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. 2021 Jul 26;12(9):1574–1584. doi: 10.1039/d1md00182e

Posttranslational modifications of α-conotoxins: sulfotyrosine and C-terminal amidation stabilise structures and increase acetylcholine receptor binding†

Thao N T Ho 1,‡, Han Siean Lee 2,‡, Shilpa Swaminathan 2, Lewis Goodwin 2, Nishant Rai 2, Brianna Ushay 2, Richard J Lewis 1, K Johan Rosengren 2, Anne C Conibear 2,✉
PMCID: PMC8459321  PMID: 34671739

Abstract

Conotoxins are peptides found in the venoms of marine cone snails. They are typically highly structured and stable and have potent activities at nicotinic acetylcholine receptors, which make them valuable research tools and promising lead molecules for drug development. Many conotoxins are also highly modified with posttranslational modifications such as proline hydroxylation, glutamic acid gamma-carboxylation, tyrosine sulfation and C-terminal amidation, amongst others. The role of these posttranslational modifications is poorly understood, and it is unclear whether the modifications interact directly with the binding site, alter conotoxin structure, or both. Here we synthesised a set of twelve conotoxin variants bearing posttranslational modifications in the form of native sulfotyrosine and C-terminal amidation and show that these two modifications in combination increase their activity at nicotinic acetylcholine receptors and binding to soluble acetylcholine binding proteins, respectively. We then rationalise how these functional differences between variants might arise from stabilization of the three-dimensional structures and interactions with the binding sites, using high-resolution nuclear magnetic resonance data. This study demonstrates that posttranslational modifications can modulate interactions between a ligand and receptor by a combination of structural and binding alterations. A deeper mechanistic understanding of the role of posttranslational modifications in structure–activity relationships is essential for understanding receptor biology and could help to guide structure-based drug design.

Cone snail venoms are richly decorated with posttranslational modifications. We show that tyrosine sulfation and C-terminal amidation increase the structural stability and binding of α-conotoxins.graphic file with name d1md00182e-ga.jpg

Introduction

Conotoxins are venom peptides from cone snails that have garnered widespread interest due to their stable structures and potent activities at neuronal receptors of therapeutic interest. They are classified according to their gene superfamily, cysteine framework or pharmacological activity.1–4 Under the pharmacological classification, the α-conotoxins are active at nicotinic acetylcholine receptors (nAChRs), which have important roles in neurotransmission at neuromuscular junctions and in the central nervous system.5,6 In addition to their promise as therapeutic agents, α-conotoxins able to distinguish between muscle and neuronal nAChR subtypes have provided valuable insight into the biological mechanisms of nAChRs.5,7–9 The majority of α-conotoxins are 12–20 residues long and have four cysteine residues with C(i)–C(iii), C(ii)–C(iv) disulfide connectivity, with variation in the number and type of residues in the cysteine-stabilised ‘loops’.

The diversity of conotoxins is further expanded by extensive posttranslational modification. Although posttranslational modifications (PTMs) are known to mediate a wide range of protein regulatory functions in cellular processes,10,11 it is unclear why cone snails invest energy and resources in decorating their venom peptides with PTMs. Aside from disulfide bonds, the most common PTM in α-conotoxins is C-terminal amidation,2 which has been shown to be important for folding and activity in some conotoxins: in ImI, C-terminal amidation facilitated folding and correct disulfide pairing;12 in molecular simulation studies on LsIA, C-terminal amidation increased intermolecular contacts and hydrogen bonds to α7 nAChR.13 Amidation is also a common strategy for increasing the stability and activity of other classes of peptide therapeutics such as peptide hormones and antimicrobial peptides.14–16 Other common PTMs of conotoxins include hydroxyproline, bromotryptophan, γ-carboxyglutamic acid and pyroglutamic acid.2 PTMs can only be characterized at the protein level and might be unstable to conditions used for venom fractionation and analysis, meaning that many important conotoxin PTMs might be missed in transcriptomic studies and activity-based screening. The PTMs of conotoxins that have been detected are annotated in the Conoserver database2 but reports of their biological functions are often conflicting and have rarely been studied systematically.17–19 The study of conotoxin PTMs is further complicated by the fact that activities of conotoxins are often tested on mammalian receptors of pharmacological interest, rather than receptors from their natural prey targets.

Tyrosine sulfation is a PTM that has been reported for only seven conotoxins (AnIA, AnIB, AnIC, EpI, PnIA, PnIB and TiIA), all within the α-conotoxin family, and involves transfer of an inorganic sulfate moiety onto the hydroxyl group of a tyrosine residue (Fig. 1).20,21 This modification is carried out by tyrosylprotein sulfotransferases, which are bound to the membrane of the Golgi and transfer sulfate from 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to newly-synthesised proteins destined for secretion or membrane insertion.22 The biological relevance of sulfotyrosine is only just emerging, with important roles in regulating extracellular protein–protein interactions in homeostasis, inflammation and some infectious diseases, particularly in chemokine receptors and anticoagulant proteins.20,22,23 Although tyrosine sulfation has only been reported for seven conotoxins, we predict that it is more prevalent than reported because sulfotyrosine is unstable at acidic pH and in positive mode electrospray mass spectrometry (ESI-MS) conditions, both of which are commonly used to fractionate and sequence conotoxins.22

Fig. 1. Sulfated α-conotoxins. a) Cone snails such as Conus episcopatus produce venom containing a cocktail of peptides that rapidly immobilize their prey. b) Whereas C-terminal amidation is a common PTM of α-conotoxins, tyrosine sulfation has been reported for only seven α-conotoxins to date. c) Sequence of α-conotoxin EpI, the first conotoxin identified to contain both C-terminal amidation and tyrosine sulfation, is shown.18 Amino acids are represented by their one-letter codes with disulfide-bonds shown by grey lines. Sulfotyrosine is shown in grey italics and disulfide-bonded cystines in bold.

