Solution structure of BmP08, a novel short-chain scorpion toxin from Buthus martensi Karsch

BBRC Biochemical and Biophysical Research Communications 330 (2005) 1116–1126 www.elsevier.com/locate/ybbrc

Solution structure of BmP08, a novel short-chain scorpion toxin from Buthus martensi Karsch q Xiang Chen, Yiming Li, Xiaotian Tong, Naixia Zhang, Gong Wu, Qi Zhang, Houming Wu * State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, PeopleÕs Republic of China Received 10 March 2005 Available online 23 March 2005

Abstract A novel short-chain scorpion toxin BmP08 was purified from the venom of the Chinese scorpion Buthus martensi Karsch by a combination of gel-filtration, ion exchange, and reversed-phase chromatography. The primary sequence of BmP08 was determined using the tandem MS/MS technique and Edman degradation, as well as results of NMR sequential assignments. It is composed of 31 amino acid residues including six cysteine residues and shares less than 25% sequence identity with the known a-KTx toxins. BmP08 shows no inhibitory activity on all tested voltage-dependent and Ca2+-activated potassium channels. The 3D-structure of BmP08 has been determined by 2D-NMR spectroscopy and molecular modeling techniques. This toxin adopts a common a/b-motif, but shows a distinctive local conformation and features a 310-helix and a shorter b-sheet. The unique structure is closely related to the distinct primary sequence of the toxin, especially to the novel arrangement of S–S linkages in the molecule, in which two disulfide bridges (Ci–Cj and Ci+3–Cj+3) link covalently the 310-helix with one strand of the b-sheet structure. The electrostatic potential surface analysis of the toxin reveals salt bridges and hydrogen bonds between the basic residues and negatively charged residues nearby in BmP08, which may be unfavorable for its binding with the known voltage-dependent and Ca2+-activated potassium channels. Thus, finding the target for this toxin should be an interesting task in the future.
2005 Elsevier Inc. All rights reserved. Keywords: BmP08; Scorpion toxin; Buthus martensi Karsch; NMR; Solution structure

Scorpion venoms are rich sources of fascinating neurotoxins, which bond with high affinity and specificity to various ion channels and thus widely serve as useful tools in probing the protein mapping of ion channels and clarifying the molecular mechanism in-

q Abbreviations: 1D, one dimensional; 2D, two dimensional; 3D, three dimensional; DQF-COSY, double-quantum-filtered shift correlated spectroscopy; ESI-MS, electrospray ionization mass spectrometry; NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser enhancement spectroscopy; RMSD, root-mean-square deviation; TOCSY, total correlation spectroscopy. * Corresponding author. Fax: +86 21 64166128. E-mail address: [email protected] (H. Wu).

0006-291X/$ - see front matter
2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.084

volved in the signal transduction and channel gating. So far, more than 100 peptidyl modulators of ion channels have been separated from various scorpion venoms. According to their primary structures and biological function, these toxins have been classified into two major groups: (1) group I mainly acts on sodium channels and covers peptides of 60–76 amino acids with 4 disulfide bonds [1]; (2) group II interacts specifically with potassium or chlorine channels and contains peptides of 20–40 amino acids cross-linked by 3–4 disulfide bridges [2–4]. Chinese scorpion Buthus martensi Karsch (BmK) is the species belonging to the Buthidae family. It has been used as traditional medicine in China for more than

X. Chen et al. / Biochemical and Biophysical Research Communications 330 (2005) 1116–1126

1000 years, especially for the treatment of neural diseases, such as apoplexy, hemiplegia, and facial paralysis [5]. During the past few years, a number of peptides from this species have been identified and characterized by systematic isolation, ESI-MS, N-terminal sequence, and toxicity. The solution structures of some peptides have been reported, such as the long-chain peptides BmK M1 [6], BmK M2 [7], BmK M4 [6], BmK M8 [5], and BmK 16 [8], and short-chain peptides BmP01 [9], BmP02 [10], BmP03 [11], BmTX1, BmTX2 [12], BmKTX [13], BmBKTx1 [14], BmTx3B [15], BmKK2 [16], and BmKK4 [17]. In our previous paper, a systematic isolation has been achieved and a total of 45 toxic peptides have been isolated and characterized from the venom of Chinese scorpion BmK [18]. In the present paper, we report purification, characterization, and sequence determination of a novel short-chain scorpion toxin BmP08. It is composed of 31 amino acids including six cysteine residues and shares less than 25% sequence identity with most known a-KTx toxins. The primary sequence of BmP08 is unique with an unclassified Cys residue arrangement, in which two disulfide bridges (Ci–Cj and Ci+3–Cj+3) covalently link a segment of 310-helix with one strand of the b-sheet structure. It is different from the signature sequence of common scorpion peptides, in which two disulfide bridges (Ci–Cj and Ci+4–Cj+2) covalently link a segment

