Supporting Information

Molecular interaction of δ-conopeptide EVIA with voltage-gated Na+ channels

Daniel Tietze#, Enrico Leipold#, Pascal Heimer, Miriam Böhm, Wadim Winschel, Diana Imhof, Stefan H.

Heinemann, and Alesia A. Tietze*

Table of Contents1. Experimental material

2. References

1

1. Experimental material

Chemical synthesis and analysis of δ-EVIA

Fig. S1. Characterization of synthetic reduced δ-EVIA by RP-HPLC, HPLC conditions were as follows: 20% - 80%

eluent B (acetonitrile, 0.1% TFA) in water (0.1% TFA) in 15 min, detection was performed at 220 nm. The inset

shows the ESI mass spectrum.

The RP HPLC purified linear precursor δ-EVIA (Fig. S1), characterized by LC-ESI MS and revealing the

desired mass peaks of reduced peptide ([M+4H]4+ at 823.9, [M+3H]3+ at 1098.2 and [M+2H]2+ at 1647.3),

was subjected to oxidative folding under following conditions: peptide concentration of 0.05 mM in buffer

solution (0.1 M Tris-HCl buffer/1 mM EDTA) containing 30% of organic solvent, primarily isopropanol.

The reaction was left while stirring at 4 °C for 16 h. The yield of the peptide dropped dramatically the

longer it was stirred in the aqueous medium (data not shown). The main fraction of oxidative folding was

purified by RP HPLC. HPLC conditions were as follows: 25% - 75% eluent B in 30 min, eluent B: 0.1%

TFA in acetonitrile detection was performed at 220 nm. The HPLC chromatogram of purified oxidized

product is represented in Fig. S2. The LC-ESI mass spectrum (Fig. S2) showed differently charged

species of desired oxidized product δ-EVIA ([M+4H]4+ at 822.8, [M+3H]3+ at 1096.8 and [M+2H]2+ at

1644.7). Further, the product was subjected to chemical analysis and MS/MS sequencing (Fig. S3).

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Fig. S2. Characterization of synthetic oxidized δ-EVIA by RP HPLC, HPLC conditions were as follows: 20% - 80%

eluent B (acetonitrile, 0.1% TFA) in water (0.1% TFA) in 15 minutes, detection was performed at 220 nm. The inset

shows the ESI mass spectrum.

Peptide analytical characterizationAll crude peptide material, linear and oxidized versions, was purified by semi-preparative RP-HPLC

using a Shimadzu LC-8A system equipped with a C18 column (Knauer Eurospher 100, Berlin,

Germany). The gradient elution system was 0.1% TFA in water (eluent A) and 0.1% TFA in

acetonitrile/water (9:1) (eluent B). The peptides were eluted with a gradient of 25% - 75% eluent B in 120

min and a flow rate of 10 mL/min. The peaks were detected at 220 nm. Collected fractions were

combined, freeze-dried and stored at –20 °C. Purity was confirmed by analytical RP-HPLC on a Waters

600 system equipped with a Multospher 120 RP 18 HP (C18, 3.1 µm particle size, 120 Å pore size, 4.6 x

60 mm). The peptides were eluted with a gradient of 20% - 80% eluent B in 15 min at a flow rate of

2 mL/min. Eluents were A: 0.1 % TFA in water and eluent B: 0.1 % TFA in acetonitrile; detection was at

220 nm.

The molecular weight of crude and purified peptide was confirmed by LC-ESI mass spectrometry on a

microTOF-Q III spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a Dionex-LC UltiMate

3000 system (Thermo Scientific, Dreieich, Germany). The observed monoisotopic masses in comparison

with the theoretically calculated molar masses were as follows: δ-EVIA (reduced, found) [M+H]+ 3,291.6

g/mol (m.i. 3,291.44 g/mol), δ-EVIA (oxidized, found) [M+H]+ 3,287.4 g/mol (m.i. 3,285.44 g/mol).

The Ellman’s test was used to confirm the fully oxidized peptide.[1, 2] For that purpose Ellman’s test was

performed for the reduced linear precursor of δ-EVIA and the product obtained by oxidative folding in

redox buffer. The measurements were carried out as described earlier.[3]

The amino acid composition of the peptides and the concentration determination prior to the

electrophysiological experiments was performed by amino acid analysis using a LC 3000 system from

Eppendorf-Biotronik (Hamburg, Germany).

A3

B

4

C

Fig. S3. Result from MS/MS sequencing of (A) linear δ-EVIA as derived from BioTools (Bruker), (B) partially

reduced and alkylated δ-EVIA (4-times carbamidomethylated): (C3–C21, C10–C25), and (C) partially reduced and

alkylated δ-EVIA (4-times carbamidomethylated): (C3–C21, C20–C29). *carbamidomethylated cysteine. “+”

indicates the charge of the observed fragment ion.