Fig. 1

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The roles of C-terminal amidation and/or tyrosine sulfation PTMs in the structure and activities of α-conotoxins have not been investigated systematically. To our knowledge, no structures of sulfated conotoxins are currently available, with the crystal structures of EpI and PnIA determined for the unsulfated forms.24–26 In early studies, both sulfated and unsulfated EpI inhibited the activity of nicotine in stimulating catecholamine release from bovine adrenal chromaffin cells (predominantly α3β4 neuronal nAChRs) but did not inhibit rat phrenic nerve–diaphragm (muscle nAChRs).18 Partial inhibition of acetylcholine-induced membrane currents in rat parasympathetic neurons of intracardiac ganglia was observed for both sulfated and unsulfated EpI and residual current was inhibited by α-bungarotoxin. These results led the authors to conclude that sulfated and unsulfated EpI primarily act on α3β2 and α3β4 nAChR subtypes.18 In two-electrode voltage-clamp electrophysiology experiments using rat nAChR subunits expressed in Xenopus oocytes, unsulfated AnIB showed two-fold and ten-fold decreased inhibitory activity compared to native AnIB at rat α3β2 and α7 nAChR subtypes, respectively.17 Biological effects of tyrosine sulfation, however, were not tested on mollusc receptors for which they would more likely be adapted as the native prey of the respective cone snails. Recently, Li and He reported synthesis and stabilities of eight sulfotyrosine-modified α-conotoxins but the effects of the PTMs on structure and activity were not studied.27

Here we systematically dissect the roles of C-terminal amidation and tyrosine sulfation in the structures and activities of three representative α-conotoxins, EpI, AnIB and PnIA, all from the A gene superfamily, which target neuronal subtypes of N-acetylcholine receptors.18 Solid phase peptide synthesis and directed disulfide formation provided access to pure forms of the twelve possible variants for structural studies using NMR spectroscopy, binding and activity assays. Activities of the variants were measured on two human nAChR subtypes (α7, and α3β4) as well as binding to two acetylcholine-binding proteins (AChBPs), soluble proteins that modulate neurotransmission in molluscs (Ls-AChBP from Lymnaea sp. and Ac-AChBP from Aplysia sp.).28,29 These studies showed that C-terminal amidation and tyrosine sulfation both increase binding to Ac-AChBP and stabilise the α-conotoxin structures. We propose that the PTMs stabilise the binding conformations and serve to subtly tune the activity and selectivity of these α-conotoxins.

Materials and methods

Solid phase peptide synthesis

Peptides were synthesised by solid phase peptide synthesis (SPPS), employing 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. For manual syntheses, Fmoc-protected building blocks (2.5 equiv.) were coupled using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU, 2.4 equiv., 0.5 M in DMF) as activator in combination with diisopropylethylamine (DIPEA, 5 equiv.) as base at room temperature for 20–30 min. N-Terminal deprotection was achieved with piperidine [20% in dimethylformamide (DMF), 2 × 5 min]. Peptides synthesized on a Liberty Prime (CEM) microwave peptide synthesizer were coupled with diisopropyl carbodiimide (DIC, 0.25 M in DMF) and OxymaPure (0.5 M in DMF), using 5 equiv. of amino acid and microwave irradiation for 1 min at 105 °C. Deprotection was carried out using pyrrolidine (25% in DMF). C-Terminally amidated peptides were synthesized on Rink amide resin and C-terminal acid peptides were synthesized on pre-loaded 2-chlorotrityl chloride (2-CtC) resin. For pre-loading of 2-CtC resin, Fmoc-Cys(trt)-OH (10 equiv.) and DIC (1 equiv.) were stirred in dry DCM for 30 min. The DCM was removed under vacuum and the activated amino acid dissolved in a minimum volume of DMF with 0.1 equiv. 4-dimethylaminopyridine (DMAP) and added to 2-CtC resin pre-swelled in DMF. After 30 min, the resin was drained, washed and dried and the loading was determined by measuring the absorbance of the dibenzofulvene product at 301 nm of a resin sample incubated in 20% piperidine in DMF for 30 min. Sulfotyrosine residues were incorporated using the Fmoc-Tyr(SO3·nP)-OH (Merck) building block30,31 and cysteines C(i) and C(iii) were incorporated as Fmoc-Cys(Acm)-OH for directed disulfide formation. After drying the resin, peptides were cleaved from the resin with trifluoroacetic acid (TFA)/dimethylsulfide (DMS)/triisopropylsilane (TIPS)/H2O 90 : 5 : 2.5 : 2.5 for 2–3 h and then precipitated with diethyl ether and extracted into water before lyophilisation. Crude peptides were purified by RP-HPLC on C18 columns (PhaseSep) using a gradient of ACN in water with 0.1% TFA. Purity was analysed by electrospray mass spectrometry in positive and negative ion mode and by analytical RP-HPLC. Electrospray mass spectrometry was carried out on an AB Sciex API 2000 instrument, calibrated to ±1 Da and running in direct injection mode at a flow rate of 0.1 mL min−1 with 30% solvent A (water + 0.05% formic acid) and 70% solvent B (90% acetonitrile, 10% water, 0.05% formic acid).

