The insecticidal spider toxin SFI1 is a knottin peptide that blocks the pore of insect voltage-gated sodium channels via a large β-hairpin loop

Spider venoms contain a plethora of insecticidal peptides that act on neuronal ion channels and receptors. Because of their high specificity, potency and stability, these peptides have attracted much attention as potential environmentally friendly insecticides. Although many insecticidal spider venom peptides have been isolated, the molecular target, mode of action and structure of only a small minority have been explored. Sf1a, a 46-residue peptide isolated from the venom of the tube-web spider Segesteria florentina, is insecticidal to a wide range of insects, but nontoxic to vertebrates. In order to investigate its structure and mode of action, we developed an efficient bacterial expression system for the production of Sf1a. We determined a high-resolution solution structure of Sf1a using multidimensional 3D/4D NMR spectroscopy. This revealed that Sf1a is a knottin peptide with an unusually large beta-hairpin loop that accounts for a third of the peptide length. This loop is delimited by a fourth disulfide bond that is not commonly found in knottin peptides. We showed, through mutagenesis, that this large loop is functionally critical for insecticidal activity. Sf1a was further shown to be a selective inhibitor of insect voltage-gated sodium channels, consistent with its `depressant' paralytic phenotype in insects. However, in contrast to the majority of spider-derived sodium channel toxins that function as gating modifiers via interaction with one or more of the voltage-sensor domains, Sf1a appears to act as a pore blocker

The insecticidal spider toxin SFI1 is a knottin peptide that blocks the pore of insect voltage-gated sodium channels via a large β-hairpin loop

Spider venoms contain a plethora of insecticidal peptides that act on neuronal ion channels and receptors. Because of their high specificity, potency and stability, these peptides have attracted much attention as potential environmentally friendly insecticides. Although many insecticidal spider venom peptides have been isolated, the molecular target, mode of action and structure of only a small minority have been explored. Sf1a, a 46-residue peptide isolated from the venom of the tube-web spider Segesteria florentina, is insecticidal to a wide range of insects, but nontoxic to vertebrates. In order to investigate its structure and mode of action, we developed an efficient bacterial expression system for the production of Sf1a. We determined a high-resolution solution structure of Sf1a using multidimensional 3D/4D NMR spectroscopy. This revealed that Sf1a is a knottin peptide with an unusually large β-hairpin loop that accounts for a third of the peptide length. This loop is delimited by a fourth disulfide bond that is not commonly found in knottin peptides. We showed, through mutagenesis, that this large loop is functionally critical for insecticidal activity. Sf1a was further shown to be a selective inhibitor of insect voltage-gated sodium channels, consistent with its 'depressant' paralytic phenotype in insects. However, in contrast to the majority of spider-derived sodium channel toxins that function as gating modifiers via interaction with one or more of the voltage-sensor domains, Sf1a appears to act as a pore blocker

Design of expression vector.

<p>(<b>A</b>) Schematic representation of the pLic-MBP expression vector using for periplasmic expression of disulfide-rich peptides in <i>E. coli</i>. The coding region includes a MalE signal sequence (MalE_SS) for targeting the fusion protein to the periplasm, a His₆ tag for affinity purification, a MBP fusion tag to aid solubility, and a codon-optimised gene encoding the target peptide, with a TEV protease recognition site inserted between the MBP and target-peptide coding regions. The locations of key elements of the vector are shown, including the upstream ribosome binding site (RBS), T7 promoter, lac operator, and key restriction sites. (<b>B</b>) Schematic of the periplasmic expression system for production of disulfide-rich peptides in <i>E. coli</i>. After translation, the fusion protein is transported to the periplasm via the Sec translocase system <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063865#pone.0063865-LycklamaaNijeholt1" target="_blank">[89]</a>. The MalE signal sequence (red tube) is removed during this process, releasing the fusion protein (orange tube) into the periplasm where the Dsb machinery (DsbA, DsbB, DsbC, and DsbD) can assist with disulfide-bond formation.</p

Summary of the diverse range of disulfide-rich venom peptides produced in our lab using periplasmic expression.

<p>Venom peptides with molecular weight (MW) ranging from 2000–8000 Da and with 2–6 disulfide (SS) bonds have been expressed from a phylogenetically diverse range of venomous animals. ¹Yield: +++, >5 mg/litre; ++, 1–5 mg/litre; +, 0–1 mg/litre; —, no correctly folded protein obtained; ²Calculated using ProtParam (<a href="http://web.expasy.org/protparam/accessed" target="_blank">http://web.expasy.org/protparam/accessed</a> 20130220); ³Number of disulfide isoforms evident in final RP-HPLC purification step. N/A  =  not available.</p

Effect of buffer, temperature, and pH on 2D ¹H-¹⁵N HSQC NMR spectra of a disulfide-rich venom peptide (Step 9).

