SHORT COMMUNICATION

Observation of Noncovalent Complexes Between Margatoxin and the K,1.3 Peptide Ligands: A Model Investigation Using Ion-Spray Mass Spectrometry Ray Bakhtiar Merck

Research

and Maria A. Bednarek

Laboratories,

Rahway,

NJ, USA

Margatoxin (MgTX), a 39 amino acid peptide present in the venom of the new world scorpion Centruroides marguritatus, is a potent inhibitor of the voltage-gated potassium channel (K,1.3) in human peripheral T lymphocytes. Peptide analogs corresponding to the amino acid segments that are located at the rat K”1.3 putative binding site for the ion channel blockers were synthesized. Gas phase noncovalent complexes of the synthetic analogs of the rat K”1.3 peptide ligands with MgTX were detected using ion-spray mass spectrometry. (J Am Sot Mass Spectrum 1996, 7, 1075-1080)

T

he K+, Na+, and Ca2+ channels represent a diverse group of integral membrane proteins [l]. Ion channels regulate the flux of inorganic ions across membranes and control the membrane potential with high selectivity. Among the most studied ion channels are those that which are permeable to Kf and are found in the membrane of most animal cells (e.g., nerves, lymphocytes, skeletal, and heart muscle cells). An important subset of K+ channels is the voltage-gated K+ channel (also designated as K VI which opens in response to a change in potential across the membranes [ll. This opening produces rapid and transient electrical excitations across the membrane and thus facilitates rapid transport of potassium ions into the cell. In recent years, molecular probes have been identified (e.g., drugs, naturally occurring peptidyl toxins) which have proven to be useful in elucidating the mechanism of action of ion channels [l]. These probes generally have been referred to as ion channel blockers and they have aided in investigation of the mechanisms that regulate ion release. For example, several of the naturally occurring peptidyl toxins have been shown to be potent inhibitors of ion channels 111. Margatoxin (MgTX), a 39-residue peptide (Figure 11, isolated from the venom of the new world scorpion Centruroides marguritutus, was found to block the voltage-gated potassium channel, K”1.3 (the numbering is according to the order in which they were

Address reprint requests to Ray B&h&x, Merck Research Laboratories, Rahway, ray [email protected] 0 1996 American Society 1044-0305/96/%15.00 PtI SlO44-0305(96)00108-0

Mail stop: RY8OL-109, NJ 07065-0900. E-maik

for Mass Spectrometry

discovered) in human peripheral T lymphocytes, with high affinity [2, 31. Recently, the purification 121, three dimensional structure [4], and structure-activity studies of MgTX were reported [3, 51. Furthermore, with the introduction of patch clamp techniques, vital information has been provided on the mechanism of inactivation of ion channels using peptidyl toxins (e.g., MgTX) 16, 71. The intact ion channel proteins are relatively large and too complex for mass spectral analysis. Thus, alternative approaches need to be explored in order to study the gas-phase noncovalent interactions of these proteins with their inhibitors. One approach is to study propensities of short peptides, corresponding to the selected sequences of the ion channel, to adopt nonco valent association with the corresponding inhibitor. Utilizing the ion-spray mass spectrometry technique (IS-MS), herein we report model studies on gas phase non-covalent [8, 91 complexes of MgTX with synthetic peptides, which correspond to the proposed high affinity binding site of the rat K”1.3. Figure 2 shows a simplified schematic diagram of one subunit of a voltage-gated K+ channel (e.g., rat K”1.3). The polypep tide chain is comprised of six transmembrane alpha helices embedded in the lipid bilayer and referred to as segments (S) 1 to 6 [ll. The amino-(N) and carboxy(Cl domains face the cytoplasmic (cell interior) side of the membrane. The following peptide fragments, corresponding to the specific domains of the rat K”1.3, were synthesized and used in this study: 402-413 (Pl), 378-384 (P2), 302-315 0’31, 221-235 (P4), and l-15 (N-terminal domain, designated as P5), Figure 2. Our gas-phase data on the binding trends between peptide fragments and toxins are in qualitative agreement with Received Accepted

April 2,196 June 24,1996

1076

J Am 8oc Mass Spectrom

BAKHTIAR

AND

BEDNAREK

MQTx

;IIN;KCTS;KQCLSPCKA;FGQSiiGAK n

1996,7,1075-1080

nn

ChlX IbTx

Figure 1. Amino acid alignment iberiotoxin (IbTX). Brackets indicate 44% sequence homology with ChTX

of toxins margatoxin (MgTXJ, charybdotoxin the locations of the internal disulfide bridges. and 41% with JbTX.