The first disulfide bond [Cys(ii)–Cys(iv)] was formed by dissolving the purified peptide in 0.1 M ammonium bicarbonate (at ∼0.5 mg mL−1) and adding 2,2′-dithiodipyridine (DPDS, 3 equiv.) dissolved in MeOH. Oxidation reactions were stirred in air overnight at room temperature and disulfide bond formation was monitored by ESI-MS. On completion of the first oxidation, the solution was acidified and purified by RP-HPLC. The second oxidation [Cys(i)–Cys(iii)] was performed concurrently with removal of the acetamidomethyl (Acm) protecting groups. Peptides were dissolved in water with 0.1% TFA (at ∼0.5 mg mL−1) and 1 M HCl (0.1 mL mg−1 peptide) was added, followed by 0.1 M iodine in 50% aq. acetic acid until the solution remained brown. Reactions were stirred at room temperature for 2–3 h and disulfide formation was monitored by ESI-MS. On completion of the second oxidation, the reaction was quenched with 1 M ascorbic acid, diluted with water and purified by RP-HPLC.

The neopentyl protecting group on sulfotyrosine was typically hydrolysed during the oxidation and HPLC purification steps, also observed by Li and He (2020),27 however, the sulfate group remained stable, as confirmed using ESI-MS in negative ion mode. In preliminary syntheses of PnIA variants, the neopentyl group was removed after final purification by dissolving the peptide in 2 M ammonium acetate, pH 7.2 at 37 °C overnight and then lyophilizing 3–4 times. After deprotection of the sulfate group, peptides were kept at neutral pH and analysed by ESI-MS in negative ion mode.

NMR data collection, processing, and structure calculations

Samples of EpI and PnIA variants for NMR data collection were prepared in ammonium acetate buffer at pH 6.0 with 10% v/v D2O (Cambridge Isotope Laboratories, DLM-4-99-100) and 10 μM 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt (DSS) as an internal reference.32 Samples of AnIB variants were prepared in water at pH ∼3 with 10% v/v D2O and 10 μM DSS. NMR spectra were acquired at the University of Queensland, Centre for Advanced Imaging on an Avance 700 MHz NMR spectrometer equipped with a cryoprobe at 298 K. Spectra acquired included 1H–1H TOCSY with an isotropic mixing time of 80 ms; 1H–1H NOESY with a mixing time of 300 ms; 1H–15N HSQC; and 1H–13C HSQC. Spectra were Fourier transformed, phased and calibrated on the DSS signal (1H at 0 ppm) in Topspin 4.0.6 (Bruker BioSpin). 15N and 13C spectra were calibrated on the unified scale according to the IUPAC recommendations,32,33 using a ratio of 0.251449530 for 13C and 0.101329118 for 15N. Spectra were assigned in CCP-NMR v2 and v3,34 or CARA.35

Structures of EpI and AnIB were calculated using interproton distance restrains determined from NOESY peak volumes and dihedral angle restraints derived from chemical shift data using TALOS-N.36 Hydrogen bond restraints were included based on amide proton temperature coefficients determined from 1H–1H TOCSY spectra recorded at 288, 293, 298, 303 and 308 K, with temperature coefficients ≥−4.6 ppb K−1 considered to indicate hydrogen bond donor residues.37 A set of 50 structures was calculated using torsion angle simulated annealing in CYANA,38 combined with automated NOESY peak assignment. Final structures were calculated and minimised in a water shell within CNS39 and 20 structures with the best MolProbity scores,40 minimum energies and no violations >0.2 Å or 2° were selected for the final ensemble. Structure ensembles were submitted to the Protein Data Bank (PDB) and chemical shifts to the Biological Magnetic Resonance Bank (BMRB) with accession numbers: EpI[Y(SO3)15Y]-OH (PDB: 7N0T, BMRB: 30913); EpI[Y(SO3)15Y]-NH2 (PDB: 7N26, BMRB: 30922); EpI-OH (PDB: 7N25, BMRB: 30921); EpI-NH2 (PDB: 7N24, BMRB: 30920); AnIB[Y(SO3)16Y]-OH (PDB: 7N23, BMRB: 30919); AnIB[Y(SO3)16Y]-NH2 (PDB: 7N22, BMRB:30918); AnIB-OH (PDB: 7N21, BMRB: 30917); AnIB-NH2 (PDB: 7N20, BMRB: 30916); PnIA-NH2 (PDB: 7N1Z, BMRB: 30915).

Cell culture

SH-SY5Y neuroblastoma cells [a gift from Victor Diaz (Max Planck Institute for Experimental Medicine, Göttingen, Germany)] were cultured at 37 °C/5% (v/v) CO2 in RPMI media containing 2 mM l-glutamine and 15% (v/v) FBS. Cells were passaged every 3–5 days using 0.25% trypsin/EDTA at a dilution of 1 : 5. Experiments were conducted over several months and spanned on average a minimum of 10–20 passages. Responses were not affected by cell passage number with consistent control responses recorded over the duration of experiments.