MS/MS analysis of disulfide connectivityThe partial reduction and alkylation protocol was adapted from an earlier report.[4] The freeze-dried

peptide was dissolved in citrate buffer (0.05 M, pH 3.0) containing 0.02 M tris(2-carboxyethyl)phosphine

(TCEP) to a final concentration of 0.5 mg/ml. The mixture was then incubated at room temperature and

samples were taken at different time points ranging from 0 sec to 3 h. The samples (one volume) were

mixed with three volumes of alkylation buffer (0.5 M tris acetate pH 8.0, 2 mM EDTA and 1.1 M

iodacetamide) to stop the reaction and perform carbamidomethylation at the free thiol groups. After

10 min the alkylation reaction was terminated by addition of one volume of 10% TFA and stored on dry

ice until analysis. HPLC analysis of the samples was carried out as described earlier, using the following

gradient: 25% eluent B for 15 min (isocratic), then linear increase of eluent B up to 58% for 20 min.

Fractions were collected and analyzed on a micrOTOF-Q III mass spectrometer (Bruker Daltonics,

Bremen, Germany). Results are shown in Fig. S3 A-C. Disulfide connectivity in the samples was

determined using derivatives containing two or four carbamidomethylated cysteine residues which were

reduced using 10 mM DTT, 10 mM phosphate buffer pH 8.5 and analyzed by MS/MS analysis using the

aforementioned micrOTOF-Q III system and BioTools (Bruker Daltonics, Bremen, Germany).

5

NMR structure determination

Table S1. Energy and structural statistics of the cis/trans δ-EVIA NMR ensemble (lowest energy

structures).

NMR distance & dihedral constraints 16 structurestrans δ-EVIA cis δ-EVIA

Distance constraints 224 216Dihedral angle restraints 46 42Structure statisticsViolations Mean (s.d.)Total restraint violation energy 12.9 kJ/mol 13.7 kJ/mol

Distance restraints 10.5 kJ/mol 6.5 kJ/molDihedral angle restraints 2.4 kJ/mol 7.2 kJ/mol

Total force field energy (Yasara) -6902 kJ/mol -5739 kJ/molInternal solute energy -3370 kJ/mol -2211 kJ/molElectrostatic solv. energy -3141 kJ/mol -3153 kJ/molVan der Waals solv. energy -391.3 kJ/mol -374.8 kJ/mol

Mean global r.m.s.d. (Å)Backbone global (residues 1-32) 1.62 (0.41) 1.91 (0.66)Heavy atoms (residues 1-32) 1.86 (0.45) 2.33 (0.71)Backbone global (residue 3-10, 20-30) 1.20 (0.31) 1.00 (0.28)Heavy atoms (residue 3-10, 20-30) 1.35 (0.34) 1.32 (0.29)

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Fig. S4. Alignments of the lowest energy conformation of the NMR ensemble of (A) cis-δ-EVIA with pdb 1G1Z [5]

(gray), (B) trans -EVIA with pdb 1G1P [5] (gray) and (C) cis-δ-EVIA (cyan, disulfide bridges are highlighted in

yellow, -sheet in red) with trans δ-EVIA (blue, disulfide bridges are highlighted in grey). Residues Leu12-Pro13

and the disulfide bonds between Cys3-21, Cys10-25, and Cys20-29 are shown as sticks (carbon – cyan, nitrogen –

blue, oxygen – red).

Homology Modeling

Fig. S5. Alignment of the pore region of domain I (DI) of the channel template Na VAB (pdb ID 3rvy) to rNaV1.4

(uniprot ID P15390) and mNaV1.7 (uniprot ID B1AYL0). Residues labeled by the blue box represent the homology-

modeled segment DI S5 and its alignment to the channel template NavAB. Additionally, residues in the SS2-S6

loop in NavAB (magenta box) are swapped according to the sequence of rNaV1.4 and mNaV1.7, since they could

be part of the toxin receptor site as shown for the scorpion -toxin LghII at rNaV1.2.[6] Sequence alignments were

created with Clustal Omega [7-9] on www.uniprot.org. Alignment figure was created with JALVIEW 2.8.[10]

A B7

Fig. S6. General production scheme for the NaV1.7 homology model, (A) alignment of the resting state DIV VSD

(green) and DI S5 (red) at the channel template NavAB (pdb 3rvy, gray) (B) final Na V1.7 homology model prior to

refinement simulation in membrane environment, which now consists only of the pore domain of pdb 3rvy, the

modeled DI S5 transmembrane domain and DIV VSD. The bulky S5-S6 loop in DIV was excluded from the final

homology model due to its unpredictable structure and, hence, poor structural quality. The same procedure was

applied for NaV1.4, 4777 and 7444 homology models.