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of a-helix with one strand of the b-sheet structure. This toxin exhibited almost no inhibitory activity towards all tested voltage-dependent and Ca2+-activated K+ channels, such as Kv channel, BK channel, and SK channel in the electrophysiological experiment (personal communication with Prof. Z. Zhou) indicating a unique member in the scorpion toxin sub-family. Hence, the 3D solution structure of BmP08 was determined by NMR spectroscopy in this article to reveal the structural features of this molecular. Materials and methods Sample preparation. Crude venom was collected by electrical stimulation of the telson of scorpion BmK bred in captivity in Henan Province, China. The peptide was purified as described previously [18]: lyophilized crude venom was dissolved in NH4HCO3 buffer (50 mM, pH 8.5) and centrifuged at 4000g for 15 min. The supernatant was loaded onto a Sephadex G-50 column (2.5 · 150 cm, Pharmacia Fine Chemicals), which was equilibrated and eluted with the same buffer (Fig. 1A). The fraction IV from the Sephadex G-50 column was loaded onto a Mono S cation exchange column (HR5/5, Pharmacia LKB Biotech.), eluted with a step gradient of solution A to solution B at pH 5.0 (Fig. 1B). Solution A contained NaAc (20 mM), and solution B contained NaAc (20 mM) and NaCl (1 M). It was followed by similar separation on another set of Sephadex G-50 column (Fig. 1C) and Mono S cation exchange column (Fig. 1D). The finial purification of BmP08 was performed by using a reverse-phase HPLC column (C18 column, 4.6 · 250 mm, 5 lm bead size, Alltech) eluted with a linear

Fig. 1. Isolation and purification of BmP08. (A) Sephadex G-50 column chromatography of the crude venom form B. martensi Karsch. (B) FPLC fractionation of the fraction IV obtained in A on a Mono S column. (C) Sephadex G-50 column chromatography of the fraction 1 in (B). (D) FPLC fractionation of the fraction 1–1 in (C) on a Mono S column. (E) RP-HPLC fraction of the fraction 1–12 in (D) on a C18 column. (F) ESI-MS spectrum of BmP08.