In vitro nAChR activation assays (FLIPR assays)

Cultured SH-SY5Y cells were plated at a density of 100 000 cells per well on black-walled 384-well imaging plates and cultured for 48 h to form a confluent monolayer. Growth media were removed, and plates were incubated for 30 min at 37 °C with component A of the calcium 4 assay kit (Molecular Devices), which contains the calcium fluorophore required for Ca2+ imaging. After incubation, the cells were transferred to the FLIPR (Molecular Devices) which measures the changes in fluorescence correlated to the intracellular calcium levels in response to choline activating α7 and nicotine activating α3β4 nAChRs endogenously expressed by the SH-SY5Y cells via a cooled CCD camera with excitation at 470–495 nm, and emission at 515–575 nm every 1 s. Camera gain and intensity were adjusted for each plate of cells yielding a minimum of 1500–2000 arbitrary fluorescence units (AFU) as a baseline fluorescence value. α-Conotoxin peptides were added 10 min before applying choline (30 μM) for α7 nAChRs or nicotine (30 μM, for α3β4). For activation measurements on the α7 nAChR subtype, N-(5-chloro-2,4-dimethoxyphenyl)-N′-(5-methyl-3-isoxazolyl)-urea (PNU-120596, 10 μM) was added to slow the channel kinetics, which would otherwise be too fast to measure on the FLIPR platform. All compounds were diluted with physiological salt solution (PSS; 5.9 mM KCl, 1.5 mM MgCl2, 1.2 mM NaH2PO4, 5.0 mM NaHCO3, 140 mM NaCl, 11.5 mM glucose, 5 mM CaCl2, 10 mM HEPES, pH 7.4). FLIPR data were normalised to the maximum nicotine (30 μM) response in the SH-SY5Y cells to yield the % Fmax. A four-parameter Hill equation was fitted to the data using GraphPad Prism 9.0. Experiments were performed in triplicate in three independent experiments. pIC50 values are reported as mean with 95% confidence interval.

Expression and purification of AChBPs

Over-expression of Ls-AChBP and Ac-AChBP was performed as previously described.41 Briefly, E. coli cultures were grown in 2× yeast tryptone (2YT) medium and protein expression was induced using 1 mM IPTG at 16 °C and 0.5 OD600 for Ls-AChBP and 0.1 mM IPTG at 16 °C and 0.2 OD600 for Ac-AChBP. Cells were harvested by centrifugation (6076 × g) and the cell pellet re-suspended in 20 mM Tris, 150 mM NaCl, 10% glycerol, pH 8.0. Cell pellets were lysed by repeated (3×) freeze-thawing of the pellets, sonication and incubation with lysozyme at a final concentration of 0.5 mg mL−1 (Sigma-Aldrich). To reduce viscosity and to prevent proteolysis of the expressed protein, DNAase (10 units per μL, Roche) and protease inhibitor tablets (1 tablet/50 mL of supernatant, cOmplete EDTA-Free Roche) were added. The lysed cells were pelleted at 39 000 × g and the supernatant purified by IMAC using HIS-select® HF nickel affinity gel (Sigma Aldrich). Theoretical molecular weights for AChBPs were calculated using ExPASy Protparam.

AChBP binding assays

The ability of peptides to displace the binding of [3H]-epibatidine to the recombinantly expressed Ls- and Ac-AChBPs was examined via a competitive radioligand binding assay. Briefly, a master mixture containing [3H]-epibatidine (fixed 1 nM final concentration) and increasing concentrations of test ligand in a final volume of 100 μL were incubated for 1 h at 4 °C in 96-well plates (Flexible PET Microplate, Perkin Elmer) pre-coated with 1 ng μL−1 of AChBP per well in binding buffer [phosphate-buffered saline (PBS), 0.05% bovine serum albumin]. Unbound ligands were washed off manually followed by the addition of 100 μL of scintillant (Optiphase Supermix, Perkin Elmer) in each well. Plates were incubated for 2 min on a shaker. The radioactivity was measured with a Wallac 1450 MicroBeta liquid scintillation counter (Perkin Elmer). Radioligand binding data were analysed by a nonlinear, least squares one-site competition fitting procedure using GraphPad Prism 9.0 (GraphPad Software Inc.). Experiments were performed in triplicate in three independent experiments and pKi values are reported as mean with 95% confidence interval. Comparison of the log[Ki] values of each variant with the respective native α-conotoxin was carried out by pairwise comparison using an extra sum-of-squares F test with P < 0.05 in GraphPad Prism 9.0.

Results and discussion

Synthesis and purification of α-conotoxin variants

Four variants of each of EpI, AnIB and PnIA were synthesised by SPPS, with sequences shown in Table 1. Note that the native peptides bearing both C-terminal amidation and sulfotyrosine PTMs are designated as e.g. EpI-NH2 while the variants in which the native sulfotyrosine is replaced by tyrosine are designated as e.g. EpI[Y(SO3)15Y]-NH2. Synthesis proceeded smoothly by SPPS, although truncation after Pro6 was a major side-product for PnIA syntheses, despite prolonged double coupling of Leu5. Whereas PnIA has consecutive proline residues at this position, EpI and AnIB only have a single proline, which is less likely to lead to hindered coupling. Nevertheless, the truncated PnIA by-product was easily separated by RP-HPLC and sufficient material (yields of 2–8 mg from 0.1 mmol scale syntheses) was obtained for NMR spectroscopy and functional assays. The native Cys(i)–Cys(iii) and Cys(ii)–Cys(iv) disulfides were formed selectively using a two-step orthogonal protection strategy; the Cys(ii)–Cys(iv) disulfide was formed first under air oxidation, followed by removal of the Acm protecting groups on Cys(i) and Cys(iii) and concurrent oxidation with iodine. Similar yields and purity were obtained with formation of the Cys(i)–Cys(iii) disulfide first, followed by Cys(ii)–Cys(iv). Although a one-pot oxidation using a similar strategy was reported while this study was in progress,27 we found that a RP-HPLC purification step between the two oxidation steps simplified purification. Preliminary syntheses of EpI followed by undirected disulfide oxidation led to multiple conformers that were difficult to separate. As selective disulfide formation was used in this study to ensure the native disulfide connectivity, we did not explore the possible effect of tyrosine sulfation and/or C-terminal amidation on the disulfide connectivity in an undirected folding experiment, or the possible effects on rate or yield of folding.