the ones reported for the intact protein ion-channel (vi& irzffa). However, it is important to point out that this communication does not take into consideration the tertiary structure, solvent interactions, and the intrinsic heterogeneity (e.g., post-translational modifications) of the intact ion channel. Furthermore, due to lack of a solution phase model of the systems described here, our results do not prove beyond doubt, that a specific association exists in solution. In an ideal case, mass spectrometry data on noncovalent interactions should be confirmed by independent studies in

PI

ERQGNGQQAMSLAI

(ChTXJ, and MgTX displays

the solution phase. Nonetheless, the aim of this effort is to explore the utility of a simplified biomimetic approach in qualitative characterization (e.g., specific vs. nonspecific associations) of peptide-peptide complexes in the gas phase.

Experimental All experiments were performed on an API III” triple quadrupole mass spectrometer (PE-Sciex, Thornhill, Ontario, Canada) equipped with an ion-spray (IS) in-

AC-S s’?F

NI

AAEPELLHDGPVVTi4 (W Figure 2. A simplified diagram of one subunit of rat voltage-gated K+ channel (e.g., K”1.3). The cylinders represent membrane spanning ammo acid sequences, which are proposed to form alpha helices. The voltage-gated K+ channel is proposed to consist of four such subunits radially arranged around a central hydrophobic pore. Shown are the regions corresponding to residues 402-413 (Pl), 378-384 (P2),302-315 (P3), 221-235 (P4), and 1-15 (N-terminal domain, designated as I’S). Synthetic analogs of these domains were used in this study. The N-terminus of the polypeptide is located in the cytosol (separated from the exterior region of the cell by plasma membrane). The pore region corresponding to the MgTX binding site, between the transmembrane linker segments S5-S6 (part of the pore wall), is indicated by an arrow. MgTX physically plugs the channel “mouth,” in part, by specific non-covalent interactions with regions on the extracellular loops, Pl and P2 fragments [2-51.

J Am

Sot Mass

Spechom

1996,7,

1075-1080

MARGATOXIN

terface. Details of the experimental conditions for observation of noncovalent complexes using an API IlI (and its up-grade Al? III+) triple quadrupole mass spectrometer have been described previously [8]. A detailed description for synthesis (using solid phase peptide synthesis), chromatographic conditions, and purification of MgTX was previously reported [lo]. The synthetic MgTX has been shown to be identical in physical and biological properties to MgTX produced bio-synthetically [lo]. Charybdotoxin (ChTX) and iberiotoxin (IbTX) were purchased from Peptide International (Louisville, KY). Synthetic analogs of the rat K”1.3 peptide ligands were prepared by standard solid phase techniques on an ABI 431A peptide synthesizer. The purity of all samples was examined by reversed-phase high performance liquid chromatography and mass spectrometry [lo]. All other chemicals and solvents were of reagent grade and were used without further purification. Stock solutions of MgTX, ChTX, and IbTX were prepared by dissolving 0.1 mg of each in 0.5 mL of 10 mM ammonium acetate (NH,OAc) buffer (pH - 6.5). Stock solutions (1

K”1.3

PEPTIDE

LIGANDS

1077

mg/mL) of each synthetic peptide were prepared in 10 mM NH,OAc and an appropriate aliquot was added to the peptidyl toxin solution to reach the desired molar ratio. Experiments were performed with 1, 5, 10, and 20 molar excess of peptide-to-peptidyl toxin. All sample mixtures were incubated for l-2 h at 37°C prior to mass spectrometric analysis.

Results and Discussion Figure 3a illustrates the positive ion IS-MS of MgTX. The spectrum contains peaks corresponding to the charge states 3 + , 4 + , and 5 + of MgTX that were used for molecular weight (MW) determination. Satellite peaks corresponding to the oxidation products [ill (e.g., methionine to methionine sulfoxide), potassium, and sodium adducts of MgTX were also evident. Addition of an approximately equal molar amount of peptides Pl or P2 (Figure 2, fragments of the outer “mouth” of the ion conductance pore) to a solution of MgTX resulted in the appearance of new peaks which were representative of a 1:l noncovalent association of AVE MW calculated AVE Mw observed