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gradient from solution C to 50% solution D in solution C at a flow rate of 1 ml/min. Solution C contained CH3CN (10%) and trifluoroacetic acid (0.1%) in H2O, and solution D contained H2O (20%) and trifluoroacetic acid (0.1%) in CH3CN. Characterization of the peptide BmP08. Native Bmp08 was subjected to amino acid composition analysis on a Beckman 6300 amino acid analyzer (Beckman, USA) after acid hydrolysis in 6 N HCl under vacuum at 110 C for 20 h. The molecular weight of BmP08 was measured on a VG Quattro spectrometer (Micromass, UK) equipped with an electrospray ion source. The N-terminal sequence of BmP08 was achieved by Edman degradation using a Beckman LF3200 Protein-Peptide sequencer (Beckman, USA). Mass spectroscopic analysis. To cleave disulfide bonds in the sequence, the sample of BmP08 was subjected to DTT reduction and iodoacetic acid derivation before MS analysis. Fifty nanomolar peptide BmP08 was reduced with a 250-fold molar excess of DTT in 0.25 M Tris–HCl buffer (pH 8.5) containing 6 M guanidine HCl and 4 mM EDTA. Reduction was carried out in the dark under nitrogen at 37 C for 1 h. Free thiols were alkylated by addition of a 500-fold molar excess of iodoacetic acid held at room temperature for 30 min in the dark. Sample was then purified by HPLC and its molecular weight was achieved by ESI-MS. Twenty-five nanomolar alkylated sample was digested with TPCK-trypsin enzyme in 50 mM NH4HCO3 buffer (pH 8.0) at 37 C for 4 h. The digested product was purified by HPLC (C18 Prosphere column, 4.6 · 150 mm, 5 lm bead size, Alltech) to give three main fragments (I–III). The fragment I was then submitted to Edman degradation. The other 25 nM alkylated sample was also digested with TPCKtrypsin enzyme and desalted into 5 ll of 4:1 CH3CN:H2O, 0.1% formic acid using C18 Zip Tips (Millipore, USA). ESI-MS and ESI-MS/MS spectra were performed using a tandem quadrupole-TOF instrument (Micromass, UK). The sample (1–2 ll) was loaded into a nanospray needle. For the recording of ESI-MS/MS spectra the quadrupole was operated as a mass filter and used to select the desired precursor ion isotope cluster. During product ion analysis the resolving quadrupole was set to transmit a ± 2 m/z window around the precursor ion. Precursor ions were directed into a hexapole collision cell. The collision gas was argon and the collision energy was optimized for each analysis. NMR experiments. The peptide BmP08 was dissolved in H2O/D2O (90/10 v/v) or 100% D2O, pH 3.02, adjusted by adding 1 ll of dilute DCl or NaOD. The final concentration of BmP08 was about 4.7 mmol/L. The amide proton exchange rate was determined after lyophilization and dissolution in 100% D2O. All the NMR experiments were recorded at 303 K on a Varian unity Inova 600 spectrometer. Quadrature detection was employed in all experiments and the carrier frequency was always maintained at the solvent resonance. Presaturation was used to suppress the water peak in all experiments. 2D DQF-COSY [19], TOCSY [20], and NOESY [21] spectra were achieved in phase-sensitive mode by using the time-proportional phase incrementation method. All 2D-NMR spectra were recorded with 4K data points in the t2 dimension and 512 data points in the t1 dimension. The TOCSY spectra were recorded using the MLEV-17 pulse sequence with mixing times of 80 and 120 ms [22]. The NOESY spectra were acquired using mixing times of 200 and 300 ms, respectively. To determine slowly exchange protons, a series of 1D spectra during the first 2 h followed by four TOCSY spectra (mixing time, 80 ms, 4 h) were recorded at 303 K immediately after the sample was dissolved in D2O. A shifted sine window function and zero filling were applied prior to Fourier transformation. All experimental data were acquired and processed using the Vnmr 6.1B program on a SUN Sparc station 4 computer. The processed data were analyzed with XEASY for NMR spectrum visualization, peak picking, and peak integration on a Silicon Graphics Indigo R 5000 computer. Assignment strategy and structure calculation. The identification of amino acid spin systems and the sequential assignment were done using the standard strategy described by Wu¨thrich [23].

The NOESY (200 ms) spectrum was used to generate the distance constraints. Dihedral angle constraints were derived from 3JHNHa coupling constants, which were obtained by analyses of the 1D 1H NMR spectrum. Additional constraints were used to enforce hydrogen bonds implicated by the H–D exchange spectra. Distance geometry calculations were performed with the target function program DYANA [24] on a Silicon Graphics Indigo II computer. The 35 structures with the lowest constraint violations were subjected to restrained energy minimization (REM) performed with the AMBER 5.0 package [25,26]. The 20 best conformers with the lowest energy were used to represent the solution conformation of BmP08. The programs PROCHECK and PROCHECK_NMR were used to evaluate the NMR structures of BmP08 [27,28]. In addition, 3D conformations were produced with the MOLMOL program [29] for visual comparison of the structures on a Silicon Graphics Indigo II computer. For pairs of conformers, RMSD values for various subsets of atoms were calculated. The mean solution structure was obtained by superimposing the 20 lowest energy AMBER conformers and then averaging the Cartesian coordinates of the corresponding atoms in the 20 superimposed conformers.

Results and discussion Purification and identification of BmP08 The soluble venom was initially separated into four fractions (I–IV) by gel-filtration chromatography on a Sephadex G-50 (Fig. 1A). Fraction IV was further separated into 13 fractions on a Mono S cation exchange column (Fig. 1B). After another separation of fraction 1 on a Sephadex G-50 column, four fractions (1–1, 1– 2, 1–3, and 1–4) were obtained (Fig. 1C). A further separation of the fraction 1–1 on a Mono S column gave five fractions (1–11, 1–12, 1–13, 1–14, and 1–15) (Fig. 1D). A pure peptide named as BmP08 (7.0 mg) was obtained after the purification of fraction 1–12 on a reverse-phase HPLC column (Fig. 1E). The ESI-MS of BmP08 showed homogeneity of the peptide and a molecular weight of 3326 Da (Fig. 1F). The amino acid composition analysis gave the results that the peptide was composed of 31 amino acids. The N-terminal sequence of BmP08 was determined by Edman degradation as TPYPVNCKTDRDCVMCGLGI. After DTT reduction and iodoacetic acid derivation, Bmp08 was purified by HPLC and the molecular weight achieved by ESI-MS was 3680 Da. Compared with the unalkylated peptide, the molecular weight increased to 354 Da, corresponding with the cleavage of three disulfide bonds and addition of six acetoxyl groups, indicating that six cysteine residues existed in the sequence of BmP08. After TPCK-trypsin enzyme digestion, alkylated sample was purified by HPLC and three main fragments (I–III) were separated. The molecular weight of the three fragments was 961, 980, and 1154 Da, respectively. The sequence of fragment I was determined by Edman degradation as NGYCTGQC with some ambiguity for the last four residues (see later) [30].