<p>(<b>A</b>) Overlays of the downfield region of 2D ¹H-¹⁵N HSQC spectra of a spider-venom peptide (46 residues, 4 disulfide bonds) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063865#pone.0063865-Lipkin1" target="_blank">[90]</a> acquired at 25°C using different buffers and pH: 20 mM MES pH 6 (pink); sodium phosphate, pH 6 (purple); 20 mM sodium acetate, pH 5 (red); 20 mM sodium citrate, pH 4 (blue). This region of the spectrum shows the sidechain ¹H-¹⁵N correlation for the single Trp residue in this peptide. (<b>B</b>) Effect of temperature on the same resonance. Spectra were acquired in 20 mM citrate, pH 4 at the following temperatures: 10°C (purple); 25°C (red); 40°C (blue). At low pH and high temperature the equilibrium is shifted towards a single conformer, compared to the three conformers apparent at lower temperature.</p

Workflow for obtaining a high yield of recombinant venom peptide.

<p>Coomassie stained SDS-PAGE gels illustrating various optimization steps. The molecular mass of the standards (in kDa) are indicated on the left or right of each gel. <b>Step 2</b>: The effect of increasing concentrations of IPTG on the level of fusion protein expression. Note that as little as 10 µM ITPG is required for induction of fusion protein expression. <b>Step 3</b>: Yields of isotopically labelled fusion protein obtaining using LB medium, minimal medium (MM), and using the dual media protocol (LB/MM). <b>Step 4</b>: Comparison of the yield of venom peptide obtained when cell pellets were obtained via periplasmic extraction (PE), French press (FP), or a constant-pressure cell disruptor (CD). <b>Step 5</b>: Ni-NTA purification of fusion protein. <b>Step 6</b>: Effect of various redox buffers on the efficiency of TEV protease cleavage of the MBP-venom peptide fusion protein. <b>Step 7</b>: RP-HPLC chromatograms comparing the efficacy of three different methods for removing His₆-tagged fusion protein (MBP) and TEV protease prior to the final peptide purification step: precipitation with 1% TFA (black); removal with a solid phase extraction (SPE) column (red); passage of the cleavage mixture through a Ni-NTA column (blue). The asterisked peak corresponds to the peptide of interest, while the dashed line shows the gradient of solvent B (0.043% TFA in 90% acetonitrile).</p

Comparison of different methods for determining protein concentration.

<p>Concentration determination was performed using five dilutions (1∶1, 1∶2, 1∶5, 1∶10 & 1∶20) of a standard peptide solution (100 µM). The lowest dilution produced unreliable readings for all methods other than the BCA assay, and was not used for calculating the concentration or the standard deviation in those methods. Only the 1∶1 and 1∶2 dilutions produced reliable readings when using the NanoDrop; for this method, all other readings were omitted when calculating the concentration and standard deviation. * dilutions below 20 µM were unreliable and excluded.</p

The insecticidal neurotoxin Aps III is an atypical knottin peptide that potently blocks insect voltage-gated sodium channels

One of the most potent insecticidal venom peptides described to date is Aps III from the venom of the trapdoor spider Apomastus schlingeri. Aps III is highly neurotoxic to lepidopteran crop pests, making it a promising candidate for bioinsecticide development. However, its disulfide-connectivity, three-dimensional structure, and mode of action have not been determined. Here we show that recombinant Aps III (rAps III) is an atypical knottin peptide; three of the disulfide bridges form a classical inhibitor cystine knot motif while the fourth disulfide acts as a molecular staple that restricts the flexibility of an unusually large β hairpin loop that often houses the pharmacophore in this class of toxins. We demonstrate that the irreversible paralysis induced in insects by rAps III results from a potent block of insect voltage-gated sodium channels. Channel block by rAps III is voltage-independent insofar as it occurs without significant alteration in the voltage-dependence of channel activation or steady-state inactivation. Thus, rAps III appears to be a pore blocker that plugs the outer vestibule of insect voltage-gated sodium channels. This mechanism of action contrasts strikingly with virtually all other sodium channel modulators isolated from spider venoms that act as gating modifiers by interacting with one or more of the four voltage-sensing domains of the channel

Production of recombinant disulfide-rich venom peptides for structural and functional analysis via expression in the periplasm of E. coli

Disulfide-rich peptides are the dominant component of most animal venoms. These peptides have received much attention as leads for the development of novel therapeutic agents and bioinsecticides because they target a wide range of neuronal receptors and ion channels with a high degree of potency and selectivity. In addition, their rigid disulfide framework makes them particularly well suited for addressing the crucial issue of in vivo stability. Structural and functional characterization of these peptides necessitates the development of a robust, reliable expression system that maintains their native disulfide framework. The bacterium Escherichia coli has long been used for economical production of recombinant proteins. However, the expression of functional disulfide-rich proteins in the reducing environment of the E. coli cytoplasm presents a significant challenge. Thus, we present here an optimised protocol for the expression of disulfide-rich venom peptides in the periplasm of E. coli, which is where the endogenous machinery for production of disulfide-bonds is located. The parameters that have been investigated include choice of media, induction conditions, lysis methods, methods of fusion protein and peptide purification, and sample preparation for NMR studies. After each section a recommendation is made for conditions to use. We demonstrate the use of this method for the production of venom peptides ranging in size from 2 to 8 kDa and containing 2-6 disulfide bonds