxitoxin, and saxitoxin congeners (9, 10, 33). In the present experiments, we employed μ-PIIIA[R14Q] and rNaV1.4. The site 1 blocker μ-PIIIA[R14Q] was first characterized at the laboratory of French and co-workers (11), who performed single-channel measurements in planar lipid bilayers to show that this peptide, like μ-GIIIA[R13Q], blocked rNaV1.4 with a significant residual current, but unlike μ-GIIIA[R13Q], which has ~100-fold lower affinity than its parent μ-GIIIA (34), the affinity of μ-PIIIA[R14Q] was only ~10-fold lower than its wild-type counterpart (Arg-13 of μ-GIIIA and Arg-14 of μ-PIIIA are homologous residues). Thus, we chose to use μ-PIIIA[R14Q] for our studies here to further explore the distinction between sites 1 and 8. In oocytes expressing rNaV1.4, μ-PIIIA[R14Q] blocked with an IC50 only 7-fold higher than that of native μ-PIIIA (0.25 versus 0.036 μm; Fig. 8A and Ref. 25, respectively). At saturating concentrations, μ-PIIIA[R14Q] blocked with an rINa of 27% (Fig. 8A), whereas the native peptide, μ-PIIIA, blocked with essentially no rINa (25). μ-GIIIA blocked the rINa of μ-PIIIA[R14Q] with a kobs much smaller than that of control INa (Fig. 8B and Table 4), consistent with the two peptides competing for the same site (site 1) on the channel. In contrast, the kobs of the block by μO§-GVIIJSSG was not affected by the presence of μ-PIIIA[R14Q] (Fig. 8C and Table 4), indicating that μ-PIIIA[R14Q] did not interfere with the block by μO§-GVIIJSSG and therefore that sites 1 and 8 are distinct. These results are consistent with those of earlier competition experiments performed with μ-KIIIA[K7A] and rNaV1.2 (4). Finally, the kobs of the dimer of μO§-GVIIJSSG was slightly, but significantly, decreased by the presence of μ-PIIIA[R14Q] (Fig. 8D and Table 4), suggesting that sites 1 and 8 are close to each other.

Previously we tested seven μO§-GVIIJSSR derivatives (where SR was a different R-group disulfide-bonded to Cys-24, including Cys-24 of another μO§-GVIIJ monomer to form the (μO§-GVIIJ)2 dimer). They all blocked rNaV1.2 with the same koff and mNaV1.6 with a koff 17-fold larger than for rNaV1.2 (5). These results led us to propose that for all seven peptides, the same peptide-channel complex was formed (5). We report here that (μO§-GVIIJ)2 blocked rNaV1.4 with a koff of 0.0025 ± 0.0009 min−1, a value essentially the same as that of GVIIJSSG, which was 0.0016 ± 0.0008 min−1 (4) (p = 0.25). Thus, we expand to rNaV1.4 our proposal that monomeric and dimeric GVIIJ (μO§-GVIIJSSR and (μO§-GVIIJ)2, respectively) block by forming the same peptide-channel complex.

Combining our structural and functional results, it appears that μO§-GVIIJ may interact with two distinct subsites on the channel. Substitution of specific residues, predominantly located in a less well defined region of the peptide (loop 2), had the greatest effects on kon (Fig. 10A). Analogs containing substitutions of important residues in this loop were still very slowly reversible but exhibited much slower on rates and subsequently lower potency compared with GVIIJSSEA. Comparison of the activity of GVIIJ[C24S] with analogs containing a cysteine at position 24 showed that the identity of the residue at this position was important for koff (Fig. 10B). The C24S replacement led to rapid reversibility of the peptide but did not prevent VGSC inhibition. Presumably, this was because the peptide was no longer capable of undergoing covalent attachment to the channel, but by retaining key residues, the analogs were still able to elicit VGSC inhibition. Previous studies with the free thiol form of μO§-GVIIJ (GVIIJSH) showed similar results (4, 5). The absence of a leaving group attached to Cys-24 prevented efficient disulfide bond formation between the channel and the peptide, which led to rapid reversibility of GVIIJSH upon washout. Likewise, the important residues in loop 2 (i.e. Lys-12, Arg-14, and Tyr-16) were retained in GVIIJSH, and therefore the peptide was still capable of block. Thus, our data suggest a “functionally bipartite” mechanism of action where disulfide bond formation through Cys-24 and interactions between Lys-12, Arg-14, and Tyr-16 and the channel both contribute to VGSC blockade (Fig. 10).

FIGURE 10.
“Functionally bipartite” mechanism of action for μO§-GVIIJ. A, locations of amino acid residues important for on rates (kon) of μO§-GVIIJ against NaV1.2. Residues with the slowest kon values are colored ...

μO§-GVIIJ and its analogs present a unique opportunity to probe the structure and function of the newly described site 8 on the sodium channel. In addition to the identification of residues that are important for VGSC blockade, SAR studies also identified potential sites for modification (i.e. residues that are noncritical for inhibition), such as residues near the C terminus (e.g. Lys-30 and Asp-31), which could be replaced by residues with reporter groups (e.g. fluorescently labeled amino acids) for the development of peptidic probes to identify the presence or absence of specific VGSC subtypes in different tissue preparations.

Given that μO§-GVIIJ is the first conotoxin found to bind by the described “tethering” mechanism, an untapped and significant opportunity now exists to identify additional ligands for site 8. Efforts are ongoing to improve the VGSC selectivity profile of μO§-GVIIJ. In addition, mining of venoms of closely related Conus species is also underway to identify other members of this peptide family with improved selectivity for pain-relevant VGSC subtypes (e.g. NaV1.3 and 1.7). From a therapeutic perspective, such efforts might prove useful in the identification and development of peptidic drug leads to combat disease states stemming from VGSC dysfunction, such as neuropathic pain, epilepsy, and multiple sclerosis (35, 36).

Author Contributions

B. R. G. designed, performed, and analyzed experiments and drafted the paper. J. G. assisted with the synthesis, purification and oxidative folding of selected μO§-GVIIJ analogs. S. C. and J. J. S. assisted with the collection and analysis of NMR spectra. M.-M. Z. conducted electrophysiology experiments of selected μO§-GVIIJ analogs and all experiments involving rNaV1.4. J. E. R. synthesized μ-GIIIA and μ-PIIIA[R14Q]. B. R. G., J. G., G. B., B. M. O., D. Y., and R. S. N. initiated the project and helped design experiments. All authors reviewed the results and assisted in preparing the manuscript.

Acknowledgments

We thank Sam Robinson for review of the manuscript and for numerous helpful discussions related to this work. We also thank William Low for MS analysis of synthetic analogs, Alan Golden for the NaV1.2 and NaV1.4 clones, and Layla Azam for preparation of rNaV1.2 and rNaV1.4 cRNA.

*This work was supported by National Institutes of Health Grant GM 048677. Syntheses of GVIIJ analogs were also supported in part by funds from Janssen Research and Development, LLC. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

4The abbreviations used are:

VGSC
voltage-gated sodium channel
RMSD
root mean square deviation
SAR
structure-activity relationship
ICK
inhibitor cystine knot
Fmoc
N-(9-fluorenyl)methoxycarbonyl
Hyp
N-α-Fmoc-O-t-butyl-l-trans-4-hydroxyproline
TOCSY
total correlation spectroscopy
CI
confidence interval.

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