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The CID MS/MS analysis of enzyme digestion products of BmP08 The desalted sample of digestion products of reduced and S-alkylated BmP08 by trypsin was submitted to ESI-MS and CID MS/MS analysis. ESI-MS spectrum of trypsin digested mixture of reduced and S-alkylated BmP08 exhibited two doubly charged ions at m/z 490.1 and 701.6, indicating the presence of two segments (denoted as peptide segment T1 and T2, respectively). These precursor ions were then selected for fragmentation in the CID MS/MS analysis. The CID MS spectrum of each precursor ion is shown in Figs. 2 and 3, respectively. The intact amino acid sequence of the corresponding precursor ion peptide was obtained straight from the b and y series fragment ions as shown in their spectra. Combination of the sequence information derived from the CID MS/MS spectra and Edman degradation results, the intact amino acid sequence of the scorpion BmP08 listed in Fig. 4A. Database searching by experimentally determined sequence information yielded a cDNA sequence corresponding to the precursor peptide of BmKX except the last four residues (TGQC in BmP08 corresponding

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to the QGCT in BmKX). The ambiguity was resolved at last by sequential NMR assignments. Strong dNN and daN NOEs were observed in Gly29-CysXX and CysXX-ThrXX, and a daN NOE was observed in Cys27-GlnXX. It was clear that the C-terminus of BmP08 should be QGCT instead of TGQC obtained by Edman degradation as described in previous paper [30]. Therefore, the amino acid sequence of BmP08 is determined as Fig. 4B. NMR assignment The spin systems were identified on the basis of the DQF-COSY and the TOCSY spectra. The fingerprint region of the DQF-COSY spectra recorded in H2O showed most of the HN–Ha cross-peaks, and the TOCSY spectra then were used to correlate the HN– Ha cross-peaks with their side chain spin systems for each residue. The spin systems were connected in sequence by virtue of daN, dNN, and dbN connectivities in well-dispersed NOESY spectra. The unique residue combination of Leu18, Ile20, and Gln28 in the sequence of BmP08 was used as the entry points for the sequential assign-

Fig. 2. The CID MS/MS spectrum of the tryptic fragment T1 of reduced and S-alkylated BmP08, and the sequence information derived from the mass differences between the b series ions and between the y series ions.

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Fig. 3. The CID MS/MS spectrum of the tryptic fragment T3 of reduced and S-alkylated BmP08, and the sequence information derived from the mass differences between the b series ions and between the y series ions.

Fig. 4. The sequence of BmP08 (A) determined by Edman degradation and CID MS/MS, (B) adjusted by NMR technique.

ment. Starting from these three amino acids, the sequential assignments of the segments Pro2-Leu18, Ley18Ile20, and Ile20-Thr31 were obtained via daN (i, i + 1), dNN (i, i + 1), and dbN (i, i + 1) connectivities. The remaining Thr residue was assigned as Thr1. The

sequential connectivities are illustrated in Fig. 5. In addition, the mediate-range NOE contacts such as daN (i, i + 2), daN (i, i + 3), and dab (i, i + 3), and coupling constants 3JHNHa, as well as the slowly exchanging NH protons are also summarized in Fig. 5.

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Fig. 5. Summary of NOE connectivities, J coupling constant 3JHNHa, the amide proton exchange rate, and CaH chemical shift index. The thickness of the bar indicates the intensity of NOEs. Asterisks represent the NH protons as slowly exchanging. J coupling constant 3JHNHa are smaller than 7.0 Hz (open circle) and bigger than 9.0 Hz (filled circle). Positive bars and negative bars in the chemical shift index indicate the CaH protons downfield-shifted and up-field-shifted by >0.1 ppm, respectively, as compared with the CaH proton chemical shifts of random-coil.