7 67 3s 1 I

AND

= 4180 Da = 4178 Da

[MgTX+5HJ5+

[M@‘X+4H14+ [MfTJL+Sl#+

/ [MgTXtSH+P2]S+ fMgTXt4H+~2~

tMgTX+JH+

I

tM@‘X+3H+F2]k

ml.2 Figure 3. (al Positive ion IS-MS of synthetically obtained MgTX in 10 mM NH,OAc (pH - 6.51 at an orifice voltage of 45 V. Oxidation products (e.g., methionine to methionine sulfoxide), potassium, and sodium adducts were observed (satellite peaks). Charge states 3 +, 4 +, and 5 + were used for molecular weight deconvolution. f.bl Normalized positive ion IS-MS of MgTX in 10 mM NH,OAc (pH - 6.5) at an orifice voltage of 45 V upon incubation with approximately equal molar amount of P2 (the I’2 fragment was the base peak in the spectrum obtained for the mixture). Qualitatively analoeous results were obtained for MelX:Pl mixture (see Table 11.

(a)

1078

BAKHTL4B

AND

J Am Sot Mass

BEDNAREK

the MgTX-peptide complexes. For example, Figure 3b shows the positive ion IS-MS of MgTX upon incubation with P2 peptide at a 1:l molar ratio. Ions corresponding to 1:l non-covalent complexes for charge states 3 + , 4 + , and 5 + were observed. These complexes underwent dissociation upon increasingly “harsh” interface conditions (e.g., orifice potential of > 90 V, an increase in the interface temperature, etc.) [9] yielding free peptide ligand and MgTX. A decrease in the solution pH (e.g., from 7 to 3 by addition of acetic acid) afforded similar results. In order to gain additional information on the nature of the noncovalent complexes between MgTX and fragments Pl and P2, several key experiments (under similar instrumental conditions) were performed. First, the peptide-to-toxin molar ratio was varied and the stoichiometry of the resulting IS-MS spectrum was examined. Of specific interest were the type of adducts obtained such as AB vs. a random aggregation like A,B, AB,, A,, etc. [9a]. Table 1 summarizes the IS-MS data obtained for noncovalent complexes between the peptidyl toxins and the rat K”1.3 peptide ligands under similar experimental conditions. Variation in the toxin-to-peptide molar ratio was investigated as outlined in Table 1. No “false positive” or nonspecific aggregation was observed even at 1:20 toxin-to-peptide ligand molar ratios and higher for Pl, P2, and MgTX. Second, MgTX was incubated separately with P3, P4, and P5 peptides and the resulting IS-MS spectrum examined. These peptide fragments are not located at the proposed high affinity binding region of the rat K”1.3 ion channel (Figure 2) and would not be expected to exhibit noncovalent complexation with MgTx. As anticipated, mixtures of MgTX with peptides P3, P4, and P5 did not result in formation of noncovalent complexes (Table 1). Third, as additional control experiments, we examined the binding specificity of P2 incubated with two other high affinity scorpion toxins, charybdotoxin (ChTX) and iberiotoxin @TX), which are similar in structure to MgTX (Figure 1, Table 1). Despite some similarities in channel modulation properties of MgTX,

Table 1. Summary of the IS-M!5 data on noncovalent and the rat K”1.3 peptide ligands Toxin Mgm ChTX IbTX

