A Distinct Functional Site in Ω-Neurotoxins: Novel Antagonists of

Oct 8, 2015 - Snake venom α-neurotoxins from the three-finger toxin (3FTx) family are competitive antagonists with nanomolar affinity and high select...
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A Distinct Functional Site in Ω‑Neurotoxins: Novel Antagonists of Nicotinic Acetylcholine Receptors from Snake Venom Varuna Hassan-Puttaswamy,† David J. Adams,‡ and R. Manjunatha Kini*,† †

Department of Biological Sciences, National University of Singapore, Singapore 117543 Health Innovations Research Institute, RMIT University, Melbourne, Victoria 3083, Australia

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S Supporting Information *

ABSTRACT: Snake venom α-neurotoxins from the three-finger toxin (3FTx) family are competitive antagonists with nanomolar affinity and high selectivity for nicotinic acetylcholine receptors (nAChR). Here, we report the characterization of a new group of competitive nAChR antagonists: Ω-neurotoxins. Although they belong to the 3FTx family, the characteristic functional residues of α-neurotoxins are not conserved. We evaluated the subtype specificity and structure−function relationships of Oh9-1, an Ω-neurotoxin from Ophiophagus hannah venom. Recombinant Oh9-1 showed reversible postsynaptic neurotoxicity in the micromolar range. Experiments with different nAChR subtypes expressed in Xenopus oocytes indicated Oh9-1 is selective for rat muscle type α1β1εδ (adult) and α1β1γδ (fetal) and rat neuronal α3β2 subtypes. However, Oh9-1 showed low or no affinity for other human and rat neuronal subtypes. Twelve individual alanine-scan mutants encompassing all three loops of Oh9-1 were evaluated for binding to α1β1εδ and α3β2 subtypes. Oh9-1’s loop-II residues (M25, F27) were the most critical for interactions and formed the common binding core. Mutations at T23 and F26 caused a significant loss in activity at α1β1εδ receptors but had no effect on the interaction with the α3β2 subtype. Similarly, mutations at loop-II (H7, K22, H30) and -III (K45) of Oh9-1 had a distinctly different impact on its activity with these subtypes. Thus, Oh9-1 interacts with these nAChRs via distinct residues. Unlike αneurotoxins, the tip of loop-II is not involved. We reveal a novel mode of interaction, where both sides of the β-strand of Oh9-1’s loop-II interact with α1β1εδ, but only one side interacts with α3β2. Phylogenetic analysis revealed functional organization of the Ω-neurotoxins independent of α-neurotoxins. Thus, Ω-neurotoxin: Oh9-1 may be a new, structurally distinct class of 3FTxs that, like α-neurotoxins, antagonize nAChRs. However, Oh9-1 binds to the ACh binding pocket via a different set of functional residues.

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endothelial cells,8 and cholinergic modulation of immune cells (reviewed in ref 9). Therefore, nAChRs are also targets for anticancer drugs. Reduced growth and proliferation of breast and lung cancer cells has been achieved by inhibiting α9 and α7 receptors, respectively (reviewed in ref 10). Similarly, blocking nAChRs has been used as a strategy to treat inflammatory diseases such as Crohn’s disease, epilepsy, rheumatoid arthritis, and ulcerative colitis (reviewed in ref 11). However, due to a lack of adequate subtype-specific ligands, our understanding of how the distribution and specific physiological roles of individual nAChRs are associated with cholinergic and noncholinergic functions is limited.12 Discovery of α-bungarotoxin from Bungarus multicinctus venom enabled the identification of the first described muscle “receptive substances,” the muscle-type nAChRs.13 Since then, a number of proteins that target nAChRs, now referred to as αneurotoxins, have been isolated from snake venoms.14 αNeurotoxins have been invaluable tools to elucidate the specific

icotinic acetylcholine receptors (nAChR) facilitate neurotransmission in the central and peripheral nervous systems.1 Their pentameric assembly is composed of α (α1− 10) and non-α subunits (β1−4, γ, δ, and ε). nAChRs can be heteromeric (adult/fetal muscle-type α1β1εδ/α1β1γδ and neuronal α3β2, α4β2, α4β4, α3β2, α3β4, α7β2, and α9α10) or homomeric (neuronal α7 and α9). They may also exhibit the same subunit composition but different subunit stoichiometries, giving molecular diversity and functional heterogeneity; for instance, alternate stoichiometries of α4 and β2 nAChR [(α4β2)2α4 and (α4β2)2β2] exhibit differing sensitivity to activation by agonists and allosteric modulators.2,3 Neuronal nAChRs are involved in regulatory processes (neurotransmitter release, cell excitability, and neuronal integration) and complex brain functions (memory, cognition, nociception, and attention). Hence, they are potential therapeutic targets for various pathophysiological conditions, including Alzheimer’s (α7, α4β2) and Parkinson’s diseases (α6, β5, α4β2), pain (α4β2, α9α10), and schizophrenia (α7).4−6 nAChRs are also involved in non-neuronal cholinergic systems. The α3, α5, α7, α9, or β2 subtypes facilitate cell adhesion and proliferation in keratinocytes (reviewed in ref 7), nAChR-mediated angiogenesis in © XXXX American Chemical Society