Hydrogen bonds Total nine (Cys7, Asp12, Cys13, Val14, Cys16, Ile20, Cys22, Lys23, and Tyr26) amide protons, which are still visible after H–D exchanging for 2 h, are considered as being engaged in hydrogen bonds (Fig. 5). The acceptors of hydrogen bonds for the slowly exchanged amide protons were identified during the structure refinement. Coupling constants Ten 3JHNHa coupling constants were measured by the well-resolved 1H NMR spectrum and were converted into dihedral angle restraints (Fig. 5). Secondary structure Secondary structural elements of BmP08 were identified using the unique NOE contacts and 3JHNHa coupling constants. A continuous set of strong dNN (i, i + 1) NOEs was observed in the segment of Asp10 to Cys16, indicating a helical element. The deduction was supported by a series of mediate-range daN (i, i + 2) and daN (i, i + 3) NOEs in this region. Further corroborative data came from 3JHNHa coupling constants and slowly exchanging amide protons. Most of the 3JHNHa coupling constants in the region of residues 10–16 are smaller than 7.0 Hz. The amide protons of the residues Asp12, Cys13, Val14, and Cys16 are slowly exchanging (Fig. 5). Besides, the chemical shifts of a-protons for most of the residues in this region move up-field. These

data further confirmed the above assignment of the helical structure. In addition, strands Cys22-Lys23 and Tyr26-Cys27 showed strong sequential daN connectivities. Meanwhile, a long-range dNN NOE was observed between residues Lys23 and Tyr26. These observations suggested the presence of an antiparallel double-stranded b-sheet. The location of the b-sheet secondary structure was also confirmed by the chemical shift index of the a-protons and the slowly exchanging amide proton data. As shown in Fig. 5, the amide protons of residues Lys23 and Tyr26 involved in the b-sheet are slowly exchanged. The hydrogen bind partner of the amide proton of Lys23 and Tyr26 was identified as being the carbonyl oxygen of Tyr26 and Lys23, respectively, during the structure refinement. The strands Cys22-Lys23 and Tyr26-Cys27 were connected by a type I b-turn comprised of residues Lys23-Tyr26. The latter was confirmed by the dNN (Asn24, Gly25), as well as dNN (Gly25, Tyr26). In addition, the disulfide bridges were established from the dipolar interactions between the b-protons of relevant Cys residues. Structure determination The input for the distance geometry calculations with the program DYANA consisted of upper distance limits derived from NOESY (mixing time 200 ms) cross-peak intensities using the program CALIBA [31], and dihedral angle constraints were obtained from an initial interpretation of the vicinal coupling constants 3JHNHa. For the calibration of proton–proton distance limits (r versus the cross-peak intensities), the dependence of

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1/r6 was used for all protons. The calibration curves were refined on the base of plotting cross-peak volume versus average proton–proton distance according to the preliminary structures. A total of 296 distance constraints were used (180 intra-residual, 66 sequential, 32 medium-range, and 18 long-range NOEs). Ten / angle constraints ( 55 ± 15 for 3JHNHa < 7.0 Hz, and 120 ± 50 for 3JHNHa > 9.0 Hz) were used for structure calculation. In addition, nine distance constraints were added for three disulfide bonds (three per bond). Nine slowly exchanging amide protons were determined from the H–D exchanging experiments. Seven of them were used to generate hydrogen bond constraints, which were imposed between slowly exchanging amide protons and their receptors on the base of the DYANA preliminary structures. For each hydrogen bond, two limit restraints were used between the NH–O (0.22 nm) and the N–O (0.32 nm) atom pairs. Totally, 329 constraints (average 10.6 constraints per residue) were obtained and used in the structure calculations of BmP08. Starting from 200 random structures, the 35 preliminary structures with the lowest target functions resulting from distance geometry calculations were subjected to simulated annealing and restrained energy minimization (REM) using the SANDER module of the AMBER 5.0 package. A cutoff radius of 0.8 nm for non-bonded interactions, with a residue-based pair-list routine, was used in all calculations. The force constants for distance restraints in EM were 500 kJ/mol/nm2. Energy minimization was performed using a combination of steepest descent and conjugate gradient algorithms with a gradient convergence norm of less than 10 4 kJ/mol/nm. After energy minimization with AMBER, the 20 best DYANA conformers with the lowest energy are used to represent the solution conformation of BmP08.