Names and sequences of α-conotoxin variants synthesised in this study.

Peptide name Sequencea
EpI[Y(SO3)15Y]-OH H-GCCSDPRCNMNNPDYC-OH
EpI-OH H-GCCSDPRCNMNNPDY(SO3)C-OH
EpI[Y(SO3)15Y]-NH2 H-GCCSDPRCNMNNPDYC-NH2
EpI-NH2 (native) H-GCCSDPRCNMNNPDY(SO3)C-NH2
AnIB[Y(SO3)16Y]-OH H-GGCCSHPACAANNQDYC-OH
AnIB-OH H-GGCCSHPACAANNQDY(SO3)C-OH
AnIB[Y(SO3)16Y]-NH2 H-GGCCSHPACAANNQDYC-NH2
AnIB-NH2 (native) H-GGCCSHPACAANNQDY(SO3)C-NH2
PnIA[Y(SO3)15Y]-OH H-GCCSLPPCAANNPDYC-OH
PnIA-OH H-GCCSLPPCAANNPDY(SO3)C-OH
PnIA[Y(SO3)15Y]-NH2 H-GCCSLPPCAANNPDYC-NH2
PnIA-NH2 (native) H-GCCSLPPCAANNPDY(SO3)C-NH2
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a

C = oxidised cysteine (cystine); Y(SO3) = sulfotyrosine; N- and C-termini are shown in small capitals representing a primary amine at the N-terminus and either carboxylic acid or amide at the C-terminus.

The sulfate group on sulfotyrosine has been reported to be readily hydrolysed under strongly acidic conditions and standard peptide coupling conditions and therefore a protection strategy is needed to incorporate sulfotyrosine into peptides by SPPS.22 The neopentyl group is stable to SPPS conditions and was used in this study.30,31 Previous syntheses of EpI used unprotected sulfotyrosine and reported hydrolysis during the resin cleavage.18 We found the neopentyl group to be stable during SPPS and peptide cleavage, but readily cleaved in aqueous solution (0.05% TFA) during dissolution and HPLC purification, as also reported by Li and He (2020).27 By the second oxidation and purification step, sulfotyrosine was fully deprotected. Despite initial concerns that the sulfate group would be hydrolysed in the acidic conditions used for HPLC, we found that all six sulfated conotoxins were stable to multiple rounds of HPLC and for up to a week at pH 3 and room temperature. Mass spectrometry analysis of the sulfated peptides in positive ion mode showed the ions of the desulfurised product, however, negative ion mode was used to confirm the presence of the sulfate PTM in the purified products and NMR samples.22 Analytical HPLC and mass spectrometry data of the 12 synthesised α-conotoxin variants are shown in the ESI† S1. These syntheses demonstrate the versatility of SPPS in introducing site-selective disulfide bonds, C-terminal amidation and tyrosine sulfation in these small and highly-modified peptides. Such precisely-modified α-conotoxin variants would be difficult to obtain using other approaches such as recombinant expression, genetic code expansion, enzymatic modification or isolation from natural sources.11

Activation of nAChR receptors and AChBPs

Activities of α-conotoxins EpI, AnIB and PnIA have been reported but it is often difficult to compare activities from the literature directly as many different assays, receptors and species have been used.17,18,42,43 The activity of EpI was originally tested using rat phrenic nerve hemidiaphragm preparations (for muscle-type nAChRs) and bovine chromaffin cells (for neuronal-type nAChRs) and membrane currents were measured in rat parasympathetic neurons.18 From these early studies, it was concluded that EpI was active at rat α3β2 and/or α3β4 nAChR subtypes and inactive at α7 and muscle nAChRs.18 In these assays, activities of the sulfated and unsulfated EpI variants were similar.18 In contrast, later experiments using oocyte-expressed rat nAChRs showed no significant inhibitory activity for EpI on α3β2 or α3β4 but nanomolar IC50 values at α7 nAChR, suggesting that selectivity and activity might differ between native and recombinant nAChRs.42 In this study, sulfated EpI was three-fold more potent than unsulfated EpI at oocyte-expressed rat α7 receptors.42 A study on AnIB found that C-terminal amidation and tyrosine sulfation of AnIB increased activity at rat α7 receptor more than α3β2 subtypes expressed on oocytes.17 PnIA was reported to be active at α3β2, α3β4 and α7 receptors in electrophysiology experiments, and amino acid substitutions led to shifts in the subtype selectivties.43,44 Here, we aimed to determine the role of the tyrosine sulfation and C-terminal amidation PTMs systematically at pharmacologically relevant human and prey-relevant mollusc receptors. To compare the activities of the PTM variants, we tested all four variants of the α-conotoxins EpI, AnIB and PnIA on two nAChR subtypes (α7 and α3β4), endogenously expressed on human SH-SY5Y cells, using a fluorescence imaging plate reader (FLIPR) calcium-influx assay. As shown in Table 2, none of the tested α-conotoxin variants was active at the human α7 nAChR subtype and only the EpI variants showed antagonist activity at human α3β4 nAChR (Fig. 2a).