Received: June 29, 2015 Accepted: October 8, 2015

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DOI: 10.1021/acschembio.5b00492 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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Figure 1. Primary structure analysis of Oh9-1. Sequence alignment of Oh9-1 with (A) the most homologous 3FTx sequences and (B) canonical short-chain α-neurotoxins. Toxin names, species, and accession numbers (ACCN) are shown. Disulfide bridges and loop regions are also shown. The number of amino acid residues and homology (sequence identity (%Id)) to Oh9-1 of each protein is shown at the end of sequences. Amino acid residues that are critical for interacting with nAChR in canonical short-chain α-neurotoxins are highlighted in gray. Asterisks indicate that OH561 and SNTX1162 have an identical mature protein sequence, but a single residue difference in their signal peptide.

physiological roles of nAChR subtypes.1,15 α-Neurotoxins belong to the three-finger toxin (3FTx) superfamily of venom proteins. These monomeric, competitive antagonists interact with different nAChR subtypes. They are classified into two subclasses: short-chain α-neurotoxins comprise 57−62 amino acid residues and four conserved disulfide bridges in the core region, and long-chain α-neurotoxins comprise 66−75 amino acid residues and a fifth disulfide bridge at the tip of loop-II (reviewed in ref 15). Although both short- and long-chain αneurotoxins block muscle-type nAChRs with similar nanomolar affinities (Kd ∼ 109−1011 M),16 only long-chain α-neurotoxins block neuronal α7, α9, and α9α10 nAChRs with high affinity (Kd ∼ 108−109 M; reviewed in ref 17). Neurotoxins with the fifth disulfide in loop-I were characterized in the past 15 years. Most of them show micromolar toxicity, which explains their classification as “weak toxins.”18 However, candoxin from Bungarus candidus blocks α7 and muscle nAChRs with nanomolar affinity.19 Therefore, we classified toxins with the fifth disulfide bond in the first loop as nonconventional toxins.20 Most characterized neurotoxins exist as monomers. However, κ-neurotoxins and haditoxin are noncovalent homodimers.21,22 κ-Neurotoxins are structurally similar to long-chain neurotoxins and bind to neuronal (α3β2 and α4β2), but not muscle, nAChRs. In contrast, haditoxin is structurally similar to short-chain neurotoxins and efficiently blocks muscle-type and α7 nAChRs. Haditoxin also interacts with α3β2 and α4β2 nAChRs at very high concentrations.23 Utkin and colleagues described homomeric and heteromeric disulfide-bonded 3FTx dimers in Naja kaouthia venom.24,25 They proposed such dimerization enhances the diversification of structure and function. In the meantime, we described a disulfide-bonded heterodimeric neurotoxin: irditoxin from Boiga irregularis (colubrid snake) venom. Irditoxin uniquely inhibits bird-specific muscle-type nAChRs.26 Thus, 3FTxs are a new class of nAChR antagonists and have diverse pharmacological profiles. Mutational studies of α-neurotoxins have delineated 10 to 12 specific functional residues, with Ser8/_, Gln10/_, Lys27/Lys23,

Trp29/Trp25, Asp31/Asp27, Phe32/Phe29, Arg33/Arg33, _/Arg36, Glu38/_, Lys47/Lys49, and _/Phe65 [erabutoxin b (ETxb)/αcobratoxin (α-CBTx) numbering] critical for their interaction with nAChRs.27,28 Along with these toxin-specific residues, long- and short-chain α-neurotoxins utilize a common binding core to interact with different nAChRs. This region consists of Lys27/Lys23, Trp29/Trp25, Asp31/Asp27, Arg33/Arg33, and Lys47/ Lys49 and establishes contact with invariant residues on the nAChRs. Mutation of the common binding core residues of αneurotoxins causes significant, ranging from 20- to 780-fold, loss of affinity toward nAChRs.27,28 Oh9-1, a 3FTx isolated from Ophiophagus hannah venom, lacks all the functionally important residues conserved in αneurotoxins. However, with an IC50 of 88 nM, it irreversibly inhibits carbachol-induced muscle contraction. This potency is only 4-fold less than that of α-bungarotoxin.29 Here, we dissect the mode of interaction of Oh9-1 with nAChRs. We describe the functional determinants, phylogenetic analysis, and, by alanine-scanning mutational analysis, delineation of the functional site of Oh9-1. The data revealed a novel functional site in Oh9-1 and related toxins. We named this new class of 3FTx with a distinct functional site “Ω-neurotoxins.” Evolutionarily, Ω-neurotoxins reside in a clade separated from those containing α-neurotoxins. Functionally, they interact with nAChRs through a distinct site, providing an illustration of unusual convergent evolution. As well as being important tools to further understand the physiological diversity of nAChRs, Ωneurotoxins may also represent new pharmacophores.