Structure description Fig. 6A represents the superposition of the polypeptide backbones of the 20 best conformers. No NOE vio˚ and no angle violations larger lations larger than 0.20 A than 5 can be found. The overall agreement among individual conformers is indicated by global rootmean-square deviation (RMSD). The final set of 20 ˚ for the structures displays an overall RMSD of 1.31 A ˚ backbone atoms and 2.03 A for all heavy atoms. The RMSD values for the backbone atoms and the heavy ˚ , if the first five atoms are decreased to 0.52 and 1.37 A residues and the last four residues of the toxin are not taken into consideration. Analysis of the ensemble of 20 structures using PROCHECK_NMR reveals that 61.7% and 32.8% of the residues lie in the most favored and allowed regions of the Ramachandran /, w dihedral angle plot, respectively (plot not shown). The structural statistics for 20 conformers of the toxin are summarized in Table 1. The structure of BmP08 with the lowest energy is shown in Fig. 6B. The molecule adopts the a/b-motif, which consists of a double-stranded antiparallel b-sheet anchored to a single 310-helix (Asp10-Cys16) by a disulfide bridge (Cys13-Cys27). The 310-helix and the b-sheet were linked by a tight turn. The b-sheet involves residues Cys22-Lys23 (strand I) and Tyr26-Cys27 (strand II), which are connected by a type I b-turn (Lys23-Tyr26). Structural features of BmP08 Despite BmP08 adopting a CS-a/b-motif, it shows distinctive local conformation from other typical K+ channel toxins (e.g., CTX and P05), especially in the b-sheet and helix structure, as well as in the N-terminal

Fig. 6. (A) Backbone superimposition of the best 20 structures of BmP08. Nt and Ct indicate N-terminus and C-terminus, respectively. (B) MOLMOL representation of the structure of BmP08 with the lowest energy. Secondary structure elements are drawn with three disulfide bridges (neons). Beginning and ending residues of each secondary structure element are labeled according to protein sequence. Nt and Ct indicate N-terminus and C-terminus, respectively.

X. Chen et al. / Biochemical and Biophysical Research Communications 330 (2005) 1116–1126 Table 1 Structural statistics for BmP08 DYANA (35 best structures) ˚ 2) Target function (A ˚) Maximum violation (A Maximum violation (degree) Average number of upper restraints ˚ /structure violations > 0.2 A Average number of angle restraints violations > 5/structure AMBER (20 best structures) Total energy (kcal/mol) Bond energy (kcal/mol) Angle energy (kcal/mol) Dihedral energy (kcal/mol) vdwaals energy (kcal/mol) Eel energy (kcal/mol) hbond energy (kcal/mol) Constraint energy (kcal/mol) ˚) Rmsd from mean coordinates (A All backbone atoms All heavy atoms Backbone atoms (residues 6–27) Heavy atoms (residues 6–27) Structure analysis (%) Residues in most favored regions Residues in additionally allowed regions Residues in generously allowed regions Residues in disallowed regions

0.0629 ± 0.0194 0.15 0.03 0.0 0.0

344.8 ± 7.1 6.0 ± 0.5 40.7 ± 3.1 64.3 ± 5.9 57.0 ± 3.4 390.0 ± 10.5 12.5 ± 1.1 3.5 ± 0.8 1.31 ± 0.31 2.03 ± 0.36 0.52 ± 0.11 1.37 ± 0.27 61.7 32.8 5.4 0.0

and C-terminal regions. The helical element is a shorter 310-helix and the two-strand b-sheet is smaller (each strand consisted of only two residues), while the angle between the 310-helix and b-sheet is quite large (45) as compared with P05 and P01. The structural features in the 3D-structure of BmP08 are closely related to this distinct primary sequence. The

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sequence alignments of BmP08 and other similar shortchain toxins are shown in Fig. 7. Compared with other short-chain K+ toxins like CTX, the most distinct feature of the primary sequence in BmP08 is the amino acid arrangements between the second and third Cys residue, and between the fifth and sixth Cys residue. It was suggested by Bontems et al. that Cys-[


]-Cys-xx-x-Cys-[


]-Gly-x-Cys-[


]-Cys-x-Cys is the signature sequence for scorpion toxins with the a/b-motif and the Gly in this sequence was conserved due to steric hindrance between the helix and the sheet [32]. The structural signature of these peptides is defined by the presence of the cysteine-stabilized a/b-motif (CS-ab), in which two disulfide bridges, Ci–Cj and Ci+4–Cj+2, covalently link a segment of a-helix with one strand of the b-sheet structure [4]. While in BmP08, the amino acid arrangement between relevant Cys residues is different: Cys-[


