Binding of Ala-scanning analogs of g-conotoxin MVIIC to N- andP/Q-type calcium channels

Kazuki Satoa;*, Cecile Raymondb, Nicole Martin-Moutotb, Toru Sasakia, Atsuko Ohtakea,Kazushi Minamia, Catherine Van Renterghemb, Jae Il Kimc, Masami Takahashia,

Michael J. Seagarb

aMitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-8511, JapanbINSERM U374, Institut Jean Roche, Faculte de Medecine Secteur Nord, Boulevard Pierre Dramard, 13916 Marseille Cedex 20, France

cKwangju Institute of Life Science and Technology, Kwangsan-ku, Kwangju 506-712, South Korea

Received 19 January 2000; received in revised form 7 February 2000

Edited by Shozo Yamamoto

Abstract gg-Conotoxin MVIIC binds to P/Q-type calciumchannels with high affinity and N-type channels with low affinity.To reveal the residues essential for subtype selectivity, wesynthesized Ala-scanning analogs of MVIIC. Binding assaysusing rat cerebellar P2 membranes suggested that Thr11, Tyr13

and Lys2 are essential for binding to both N- and P/Q-typechannels, whereas Lys4 and Arg22 are important for binding toP/Q-type channels. These results suggest that MVIIC interactswith P/Q-type channels via a large surface, in good agreementwith previous observations using chimeric analogs.z 2000 Federation of European Biochemical Societies.

Key words: g-Conotoxin MVIIC; Calcium channel;Ala-scanning

1. Introduction

Voltage-gated calcium channels play crucial roles in regu-lating intracellular calcium concentrations in a wide variety ofcells and have been classi¢ed into several subtypes accordingto their electrophysiological and pharmacological properties[1^3]. Among them, N- and P/Q-type channels are essentialfor the regulation of neurotransmitter release from a widevariety of neurons. Various speci¢c peptide ligands havebeen used for the pharmacological distinction of calciumchannel subtypes, including the g-conotoxins isolated fromthe venom of marine Conus snails. The de¢ning ligands forN-type calcium channels are g-conotoxin GVIA and MVIIA,while P/Q-type channels are blocked by g-conotoxin MVIIC(Fig. 1) [4]. Because MVIIC retains weak a¤nity for N-typechannels, it is important to develop more selective blockers

for P/Q-type channels. We reported previously that theN-terminal half of MVIIC is important for recognition ofN-type channels, whereas essential residues for P/Q-type chan-nel recognition are located on both N- and C-terminal halvesbased on results with two chimeric analogs of MVIIA andMVIIC [5].

In the present study, a series of analogs of MVIIC weresynthesized by replacing each amino acid residue except forGly, Ala and Cys with Ala in order to identify the essentialresidues for binding to N- and P/Q-type calcium channels.Results of binding assays suggested that a limited numberof residues are essential for binding to N-type channels,whereas the essential residues for recognition of P/Q-typechannels are widely spread over the MVIIC molecule, ingood agreement with the previous observations using chimericanalogs.

2. Materials and methods

2.1. Materials9-Fluorenylmethoxycarbonyl (Fmoc)-amino acids, Fmoc-NH-resin

and other reagents used on a synthesizer were obtained from PerkinElmer-Applied Biosystems (Chiba, Japan). Other reagents were ob-tained from Watanabe Chemical Industries (Hiroshima, Japan), Pep-tide Institute (Osaka, Japan) or Kokusan Chemical Works (Tokyo,Japan). Lysyl endopeptidase and thermolysin were purchased fromWako Pure Chemicals (Osaka, Japan).

2.2. Synthesis and puri¢cation of peptidesSolid phase peptide synthesis was performed on a Perkin Elmer-

Applied Biosystems 431A peptide synthesizer. Amino acid analyseswere performed on a Beckman System Gold amino acid analyzer afterhydrolysis in 6 M hydrochloric acid at 110³C for 24 h and derivatiza-tion by 4-dimethylaminoazobenzene-4P-sulfonyl chloride. Matrix as-sisted laser desorption/ionization time-of-£ight mass spectrometry(MALDI-TOF-MS) was carried out with a PerSeptive BiosystemsVoyager Linear DE mass spectrometer using K-cyano-4-hydroxy-cin-namic acid as a matrix. Analytical high performance liquid chroma-tography (HPLC) was conducted on a Shimadzu LC-6A system withan ODS column Shim-pack CLC-ODS (4.6U250 nm, Shimadzu).Preparative HPLC was performed on a Shimadzu LC-8A systemwith an ODS column Shim-pack PREP-ODS (H) (20U250 nm, Shi-madzu).