Activities and binding affinities of α-conotoxin variants at nAChRs (human α7 and α3β4 subtypes) and mollusc AChBPs (Ac-AChBP and Ls-AChBP)a.

Peptide name pIC50 pKi
α7 nAChR α3β4 nAChR Ac-AChBP Ls-AChBP
EpI[Y(SO3)15Y]-OH NAb 5.87 (6.05–5.67) 6.00 (6.29–5.77) <4.00
EpI-OH NA 6.30 (6.47–6.13) 6.50 (6.77–6.24) <4.00
EpI[Y(SO3)15Y]-NH2 NA 6.46 (6.57–6.36) 7.57 (7.70–7.44) <4.00
EpI-NH2 (native) NA 7.19 (7.24–7.14) 8.38 (8.51–8.23) <4.00
AnIB[Y(SO3)16Y]-OH NA NA 8.00 (8.19–7.79) <4.00
AnIB-OH NA NA 8.66 (8.85–8.44) <4.00
AnIB[Y(SO3)16Y]-NH2 NA NA 8.96 (9.10–8.82) 4.61 (4.99–4.15)
AnIB-NH2 (native) NA NA 9.40 (9.52–9.30) 5.27 (5.51–5.03)
PnIA[Y(SO3)15Y]-OH NA NA 7.51 (7.60–7.42) <4.00
PnIA-OH NA NA 8.33 (8.43–8.23) <4.00
PnIA[Y(SO3)15Y]-NH2 NA NA 8.85 (8.96–8.77) 5.04 (5.49–4.57)
PnIA-NH2 (native) NA NA 8.92 (9.05–8.80) 5.42 (6.21–4.68)
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a

Data represent mean with 95% confidence interval.

b

NA = not active: no inhibition of calcium influx was observed at the highest concentration tested (10 μM).

Fig. 2. Activity of EpI variants on nAChR α3β4 subtype and Ac-AChBP. a) Inhibition of nicotine activation of α3β4 nAChR by EpI variants, measured in a FLIPR Ca2+ influx assay on SH-SY5Y cells. b) Displacement of [3H]-epibatidine from Ac-AChBP by EpI variants. c) Concentration–response curves for nicotine alone (black curve) and in the presence of increasing concentrations of EpI variants i) EpI-NH2, ii) EpI[Y(SO3)15Y]-NH2 and iii) EpI-OH. Data represent mean ± SEM of triplicate data from three independent experiments.

Fig. 2

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At the α3β4 nAChR subtype, the amidated variants EpI-NH2 and EpI[Y(SO3)15Y]-NH2 showed higher inhibitory activity than the corresponding acids, EpI-OH and EpI[Y(SO3)15Y]-OH (Table 2 and Fig. 2a). Likewise, the sulfated variants EpI-NH2 and EpI-OH showed higher inhibitory activity than their corresponding unsulfated counterparts EpI[Y(SO3)15Y]-NH2 and EpI[Y(SO3)15Y]-OH. Interestingly, EpI[Y(SO3)15Y]-NH2, EpI-OH and EpI[Y(SO3)15Y]-OH failed to fully inhibit α3β4 nAChR responses at concentrations up to 10 μM but fully displaced 3H-epibatidine binding (Fig. 2b). Inhibition of α3β4 nAChR by EpI-NH2 is almost complete at 10 μM but the confidence interval did not overlap with 0% nicotine response. These data, and competition studies between nicotine and increasing concentrations of EpI-NH2, EpI[Y(SO3)15Y]-NH2 and EpI-OH (Fig. 2c), suggest a possible insurmountable antagonism by EpI and its variants. This might arise if the EpI analogues act as functional allosteric or partial inhibitors at nAChRs, potentially confounded by functional experiments, which are by their nature measuring a hemi-equilibrium state.45 Further studies are required to better characterise these phenomena.

Since the natural prey of the cone snails producing EpI, AnIB and PnIA are molluscs, we tested the binding of the EpI, AnIB and PnIA variants at two acetylcholine-binding proteins (AChBPs), which are soluble proteins that modulate neurotransmission in molluscs and worms by sequestering acetylcholine released in the synapse.28,29 AChBPs form homopentameric assemblies of subunits, which are homologous to the N-terminal, extracellular ligand-binding domain of nAChRs. In particular, the aromatic cage residues responsible for orthosteric ligand recognition in nAChRs are conserved in AChBPs.46–48 Several co-crystal structures of AChBPs with various ligands have been reported, providing insights into ligand–receptor interactions and serving as a basis for homology models of nAChRs.49,50 We measured binding of the four variants of each of the α-conotoxins EpI, AnIB and PnIA to AChBPs from Aplysia californica (Ac) and Lymnae stagnalis (Ls) in a displacement assay that detects displacement of the tritiated ligand [3H]-epibatidine from immobilised AChBPs. As shown in Table 2, Fig. 2 and ESI† S2, all variants showed binding to Ac-AChBP, whereas only weak binding to Ls-AChBP was observed for the amidated variants of AnIB and PnIA. This reinforces the high selectivity of α-conotoxins for AChBP from different species and for different nAChR subtypes, due to shifts in specific pair-wise interactions in the binding pocket. For all three α-conotoxins, a trend emerged of increasing binding with addition of the native PTMs; the sulfated and C-terminal amidated variants of all three α-conotoxins have the lowest Ki values of the four variants. Using a pairwise comparison of the Ki values for binding to Ac-AChBP, all the variants are significantly (p = <0.05) different to the native variant. This result shows that C-terminal amidation and tyrosine sulfation act in an additive manner to increase the activity or binding of the α-conotoxins.