]-Cys-x-x-Cys-[


]-ILe-x-Cys-[


]-Cysx-x-Cys. It is obvious that the number of residues between the second and third Cys reduced to two, on the contrary, that between the fifth and sixth Cys increased to two. It features a disulfide bridge pattern (Ci–Cj and Ci+3–Cj+3), which covalently link a segment of 310-helix with one strand of the b-sheet structure. In order to form two disulfide bridges in Ci–Cj and Ci+3– Cj+3, the toxin has to adopt a tight 310-helix instead of a-helix commonly found in other short-chain scorpion toxins. Meanwhile, the b-sheet element is smaller (each strand consisted only two residues). In the most short-chain scorpion toxins, the Gly residue in the signature sequence is highly conserved. Later studies showed that an Ala residue in this position is also acceptable, as in toxin NTX, MTX, and P01, and the methyl side chain of the Ala residue could be accommodated when the helix is bent slightly [33]. The replace-

Fig. 7. Sequence alignment of BmP08 with those of other short-chain scorpion toxins. The amino acid sequences are aligned according to their cysteine residue and the cysteines are boxed. An asterisk indicates amidation at the C-terminus. Gaps are presented as dashed. CTX, charybdotoxin from Leiurus quinquestriatus hebraeus. KTX, kaliotoxin from Androctonus mauretanicus mauretanicus. NTX, noxiustoxin from Centruroides noxius. MTX, maurotoxin from Scorpio maurus. ScTX, scyllatoxin from L. q. hebraeus. P01 from A. m. mauretanicus. TSK, TsKapa from Buthidae Tityus serrulatus. CoTX1, cobatoxin from Centruroides noxius. Tc1 from Titytus cambridgei.

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ment of Gly by Ile at position 20 of the toxin BmP08 should give big influences on the solution conformation of the molecule. In BmP08, the non-b-sheet conformation of the residue Ile20 and Ser21 made it become acceptable by moving their bulky side chains away from the hydrophobic core structure. This kind arrangement of side chains minimized the overlapped surface with helix, where two residues with large side chain (Val14 and Met15) are located, and reduced the energy cost of such an overlapping. Meanwhile, it increased the angle between the helix and b-sheet elements.

On the other hand, in the typical short-chain scorpion toxins the residue preceding the fourth Cys residue usually is a Lys residue, which is crucial for the pore-block type interaction and plugs into the pore and selective filter of the K+ channels. However, in BmP08 a Ser residue appears at this position to replace the conserved basic residue. This is very unusual in short-chain scorpion toxins. Compared with the Lys residue, the side chain of Ser residue is shorter and neutral. So it is likely that BmP08 is not a good blocker for the pore-block interaction with K+ channels due to the changes in the

Fig. 8. (A,B) MOLMOL representation of the structure of BmP08 with the lowest energy. Nt and Ct indicate N-terminus and C-terminus, respectively. (C–H) Electrostatic potentials of the water-accessible surfaces of three short-chain scorpion toxins: simple charge electrostatic potentials associated to the water-accessible molecular surfaces of scorpion toxins calculated using MOLMOL. Positively charged residues and negatively charged residues are shown in blue and in red, respectively. Molecules in (C,E,G) are in the same orientation of a, and molecules in (D,F,H) are in the same orientation of (B). (I,J) Ca wire representation of BmP08 view by WebLab ViewerPro. The salt-bridges between Lys8 and Asp12, Arg11 and Asp12 are described in (I); the hydrogen bond between Lys23 and Gln28 is described in (J). The side chains of Lys8, Arg11, Asp12, Lys23, and Gln28 are shown. Three disulfide bridges of the toxin are labeled and their side chains are also shown.

X. Chen et al. / Biochemical and Biophysical Research Communications 330 (2005) 1116–1126

key residue. These unique structural features are consistent with its inactivity against K+ channels. For comparison, the surface electrostatic charge distribution on both a- and b-face of BmP08, CTX (a pore blocker), and P05 peptides (a turret blocker) is shown in Fig. 8. CTX contains a dense positively charged region on the solvent-exposed surface of b-sheet, and the central basic residue Lys27 plugs into the pore upon binding to Kv or BK channel with its b-face (b-type binding). While, P05 possesses positively charged patch at the solvent-exposed surface of a-helix and blocks the vestibule of SK channel with its a-face (a-type binding) [16,34,35]. It is distinct for BmP08 that on both solventexposed a- and b-face of the toxin, the positively charged residues are fewer and always neighbor negatively charged area (Figs. 8E and F). As shown in Figs. 8I and J, the basic residue Lys8 and Arg11 on a-face of BmP08 forms salt-bridges with the negatively charged residue Asp12; the basic residue Lys23 on the b-face is located in the region which could form hydrogen bond with the side chain of Gln28. These salt-bridges and hydrogen bond should be unfavorable for the binding of BmP08 with K+ channels due to fixed orientation and lower basic property of side chains of the basic residues involved in the salt-bridges and hydrogen bond. During the preparation of this article, the physiological function and solution structure of a synthesized BmKX peptide have been reported by Wang et al. [36]. The results from their work based on a synthesized peptide might be coincident with our results on the natural one as described in this article.