Docking and molecular dynamics simulations

8

Tab. S2. Summary of the VINA docking runs. Docking results are scored according to the binding energy between

δ-EVIA and NaV channel.

best in cluster cluster [kJ/mol]members/cluster

[kJ/mol] mean SD NaV1.7 37.71 34.72 2.24 3

36.15 34.17 1.34 934.33 33.25 0.81 334.00 34.00 132.67 32.67 132.61 32.61 131.96 31.96 131.65 31.65 1

NaV1.4 29.70 28.84 0.63 728.71 28.50 0.21 228.69 28.69 128.67 28.30 0.22 428.57 28.38 0.18 228.52 28.52 128.17 28.09 0.08 227.80 27.80 1

4447 36.10 35.17 0.94 235.11 33.97 0.59 634.17 33.23 0.47 533.35 32.93 0.38 532.44 32.44 132.37 32.37 1

7774 34.10 31.47 1.50 1132.07 31.10 0.97 231.46 30.83 0.50 429.78 29.78 129.63 29.36 0.26 2

Calculated binding energies (best results shown) as a result of 24 independent docking runs.

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Fig. S7. (A, B) In silico mutagenesis study. For each mutation step, toxin binding energy (calculated as free energy

of binding incl. solvation effects) was calculated individually and plotted as an increase or decrease with respect to

the binding energy calculated from the wild-type channel.

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Fig. S8. Calculated root mean square fluctuations (r.m.s.f.) from the 50-ns refinement simulations in a membrane

environment for (A) NaV1.4 homology model, the modeled NaV1.4 – δ-EVIA complex and the modeled 7444 – δ-

EVIA complex as well as for (B) the NaV1.7 homology model, the modeled NaV1.7 – δ-EVIA complex and the

modeled 4447 – δ-EVIA complex.

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Fig. S9. (A) Root mean square deviation (r.m.s.d.) of Cα atoms and (B) binding energies of δ-EVIA at the receptor

site calculated from the 50-ns refinement simulation in membrane environment. Average binding energies were

calculated from the last 5 ns of the 50-ns refinement simulation in membrane environment including solvation

effects in order to estimate individual contributions of DI S5, DIV S1-S2, and DIV S3-S4 to δ -EVIA binding depicted

in Fig. 5A.

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Fig. S10. Energy minimized final structural models of cis-δ-EVIA binding to (A) 4447 and (B) 7444 after 50 ns

refinement simulation in a phospholipid environment. Hydrogen bonds are indicated as yellow dashed lines. Toxin

residues are represented in 3-letter code, while channel residues are indicated in single-letter code.

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Fig. S11. Sequence alignments of NaV isoforms and chimeras with highlighted residues involved in δ-EVIA binding

according to molecular docking results. Sequences alignment of the domain I S5 and domain IV VSD region (S1-

S4) of NaV channel isoforms: (A) hNaV1.5, rNaV1.4, mNaV1.7, rNaV1.2, mNaV1.6 and hNaV1.7. Residues that

comprise the δ-EVIA binding site are highlighted (mNav1.7 – blue, rNav1.4 – yellow); (B) Na V chimeras 4447 and

7444. Toxin residues that implicate binding to NaV are highlighted (7444, bold; 4777, underlined). Residues that are

involved in hydrogen bonding are indicated with an asterisk. Sequence alignments were created with Clustal

Omega on www.uniprot.org.[7-9]

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Fig. S12. Voltage dependence of activation of the NaV channel constructs used in this study. (A) Normalized mean

peak currents as a function of step depolarization for the indicated constructs before (gray) and after application of

15

150 µM δ-EVIA (red). The continuous curves are fits according to a standard Hodgkin & Huxley formalism as

described in Chen et al.([11] eq. 1). (B) Voltage dependence of channel activation, characterized by the half-

maximal activation voltage, Vm, and the corresponding slope factor, km, for the indicated constructs without (gray)

and with 150 µM δ-EVIA (red); the values for the wild types NaV1.4 and NaV1.7 are shown as vertical lines. Data

points are mean ± SEM (n in parentheses).