Toxin:Peptide

Pl

P2

1:l 1:n 1:l

+ +

+ +

+ + -

+ + -

1 :n 1:l 1 :n

a

Spectrom

1996,7,1075-1080

IbTX, and ChTX, there are fundamental differences in their specific mode of action. ChTX, purified from venom of the scorpion Leiurus quinquestriutus, blocks with high ‘affinity both the Ca’+-activated and the voltage-gated K+ channels (e.g., K “1.3). In contrast, IbTX, isolated from venom of the scorpion Buthus tanzulus, binds selectively to the high conductance Ca’+-activated K+ channel but does not block the K”1.3 ion-channel even at high (FM) concentrations [12-171. Therefore, lbTX would not be expected to form a noncovalent complex with any of the synthetic peptide ligands (in particular with Pl and P2). Accordingly, as shown in Table 1, the IS-MS spectrum of a mixture (1:l molar ratio) of IbTX with Pl and P2 did not reveal the formation of a noncovalent complex. Figures 4a and 4b illustrate the IS-MS of ChTX and ChTXP2 (1:l molar ratio), respectively. For the sake of clarity, the inset in Figure 4b depicts an expanded m/z region corresponding to [ChTX + 4H14+ and [ChTX + I’2 + 4H14+ peaks. Ions corresponding to the side products (e.g., generated during the synthesis of peptide ligands) that were not easily separated under liquid chromatography conditions were also detected (annotated with an asterisk in Figure 4b). Fourth, binding of MgTX to a modified P2 ([Lys5]P2) was examined. An alteration in the IS-MS spectra upon subtle structural changes in the Pl or P2 peptide could yield additional qualitative information on the binding specificity of a MgTX-peptide complex. The site-directed mutation of the voltage-gated K+ channels has been one of the key experiments in mapping the binding site for the pore-blocking scorpion toxins in the solution phase 1181. Thus, the rationale for synthesis of [Lys5]-P2 fragment was based on the findings reported by McKinnon et al. [18a], where LYS~*~ of the Shaker K+ channel (voltage-activated K channel in fruit fly Drosophila melunoguster, structurally related to the rat and human K”1.3 channels) was replaced by an asparagine (which occupies an equivalent position in rat K”1.3) or glutamic acid. Position 5 of P2 corresponded with position 382 of the rat K”1.3 channel and with position 427 of the Shaker K+ channel. Due to the electrostatic interactions, basic ChTX binds to

complexes [Lys51-P2 a a -

behveen

the peptidyl

toxins

P3

P4

-

-

P5 -

-

-

-

In) molar excess of peptide equals 5, 10 and 20 in separate experiments. (+I 1:l stoichiometry for noncovalent complexation observed. ( - 1 noncovalent complexation was not observed at 1 :l , 1:5, 1 :lO, and 1:20 toxin-to-peptide ligand molar ratio. (a) multiple nonspecific adduction (e.g., up to 2 peptide ligands) for noncovalent complexation observed (“false positive”) at 1:20 toxin-to-peptide ligand molar ratio.

J Am

Sot Mass

Spectrom

1996,7,

1075-1080

MARGATOXIN

AND

PEPTIDE

LIGANDS

1079

(a)

AVE MW ache AVEMW

K”1.3

= 4297 Da observed = 42% Da -

+Na

rchTx+4EJ+)

11 Molecular

+K

Weight

[chTx+3Ep

19.2[ChTx+m4HJ4+

1160

1150

1200

1256

m/z

[chTx+3Hp+

Figure 4. (a) Positive ion E-MS of commercially obtained ChTX in 10 mM NH,OAc fpH - 6.51 at an orifice voltage of 45 V. Charge states 3 +, 4 + and 5 + were used for MW deconvolution (inset). tb) Normalized positive ion IS-MS of ChTX in 10 mM NH,OAc (pH - 6.5) at an orifice voltage of 45 V, upon incubation with approximately equal molar amount of P2 (the P2 fragment was the base peak in the spectrum obtained for the mixture). The inset is an expanded region of the spectrum from m/z 1050-1300. Qualitatively similar results were obtained for ChTXzPl mixture (see Table 11. Annotated with asterisks are the ions that were also observed in the spectrum (control experiment) of a stock solution of P2 with no toxin present.

the modified Shaker channel with higher affinity when lysine-427 is replaced by an acidic or neutral amino acid. Consequently, a lower binding affinity between the peptidyl toxin and the outer “mouth” of the ion conductance pore of wild-type Shaker channel was

attributed to a simple through-space electrostatic rep&ion [lBa]. The P2 peptide fragment of rat K”1.3 was synthesized with asparagine in position 5 replaced by a lysine residue, [Lys51-P2, incubated with each toxin, and analyzed by IS-MS (Table 1). As expected,

1080

BAKHTL4R

AND

J Am

BEDNARJZK

the ISMS spectrum of a mixture (1:l molar ratio) of MgTX with [Lys’]-P2 did not show the formation of a specific noncovalent complex.

Conclusions Ion-spray mass spectrometry was utilized to observe non-covalent complexes between MgTX and rat K”1.3 peptide ligands. The data presented in this study indicated that both specific and nonspecific interactions were operative. Similar results were obtained for Pl and P2 fragments, which displayed specific 1:l noncovalent association with MgTX. Generally, concentration-dependent nonspecific interactions were observed in cases where the peptide ligands were not related to the Kv1.3 high affinity binding site (the pore region) (Table 1). Presently, due to the lack of a solution phase model of the aforementioned peptides, the application of this approach to unknown systems requires suitable control experiments in order to distinguish between nonspecific and specific complexes [ 191. Although the binding trends discussed in OUT findings are in qualitative agreement with the studies performed on the intact ion-channel proteins, caution should be exercised in relying solely on the IS-MS data in the absence of independent studies in the solution phase.