All the analogs were synthesized by a procedure similar to thatdescribed previously for the synthesis of MVIIC and its analogs[5,6]. Brie£y, linear precursors of MVIIC analogs were synthesizedby solid phase methodology of Fmoc chemistry. After tri£uoroaceticacid cleavage, crude linear peptide was diluted to the ¢nal peptideconcentration of 0.05 mM and subjected to oxidative disul¢de bondformation at 4³C for 3^5 days in 1 M ammonium acetate bu¡er (pH7.8) containing reduced/oxidized glutathione (molar ratio of peptide:GSH:GSSG was 1:100:10). The folding reaction was monitored by

0014-5793 / 00 / $20.00 ß 2000 Federation of European Biochemical Societies. All rights reserved.PII: S 0 0 1 4 - 5 7 9 3 ( 0 0 ) 0 1 2 6 3 - 1

*Corresponding author. Fax: (81)-42-724 6317.E-mail: kazuki@libra.ls.m-kagaku.co.jp

Abbreviations: Fmoc, 9-£uorenylmethoxycarbonyl; GSH, reducedglutathione; GSSG, oxidized glutathione; GVIA, g-conotoxinGVIA; HPLC, high performance liquid chromatography; [125I]GVIA,[125I]g-conotoxin GVIA; [125I]MVIIC, [125I][Nle12]g-conotoxinMVIIC; MALDI-TOF-MS, matrix assisted laser desorption/ioniza-tion time-of-£ight mass spectrometry; MVIIA, g-conotoxin MVIIA;MVIIC, g-conotoxin MVIIC; ODS, octadecylsilane; TFA, tri£uoro-acetic acid; Analogs are designated by a letter and number indicatingthe identity and position of the substituted amino acid, followed by aletter indicating the identity of the replacement residue; for example,K2A indicates an analog in which Lys2 is replaced with Ala

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HPLC and stopped by lowering the pH of the solution to 3^4 withAcOH. The crude cyclic products were puri¢ed by successive chroma-tography on Sephadex G-50F, CM-cellulose CM-52 and preparativeHPLC with an ODS column. The structure and purity of syntheticpeptides were con¢rmed by analytical HPLC, amino acid analysis andMALDI-TOF-MS measurements.

2.3. Enzymatic digestion for the determination of disul¢de bondcombination

To a solution of synthetic peptide (0.4 mg) in 100 Wl of 0.1 mMphosphate bu¡er (pH 6.5) was added a solution of lysyl endopeptidase(10 Wg) in 20 Wl of the same bu¡er. The mixture was incubated at 37³Cfor 1.5 h and subjected to HPLC separation and MALDI-TOF-MSmeasurements. The major fragment was lyophilized and dissolved into0.4 ml of 0.1 M ammonium formate bu¡er (pH 6.5). To 100 Wl of thissolution were added a solution of thermolysin (20 Wg) in 20 Wl of thesame bu¡er and 80 Wl of CaCl2 solution (2.5 mM in the same bu¡er).The mixture was incubated at 37³C for 3 h and subjected to HPLCseparation and MALDI-TOF-MS measurements.

2.4. CD measurementsCD spectra were recorded on a JASCO J-600 spectropolarimeter in

H2O solution (0.01 M sodium phosphate, pH 7.0) at 20³C, using aquartz cell of 1 mm path length. The spectra are expressed as molarellipticity (a).

2.5. Binding assayRat cerebellar P2 membranes (10 Wg) in 0.1 ml of 25 mM Tris,

150 mM NaCl, 0.1% bovine serum albumin adjusted to pH 7.4 withHCl (TBSA) were incubated with 0.5 nM [125I]GVIA or [125I]MVIICfor 1 h at 30³C. Membrane-bound radioactivity was measured afterrapid ¢ltration and washing on GF/C (Whatman) ¢lters treated with0.3% polyethyleneimine as described previously [7].

3. Results

3.1. Synthesis and puri¢cation of MVIIC analogsAll the linear precursors of MVIIC analogs were success-

fully assembled by solid phase methodology. Air oxidation ofthe crude linear precursors of most analogs a¡orded peptideswith proper disul¢de bondings as the major products, withgood overall isolation yields. However, cyclization of twoanalogs, P7A and D14A, in which Pro7 and Asp14 were re-placed with Ala, respectively, gave mixtures of several prod-ucts with a variety of retention times in HPLC analysis (Fig.2).