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RESULTS AND DISCUSSION

Functional Diversity among 3FTxs. The members of the 3FTx toxin family share similar protein-folding patterns. They have three β-stranded loops extending from a central core that is stabilized by four conserved disulfide bonds.15 Despite the overall similarity in structure, the polypeptides differ markedly in their biological targets. Neurotoxins interact with nAChRs (reviewed in refs 15, 17). Cardiotoxins or cytotoxins interact with phospholipids and cell membranes,30 and aminergic or B

DOI: 10.1021/acschembio.5b00492 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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Figure 2. Expression, purification, and secondary structural characterization of Oh9-1. (A) Oh9-1 expression and preparation of inclusion bodies in Escherichia coli was analyzed by SDS-PAGE. M, Protein standards with molecular masses denoted on the left; UI, uninduced; In, induced at 37 °C for 4 h or 16 °C for 16 h; Su, soluble fraction; Pe, pelleted insoluble fraction; W1−3, three stages of washing; IBs, prepared IBs. (B) Purification of denatured and reduced Oh9-1 on a Jupiter C18 (5 μ, 300 Å, 10 mm × 250 mm) semipreparative column equilibrated with solvent A (0.1% TFA). The protein was eluted with a linear gradient of 32−42% of solvent B (80% ACN in 0.1% TFA) over 100 min at a flow rate of 2 mL/min. The dotted line indicates the gradient of buffer B. Arrow indicates the peak containing reduced Oh9-1. (C) ESI-MS spectrum of purified reduced Oh9-1. Inset, reconstructed mass spectrum of Oh9-1 showing a single homogeneous protein with a mass of 6647.36 ± 0.89 Da. (D, E) Purification of refolded Oh9-1 from purified, reduced Oh9-1 and solubilized IBs, respectively. The refolded proteins were eluted with a linear gradient of 22−40% solvent B over 60 and 100 min at a flow rate of 1 mL/min and 2 mL/min on Jupiter C18 (5 μ, 300 Å, 4.6 mm × 250 mm) analytical and (5 μ, 300 Å, 10 mm × 250 mm) semipreparative columns, respectively. Arrow indicates the major conformer of refolded Oh9-1. (F) ESI-MS spectrum of refolded Oh9-1. Inset, reconstructed mass spectrum of refolded Oh9-1 showing a single homogeneous protein with a mass of 6639.67 ± 0.53. (G) Far-UV circular dichroism spectra of Oh9-1 and ETxb. The proteins were dissolved in Milli-Q water (20 μM) separately, and the CD spectra were recorded using a 0.1 cm path-length cuvette. (H) Refolded Oh9-1 (30 μM) was loaded onto a Superdex 75 column (1 × 30 cm), equilibrated with 50 mM ammonium bicarbonate buffer (pH = 7.4), and eluted with the same buffer at a 1 mL/min flow rate. Inset, the standard curve of elution volumes of proteins plotted against their masses; I = bovine serum albumin, II = carbonic anhydrase, III = cytochrome c, IV = aprotinin. The mass of refolded Oh9-1 was determined by its elution volume (dotted lines). C

DOI: 10.1021/acschembio.5b00492 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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Figure 3. Pharmacology of recombinant Oh9-1 on chick biventer cervicis muscle (CBCM). (A) Representative muscle contraction traces showing the effect of Oh9-1 (100 μM) and (B) reversibility of the neuromuscular block in CBCM preparations. Contractions were produced by exogenously added ACh (300 μM), CCh (10 μM), and KCl (30 mM). The horizontal black bar indicates the electrical field stimulation (EFS). The points of washes with a Krebs solution in reversibility studies are indicated as “Wash.” The block is calculated as a percentage of the control twitch responses to supramaximal nerve stimulation. (C) Traces showing the effect of Oh9-1 (10 μM and 30 μM) on carbachol (CCh)-induced contraction of CBCM. (D) Concentration−response curves of Oh9-1 toward electric field stimulation (EFS) and CCh in CBCM. Each data point is the mean ± SEM of at least three experiments.