NMR spectroscopy of α-conotoxin variants

The differences in biological activity (binding and inhibition) of the differently-modified α-conotoxin variants prompted us to investigate whether the PTMs cause structural changes to the α-conotoxins that would alter their interactions with the receptors or interact directly with the receptors. The small size of the α-conotoxin variants synthesised makes them ideal for NMR spectroscopy and 1H, 1H–1H TOCSY, 1H–1H NOESY, 1H–13C HSQC and 1H–15N HSQC spectra were acquired on a 700 MHz NMR spectrometer equipped with a cryoprobe for all variants at natural isotope abundance.51 Resonances were assigned using the sequential assignment protocol and the secondary Hα-chemical shifts of the four variants of each of the three α-conotoxins are shown in Fig. 3. The random coil shifts of sulfotyrosine were obtained from an earlier study on the random coil shifts of posttranslationally modified amino acids.52 Stretches of negative Hα shifts are indicative of the presence of α-helices and can be observed between residues 6–12, as is typical for α-conotoxins, showing that all variants are folded in the native globular form. In all three α-conotoxins, the secondary Hα shifts of the C-terminally amidated variants are greater in magnitude than those of the corresponding C-terminal acids, indicating that C-terminal amidation stabilises the secondary structures. Multiple minor conformations of the C-terminal acid variants were observed as low intensity peaks in the NMR spectra. In particular, the PnIA C-terminal acids showed multiple conformations and had secondary shifts <0.1 ppm (Fig. 3c), indicative of poor structural definition. Two distinct conformations could be observed and sequentially assigned for PnIA[Y(SO3)15Y]-NH2 and the secondary shifts are shown in ESI† Fig. S3. All proline residues in EpI, AnIB and PnIA[Y(SO3)15Y]-NH2 (major conformation, ∼65%) and PnIA-NH2 are predominantly in the trans-configuration, based on 13C chemical shifts and 1H–1H NOESY interactions; however, for PnIA[Y(SO3)15Y]-OH, PnIA-OH and PnIA[Y(SO3)15Y]-NH2 (minor conformation), Pro7 is in the cis-conformation, suggesting that the presence of the C-terminal amide and sulfotyrosine PTMs helps to determine the proline conformation.

Fig. 3. Secondary Hα chemical shifts of each of the four variants of a) EpI, b) AnIB and c) PnIA calculated with reference to the respective random coil chemical shifts.52,53 Stretches of negative Hα secondary shifts indicate α-helical regions and relative magnitudes indicate that the amidated and sulfated variants are more structured than their corresponding acids and unsulfated variants.

Fig. 3

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The three-dimensional structures of the four EpI variants, four AnIB variants and PnIA-NH2 were calculated from interproton distance restraints and dihedral angles predicted from chemical shifts. Hydrogen bond restraints were also added for the EpI variants and PnIA-NH2. Particularly for the non-native (unmodified) variants of PnIA, multiple conformations were observed, making it difficult to assign NOE cross-peaks unambiguously so structures of the non-native PnIA variants were not calculated. The structure calculation parameters and statistics are shown in ESI† Tables S1–S3 and the structures have been deposited in the Protein Data Bank. Structure ensembles of the EpI, AnIB variants and PnIA-NH2 are shown in Fig. 4, and ESI† Fig. S4 and S5, respectively. To our knowledge, these structures are the first structures of conotoxins bearing native tyrosine sulfation PTMs.

Fig. 4. Ensembles of 20 structures of EpI variants with lowest energies and best MolProbity scores in (a) cartoon representation showing the α-helical regions (figures generated in MolMol) and (b) stick representation with backbone in grey, (sulfo)tyrosine in green and the C-terminal carboxylic acid/amide coloured by element type (figures generated in Pymol).

Fig. 4

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All structures contain an α-helix from residues 6–11, which is typical of α-conotoxins.5 Although the PTMs do not cause major overall structural differences, the variants bearing C-terminal amidation and tyrosine sulfation PTMs have better-defined turns at the N- and C-termini as well as more defined ensembles (Fig. 4a). More long-range distance restraints were identified for the amidated variants of EpI (ESI† Table S1), further supporting that these variants sample less conformational space. C-Terminal amidation stabilises the helical turn at the C-terminus by counterbalancing the partial negative charges of the three backbone carbonyl oxygens of residues 14–16 (Fig. 4b). In contrast, the C-terminal acids bend away from these carbonyl oxygens. As previously noted for the crystal structures of unsulfated EpI and PnIA,25,54 the tyrosine side-chain is exposed on the surface of the structure, making it accessible for modification but unlikely to influence the overall backbone conformation. This is confirmed in the structures of all EpI, AnIB and PnIA structures. Although exposed on the surface, interactions between the aromatic protons of Tyr15 (Tyr16 in AnIB) and N-terminal residues (Cys2 or Cys3) were observed and in the sulfated variants, the negatively-charged sulfotyrosine moves closer to the positively-charged N-terminus (Fig. 4b). In the AnIB variants, which contain an additional N-terminal glycine in the native sequence, the sulfotyrosine is positioned near the N-terminus, suggesting an ionic interaction (ESI† Fig. S4). Considering these structural features alongside the activity/binding data (Fig. 2), we propose that C-terminal amidation and tyrosine sulfation stabilise the binding conformation of EpI, AnIB and PnIA, thereby lowering the entropic penalty for binding and activation of their respective acetylcholine receptors.