3.2. Disul¢de bond combinationAccording to the method described for MVIIC by Kubo et

al. [8], we successively digested most analogs with lysyl endo-peptidase and thermolysin. Fig. 3 shows the cleavage sites of

Fig. 1. Amino acid sequences and disul¢de bonds of g-conotoxinMVIIC, MVIIA and GVIA.

Fig. 2. HPLC pro¢les of the oxidative folding reaction of MVIICanalogs. Each chromatogram shows the HPLC pro¢le of reactionmixture after 3 days. Large peaks at around 4 min are due to thesolvent, GSH or GSSG. Column: Shim-pack CLC-ODS (4.6U250mm, Shimadzu). Solvent: linear gradient from 5 to 35% CH3CN in0.1% TFA for 30 min. Flow rate: 1 ml/min. Monitoring: absor-bance at 230 nm (intensity is not scaled).

Fig. 3. Scheme of the enzymatic digestion of MVIIC and HPLCpro¢les of fragment A derived from MVIIC and T11A. Same condi-tions as in Fig. 2 except for isocratic elution with 3% CH3CN in0.1% TFA.

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the peptide bonds in MVIIC and the HPLC comparison ofdigestion products of MVIIC and T11A. The result indicatedthat T11A, the weakest analog (described below), has thesame disul¢de bond pairings as native MVIIC. All the analogsexcept for K2A, Y13A, D14A, S17A and K25A were digestedand gave the same fragment A as native MVIIC.

3.3. CD spectraCD spectra of all the analogs including K2A, Y13A, D14A,

S17A and K25A were similar to that of MVIIC with positiveCotton E¡ects around 230 nm and negative ones around 203nm, suggesting that the conformations of the analogs are sim-ilar to that of MVIIC. Certain CD spectra are illustrated inFig. 4 as examples.

3.4. Biological activityThe ability of the analogs to inhibit binding of [125I]GVIA

and [125I]MVIIC to rat cerebellar P2 membranes were com-pared to displacement by MVIIA and MVIIC. The IC50 val-ues of the analogs are summarized in Fig. 5. We have previ-

ously reported that Tyr13 is important for the binding ofMVIIC to P/Q-type calcium channels [6]. For binding toP/Q-type channels, substitution at several other residues re-sulted in a signi¢cant loss of a¤nity. Among these, Thr11 wasthe most marked with an increase in the IC50 value of the Alasubstituted analog to almost 500 times that of MVIIC. Lys2,Lys4 and Arg22 were also implicated, suggesting that ionicinteraction is important for binding. For the binding toN-type channels, only three residues, Lys2, Thr11 and Tyr13,were essential.

4. Discussion

Oxidative disul¢de bond formation is a key step for thesynthesis of peptides with multiple disul¢de bonds. Hyp resi-dues were essential for the folding of W-conotoxin GIIIA dueto its cyclic structure [9] and Asn20 and Thr23 residues for g-conotoxin GVIA probably due to intramolecular hydrogenbonds involving their side chains [10,11]. In the present study,replacement of Asp14 in MVIIC had the most signi¢cant e¡ecton the folding reaction, as suggested by Fig. 2. Because Asp14

is close to Lys2 in the three dimensional structure [12,13],ionic interaction may be important for the folding in thecase of MVIIC (Fig. 6).

For the structure^activity relationship study of peptideswith multiple disul¢de bonds, it is essential to con¢rm thatthe analogs have the same disul¢de pairings as the nativepeptide. In most analogs, a comparison of their enzymaticdigestion products to that of native MVIIC clearly showedthat they possess the same disul¢de pairings as native MVIIC.However, this method was not applied to analogs K2A, Y13A,D14A, S17A and K25A, in which replacement might a¡ecteither enzyme speci¢city or the structure of digestion prod-ucts. Therefore, we measured the CD spectra of the analogs tocon¢rm indirectly that the analogs had a similar conformationto MVIIC. All the analogs showed the similar CD spectra tothat of MVIIC, suggesting that they have similar conforma-tion including common disul¢de bond pairings.