Fig. S13. Voltage dependence of inactivation of the NaV channel constructs used in this study. Steady-state

inactivation data obtained with 500-ms conditioning pulses (see Fig. 3) without toxin were described with single

Boltzmann functions according to Chen et al.([11] eq. 2) and yielding the half-maximal inactivation voltage, Vh, and

the corresponding slope factor, kh (gray symbols). Data obtained in the presence of 150 µM δ-EVIA were described

by the sum of two Boltzmann functions, the first one describing the channels without toxin and thus using the same

Vh and kh values as determined for the control, the second one representing the toxin-occupied channels with a

new set of Vh and kh values; Ptox denotes the relative contribution of the latter component. Data points are mean

± SEM (n values as in Fig. S12).

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Fig. S14. Activation and inactivation properties of mutants in the DVI S3-S4 linker of NaV1.4. (A) Current traces at –

20 mV without (black) and with 150 µM δ-EVIA (red) for the indicated constructs. (B) Steady-state inactivation for

the DVI S3-S4 mutants, analyzed as shown in Fig. 3. Mean effect of δ-EVIA on inactivation at –20  mV (C) and on

the slope of the steady-state inactivation between –140 and –120 mV (D); horizontal dashed lines indicate the

values of the wild-type channels. Data points are mean ± SEM (n in parentheses).

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Fig. S15. Structure alignment of NaV1.7 homology model (yellow) and cryo-EM structure of the rabbit voltage-gated

Ca2+ channel CaV1.1 (grey). Alignment of S1-S4 DIV with MUSTANG[12] resulted in r.m.s.d. of 1.93 Å over 68

aligned residues with 39.7% sequence identity and S5-S6 DIV in RMSD of 1.87 Å over 84 aligned residues with

28.6% sequence identity.

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2. References

[1] G.L. Ellman, A Colorimetric Method for Determining Low Concentrations of Mercaptans, Arch. Biochem. Biophys., 74 (1958) 443-450.[2] G.L. Ellman, Tissue Sulfhydryl Groups, Arch. Biochem. Biophys., 82 (1959) 70-77.[3] A. Miloslavina, C. Ebert, D. Tietze, O. Ohlenschlager, C. Englert, M. Gorlach, D. Imhof, An unusual peptide from Conus villepinii: synthesis, solution structure, and cardioactivity, Peptides, 31 (2010) 1292-1300.[4] P. Heimer, A.A. Tietze, M. Bohm, R. Giernoth, A. Kuchenbuch, A. Stark, E. Leipold, S.H. Heinemann, C. Kandt, D. Imhof, Application of room-temperature aprotic and protic ionic liquids for oxidative folding of cysteine-rich peptides, ChemBioChem, 15 (2014) 2754-2765.[5] L. Volpon, H. Lamthanh, J. Barbier, N. Gilles, J. Molgo, A. Menez, J.M. Lancelin, NMR solution structures of delta-conotoxin EVIA from Conus ermineus that selectively acts on vertebrate neuronal Na+ channels, J. Biol. Chem., 279 (2004) 21356-21366.[6] J. Wang, V. Yarov-Yarovoy, R. Kahn, D. Gordon, M. Gurevitz, T. Scheuer, W.A. Catterall, Mapping the receptor site for -scorpion toxins on a Na+ channel voltage sensor, Proc. Natl. Acad. Sci. U. S. A., 108 (2011) 15426-15431.[7] H. McWilliam, W. Li, M. Uludag, S. Squizzato, Y.M. Park, N. Buso, A.P. Cowley, R. Lopez, Analysis Tool Web Services from the EMBL-EBI, Nucleic Acids Res., 41 (2013) W597-W600.[8] F. Sievers, A. Wilm, D. Dineen, T.J. Gibson, K. Karplus, W. Li, R. Lopez, H. McWilliam, M. Remmert, J. Söding, J.D. Thompson, D.G. Higgins, Fast, scalable generation of high‐quality protein multiple sequence alignments using Clustal Omega, 2011.[9] M. Goujon, H. McWilliam, W. Li, F. Valentin, S. Squizzato, J. Paern, R. Lopez, A new bioinformatics analysis tools framework at EMBL–EBI, Nucleic Acids Res., 38 (2010) W695-W699.[10] A.M. Waterhouse, J.B. Procter, D.M. Martin, M. Clamp, G.J. Barton, Jalview Version 2--a multiple sequence alignment editor and analysis workbench, Bioinformatics, 25 (2009) 1189-1191.[11] H. Chen, D. Gordon, S.H. Heinemann, Modulation of cloned skeletal muscle sodium channels by the scorpion toxins Lqh II, Lqh III, and LqhIT, Pflugers Arch., 439 (2000) 423-432.[12] A.S. Konagurthu, J.C. Whisstock, P.J. Stuckey, A.M. Lesk, MUSTANG: a multiple structural alignment algorithm, Proteins, 64 (2006) 559-574.

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