Sot Mass

Spactrom 1996,7,1075-1080

3. Knaus, H-G.; Koch, R. 0. A.; Eberhart, A.; Kaczorowski, G. J.; Garcia, M. L.; Slaughter, R. S. Biochemistry 1995, 34, 13627-13634.

4. Johnson, B. A.; Stevens, S. P.; Williamson, J. M. Biochemistry 1994, 33,15061-15070. 5. Boltz, R. C.; Sirotina, A.; Blake, T.; Kath, G.; Uhrig, B.; McKeel, J.; Quinn, C. Cytometry 1994, 17, 128-134. 1995, 48, 1004-1009. 6. Giebisch, G. Kidney International 7. Aiyar, J.; Withka, J. M.; Rizzi, J. P.; Singleton, D. H.; Audrews,

G. C.; Lin,

W.; Boyd,

J.; Hanson,

D. C.; Simon,

M.;

Dethlefs, B.; Lee, C-L.; Hall, J. E.; Gutman, G. A.; Chandy, K. G. Neuron 1995, 15, 1169-1181. 8. Ganem, B.; Henion, J. D. Chemtracts: Org. Chem. 1993, 6, l-22. 9. For review articles see; (a) Smith, D. L.; Zhang, Z. Mass Spectrom. Rev. 1994, 23,411~429.; (bl Przybylski, M.; Glocker, M. 0. Angew. Chem. Int. Ed. En@. 1996, 35, 806-826 and

references cited therein.; (cl Smith, R. D.; Cheng, X.; Schwartz, B. L.; Chen, R.; Hofstadler, S. A. In Biochemical and Biotechnological Applications

10. 11. 12. 13.

of Electrospray

ionization

Mass Spectromety;

Snyder, A. P., Ed.; ACS Symposium Series 619, 1995, Chap. 15.; (d) Loo, J. A. Bioconjugate Chem. 1995, 6, 644-665. Bednarek, M. A.; Bugianesi, R. M.; Leonard, R. J.; Flex, J. P. Biochem. Biophys. Res. Commun., 1994, 198, 619-625. Li, S.; Schoneich, C.; Borchardt, R. T. Biotech. Bioeng. 1995, 48, 490-500. Harvey, A. L.; Vatanpour, H.; Rowan, E. G.; Pinkasfeld, S.; Vita, C.; Menez, A.; Martin-Eauclaire, M. F. Toxicon. 1995, 33, 425-436. Knaus, H-G.; Eberhart, A.; Glossmann, H.; Munujos, P.; Kaczorowski, G. J.; Garcia, M. L. Cellular Signalling 1994, 6, 861-870.

Acknowledgments The authors thank Dr. R. A. Steams and Dr. L. Chiu for support of this collaborative research project. The authors gratefully acknowledge Dr. S. A. Hofstadler (Battelle Pacific Northwest Lab+ ratoriesl, Dr. J. Liesch, Dr. L. Colwell, and Dr. P. R. Griffin for helpful discussions.

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to S.

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Cl-ClO.

16. Leonard, R. J.; Garcia, M. L.; Slaughter, R. S.; Reuben, J. P. Proc. Natl. Acad. Sci. USA, 1992, 89, 10094-10098. 17. Knaus, H-G.; Eberhart, A.; Glossmann, H.; Munujos, P.; Kaczorowski, G. J.; Garcia, M. L. Cellular Signalling 1994, 6, 861-870. 18. (a)MacKinnon, R.; Heginbotham, L.; Abramson, T. Neuron 1990, 5, 767-771. (b) MacKinnon, R. Miller, C. Science 1989, 245, 1382-1385. fc) MacKinnon, R.; Yellen, G. Science 1990, 250, 276-278. 19. (al Aplin, R. T.; Robinson, C. V.; Schofield, C. J.; Westwood, N. J. 1. Chem. Sot., Chem. Commun. 1994, 2415-2417; fb) Cunniff, J. B.; Vouros, P. 1. Am. Sot. Mass Spectrom., 1995, 6, 437-447; (cl Ding, J.; Anderegg, R. J. J. Am. Sot. Mass Spectrom. 1995, 6, 159-164; fd) Stefansson, M.; Sjoberg, P. J. R.; Markides, K. E. Anal. Chem. 1996, 68, 1792-1797.