The mechanism of tyrosine sulfation of conotoxins is not yet reported, to our knowledge, and it is not known whether sulfation occurs before or after processing of the precursor peptide and disulfide bond formation. In humans, protein sulfation by tyrosylprotein sulfotransferases involves interactions with residues flanking the tyrosine site (particularly the −1 position) and substrates have to pass through a deep cleft in the enzyme to reach the active site.55 Most tyrosine sulfation therefore occurs in disordered regions and would suggest that tyrosine sulfation in α-conotoxins occurs prior to disulfide formation. Although tyrosylprotein sulfotransferases do not have a rigid sequence motif for recognition and sulfation, acidic residues flanking the tyrosine are important for interactions with the positively-charged active site.22 All of the conotoxins reported to have a sulfotyrosine PTM contain the -DYC- motif, which fulfils the requirement for an acidic flanking residue and could be used to predict the presence of sulfotyrosine in other conotoxins. For example, a sequence search for -DYC-NH2 in the Conoserver database reveals that this motif is present in AuIA and might be sulfated,17 in addition to the seven α-conotoxins already reported to contain this PTM.

Co-crystal structures of conotoxins with AChBPs and docking studies of conotoxins with homology models of nAChRs have provided insights into the ligand binding sites and mechanisms of these pharmacologically important receptors.56–58 For example, a co-crystal structure of an unsulfated PnIA mutant [PnIA(A10L, D14K)] with Ac-AChBP, which resembles the α7 nAChR receptor in terms of the ligand-binding site at the subunit interface.56 This co-crystal structure showed that the PnIA mutant occupied all five binding sites, with the central helix protruding into the binding site (ESI† Fig. S6). The C-terminus was located near the outside of the binding site,56 suggesting that the amidated C-terminus and sulfated tyrosine near the C-terminus would protrude out of the binding site. Interestingly, electron density for a buffer sulfate molecule was observed, making contact with Cys188-NH in Ac-AChBP and suggesting that a sulfate moiety might contribute to the toxin-receptor interaction (ESI† Fig. S6).56 Our structures of the native sulfated forms EpI, AnIB and PnIA will be used in future co-crystal and docking studies to provide further insights into interactions with and specificities of α-conotoxins for their receptors. Such studies have been used, for example, to rationally design α-conotoxin analogues with specific α3β4 activity.57

Conclusions

For their size, α-conotoxins contain an extraordinary number of PTMs, however, few studies have investigated their structural and functional roles systematically. We used solid phase peptide synthesis to make all PTM variants of three α-conotoxins EpI, AnIB and PnIA and show that the C-terminal amidation and tyrosine sulfation PTMs act synergistically to stabilise the secondary structure and increase activity and binding at nAChRs and AChBPs, respectively. We propose that the C-terminal amidation and tyrosine sulfation PTMs increase activity and binding predominantly by stabilising the binding conformation, rather than interacting directly with the binding sites. Stabilisation of the binding conformation in solution would reduce the entropic cost of binding and thereby increase the proportion of bound α-conotoxin molecules. Structure stabilisation could therefore be an important consideration in designing potent and selective α-conotoxins for therapeutic applications and in studying the pharmacology of nAChRs.

In addition to increasing our knowledge of receptor biology, this study also demonstrates how conotoxins can provide a useful model system for understanding individual and combinatorial effects of PTMs. The synthetic accessibility of conotoxins bearing PTMs by solid phase peptide synthesis and their amenability to solution NMR spectroscopy without the need for isotope enrichment make them valuable small structured proteins to study the structural and functional roles of PTMs. Small modified proteins such as conotoxins could help to bridge the gap between random coil peptides bearing PTMs52 and larger proteins bearing PTMs, which typically require specialised chemical biology tools to access and would require isotope labelling for NMR structural studies.11 Studies such as this will therefore help to build up a picture of the mechanisms by which PTMs modulate protein function, either by causing structural change, or by altering binding interactions, or both. The benefit of insights into how proteins are regulated by PTMs could extend into the biomedical sciences, ultimately helping to understand disease processes and guiding drug design.

Conflicts of interest

The authors declare no conflict of interest.

Supplementary Material

MD-012-D1MD00182E-s001

Acknowledgments

We are grateful to Gregory Pierens for assistance with acquiring NMR data and to Sarah Kuschert and Mehdi Mobli (the University of Queensland, Centre for Advanced Imaging) for the sulfotyrosine (TYS) and C-terminal amide (CAM) CYANA library parameters.

Anne Conibear is supported by a University of Queensland Development Fellowship (Project 613982) and an Early Career Researcher grant (Project 616535) from the University of Queensland. Shilpa Swaminathan and Lewis Goodwin were supported by a UQ Summer Research Program stipend.

†

Electronic supplementary information (ESI) available: Analytical MS and HPLC data, structural statistics and additional assay data are available in the attached ESI. See DOI: 10.1039/d1md00182e

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MD-012-D1MD00182E-s001
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