The three-dimensional structures of GVIA [14^17], MVIIA[18,19] and MVIIC [12,13] have been determined by NMRanalysis. Despite di¡erences in primary amino acid sequences,

Fig. 4. CD spectra of MVIIC and its analogs in H2O solution(0.01 M sodium phosphate, pH 7.0) at 20³C.

Fig. 5. Inhibition (IC50) by MVIIC analogs of [125I]GVIA or[125I]MVIIC binding to rat cerebellar P2 membranes. Numbers incircles indicate the residues replaced with Ala. Letters A and C incircles indicate the IC50 of native MVIIA and MVIIC, respectively.

Fig. 6. Location of certain residues essential for the activity or oxi-dative folding reaction. The model was drawn by using the cord1OMN of the Protein Data Bank [13].

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K. Sato et al./FEBS Letters 469 (2000) 147^150 149

the polypeptide chain framework is conserved in all of theg-conotoxins. Thus the nature of the amino acid side chainsmay have a dominant role in determining the toxin selectivity.

Previously, we showed that Tyr13 is essential for the activityof GVIA and that Lys2 is the second most important residue,based on the results of systematic single Ala substitution[20,21]. Because replacement of other residues by Ala didnot a¡ect binding, we proposed a two-point binding modelbetween GVIA and the N-type calcium channel [20]. Replace-ment of Tyr13 of MVIIA by Ala also resulted in a signi¢cantloss of a¤nity, whereas substitution of Lys2 did not a¡ect thebinding [22]. Loss of the basic side chain of Lys2 may becompensated by other basic residues such as Lys7 [18], whichis substituted by a neutral amino acid in GVIA and MVIIC.Nadasdi et al. also reported that the replacement of Tyr13 inMVIIA by Phe reduced a¤nity to 0.5% of that of nativeMVIIA and that the elimination of certain positive chargesalso perturbed binding [23]. These previous observations arein good agreement with the present results that only threeresidues in MVIIC, Lys2, Thr11 and Tyr13, were essential forbinding to N-type channels. These three residues are locatedon the same side of MVII molecule (Fig. 6). It will be inter-esting to further explore the role of Thr11 of MVIIC, since N-type speci¢c MVIIA and GVIA have Leu and Thr residues atthe 11th position, respectively.

For the binding of MVIIC to P/Q-type channels, we pre-viously reported that the replacement of Tyr13 by Ala signi¢-cantly reduced the a¤nity, suggesting that Tyr13 is a commonbinding motif in g-conotoxins irrespective of the calciumchannel subtypes that they target [6]. However, replacementof Thr11 showed the largest reduction of a¤nity as shown inFig. 5. Furthermore, the replacement of many other residuesalso resulted in a large reduction in a¤nity. Consequently, theresults of single alanine substitutions of MVIIC agreed wellwith the prediction that MVIIC has a broad binding surfacewith P/Q-type calcium channels, based on the data from chi-meric analogs (Fig. 6). To develop more P/Q speci¢c conotox-ins, it will be necessary to examine combined e¡ects of simul-taneous multiple substitutions.

Voltage-gated calcium channels are complex membraneproteins consisting of multiple subunits [1^3]. A central chan-nel pore is formed by an K1 subunit that has four motifs,I^IV, each having six transmembrane segments, S1^S6. Apore-lining segment H5 between segments 5 (S5) and 6 (S6)is thought to be essential for the ion selectivity. The K1 sub-units of N- and P/Q-type calcium channels have been clonedand designated as K1B and K1A, respectively, according to thenomenclature of voltage-gated calcium channels [24]. Theamino acid sequences of H5 segments are almost identicalbetween N- and P/Q-type calcium channels [25,26]. Cloning,mutagenesis and expression of K1 subunits showed that themost dramatic e¡ects on the interaction between GVIA andN-type calcium channel involved at a single cluster of residuesin the large extracellular loop between IIIS5 and IIIH5, con-sistent with a direct pore-blocking mechanism [27]. A combi-nation of mutational studies on both ion channels and theirspeci¢c blockers will provide knowledge of the architecture ofthe outer vestibules of the channel pores. In combination withthe comparison of amino acid sequences of H5 segments ofcalcium channels, studies on the blocking mechanism of g-conotoxins may enable the design and synthesis of novelblockers with appropriate speci¢cities.

Acknowledgements: This work was supported in part by a projectgrant from the Japan Health Science Foundation, Program for Pro-motion of Fundamental Studies in Health Sciences of Organizationfor Drug ADR Relief, RpD Promotion and Product Review of Ja-pan.

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