SHuffle T7 Express lysY system, recombinant Oh9-1 (rec-Oh91) was expressed as an inclusion body (IB). IBs exhibited a molecular mass of ∼11 000 Da (Figure 2A, lane 6). The IB pellet was solubilized, reduced in denaturing buffer, and purified by reverse-phase high-performance liquid chromatography (RP-HPLC) on a C18 column (Figure 2B). Purified, reduced Oh9-1 exhibited a mass of 6647.36 ± 0.89 Da, consistent with the presence of an additional N-terminal methionine. The slow mobility on the gel (Figure 2A, lane 6) could be attributed to the 10 basic residues in Oh9-1.41 Refolding of Oh9-1 from either purified, reduced Oh9-1 or solubilized IBs yielded the same major conformer (Figure 2D,E). The mass of the refolded rec-Oh9-1 was determined as 6639.67 ± 0.5 Da (Figure 2F inset), indicative of the loss of eight protons from the formation of four disulfide bonds. At 10 (data not shown) or 30 μM, Oh9-1 eluted as a monomer on a Superdex 75 column (Figure 2H). Rec-Oh9-1 shows a characteristic β-sheet secondary structure compatible with a 3FTx fold (Figure 2G). Pharmacological Characterization. Rec-Oh9-1 (100 μM) produced a neuromuscular blockade of EFS-evoked twitch responses in chick biventer cervicis muscle (CBCM) preparations (Figure 3A). It inhibited the contractile response to the exogenous agonists, acetylcholine (ACh) and carbachol (CCh), whereas response to exogenous KCl and twitches evoked by direct muscle stimulation (not shown) were not inhibited. Neuromuscular blockade of EFS- and CCh-evoked responses in CBCM was reversible (Figure 3B,C). Rec-Oh9-1 exhibited a concentration-dependent blockade, with IC50 values of 31.03 μM (95% Cl 25.5−37.8) and 7.2 μM (95% Cl 6.2− 8.5) on EFS- and CCh-evoked responses, respectively (Figure

muscarinic toxins interact with biogenic amine receptors and muscarinic acetylcholine receptors.31,32 Fasciculins inhibit acetylcholinesterases.33 Calciseptines interact with Ca2+ channels.34 Dendroaspins interact with integrins.35 Hemextins inhibit factor VIIa.36 Mambalgins block acid-sensing ion channels,37 and micrurotoxins interact with GABAA receptors.38 Thus, small alterations in the 3FTx amino acid sequence do not change the overall protein scaffold but elicit diverse pharmacological functions.15 Understanding these subtle structure−function relationships is challenging but will provide important insights into the specific determinants of biological activity. Oh9-1 is similar (77−98% identity) to eight 3FTxs from O. hannah venom. It exhibits 51% identity to CM-1b from Hemachatus hemeachatus venom39 and 46% identity to mambalgin-1 from Dendroaspis polylepis venom37 (Figure 1A). Oh9-1 shares less than 33% sequence identity with αneurotoxins (Figure 1B); it has much shorter loops-I and -III (five- and three-residues shorter, respectively) and an extra residue in loop-II. Further, the functional residues of αneurotoxins (Figure 1B, shaded gray) are not conserved in Oh9-1. These structural differences provided the impetus for our detailed characterization of its functional site and action. Recombinant Expression. Although Oh9-1 has been reported to be present in small quantities in king cobra venom,29,40 it was not detected in the king cobra venoms we obtained from Thailand, Indonesia or Kentucky Reptile Zoo, USA (unpublished observations). To facilitate its detailed characterization, Oh9-1 was expressed as a fusion protein (without any recombinant tags) with an extra N-terminal methionine at the start of the open reading frame. In the E. coli D

DOI: 10.1021/acschembio.5b00492 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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Figure 4. Inhibition of nAChR subtypes expressed in Xenopus oocytes by Oh9-1. (A) Representative superimposed traces of inhibition of AChevoked currents at EC50 concentrations recorded in the absence () or presence (---) of Oh9-1. Determined ACh EC50s of hα7, rα3β2, rα1β1δγ (fetal), and rα1β1δε (adult) receptors are 100, 10, 1, and 1 μM, respectively. (B) Bar graph illustrating the nAChR subtype selectivity of Oh9-1. (C) Concentration−response curves of Oh9-1 on selective nAChR subtypes gave IC50’s of 3.1 μM (95% Cl 2.6−3.6), 5.6 μM (95% Cl 4.1−7.5), and 50.2 μM (95% Cl 44.9−56.2) for rα1β1δε (adult), rα1β1δγ (fetal), and rα3β2 receptors, respectively. Each data point is the mean ± SEM of at least three experiments.

(Figure S1). Interestingly, concentration-independent potentiation was observed on GlyRα1 receptors from 100 μM to