Conclusion The solution structure of BmP08, a novel short-chain scorpion toxin, has been determined by 2D-NMR spectroscopy and molecular modeling technique. The most unique structural feature in the 3D-structure of BmP08 is a 310-helix and a shorter b-sheet, and bigger angle between these two secondary structures. These structural features are closely related to the distinct primary sequence containing an unclassified Cys residue arrangement, in which two disulfide bridges (Ci–Cj and Ci+3–Cj+3) link covalently a segment of 310-helix with one strand of the b-sheet structure. Furthermore, BmP08 shows no inhibitory activity towards all tested voltage-dependent and Ca2+-activated K+ channels in the electrophysiological experiment. The electrostatic potential surface analysis of the toxin reveals neither condensed positively charged b-face with a free Lys residue preceding the fourth Cys residue like in CTX (pore-block type), nor a positively charged patch on the a-face of the molecule like in P05 (turret block type). These facts well account for its non-inhibitory activities towards all tested K+ channels and imply that

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BmP08 could not be classified into any short-chain scorpion toxin sub-family which existed. Therefore, finding the target channel for this toxin would be an interesting work in the future. Data bank accession number The atomic coordinates of the 20 energy-minimized conformers used to represent the solution structure of BmP08 have been deposited in the Brookhaven data bank, together with the input of conformational restrains used for the structure calculation under Accession No. 1WT8.

Acknowledgments This work was supported by the National Science Foundation of China (Grant No. 20132030) and Chinese Academy of Sciences (Grant No. KGCX2-SW213-05). We thank the Institute of Molecular Biology and Biophysics, ETH-Ho¨nggerberg Zu¨rich, Switzerland, for the programs DYANA (version 1.5) and XEASY, Prof. Bertini of Florence University, Italy, for the program CALIBA, and Prof. James W. Caldwell of the University of California, San Francisco, for the program AMBER. We also thank Prof. Simon J. Gaskell and Dr. Michael Chalmers of UMIST, UK, for the work of CID MS/MS spectra. We are grateful to Prof. Zhou Zhuan of Peking University, China, for the electrophysiological experiment of BmP08, and Prof. Li Wenxin of Wuhan University, China, for the helpful discussion about the sequence of BmP08.

References [1] L.D. Possani, B. Becerril, M. Delepierre, J. Tytgat, Scorpion toxins specific for Na+-channels, Eur. J. Biochem. 264 (1999) 287– 300. [2] T. Olamendi-Portugal, F. Gomez-Lagunas, G.B. Gurrola, L.D. Possani, A novel structural class of K+ channel blocking toxin from the scorpion Pandinus imperator, Biochem. J. 315 (1996) 977–981. [3] J. Tytgat, K.G. Chandy, M.L. Garcia, G.A. Gutman, M.F. Martin-Eauclaire, J.J. Van der Walt, L.D. Possani, A unified nomenclature for short-chain peptides isolated from scorpion venoms: alpha-KTx molecular subfamilies, Trends Pharmacol. Sci. 20 (1999) 444–447. [4] R.C. Rodriguez de la Vega, L.D. Possani, Current views on scorpion toxins specific for K+-channels, Toxicon 43 (2004) 865– 875. [5] H.M. Li, D.C. Wang, Z.H. Zeng, L. Jin, R.Q. Hu, Crystal structure of an acidic neurotoxin from scorpion Buthus ˚ resolution, J. Mol. Biol. 261 martensi Karsch at 1.85 A (1996) 415–431. [6] X.L. He, H.M. Li, Z.H. Zeng, X.Q. Liu, M. Wang, D.C. Wang, Crystal structures of two alpha-like scorpion toxins: non-proline cis peptide bonds and implications for new binding

1126

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

X. Chen et al. / Biochemical and Biophysical Research Communications 330 (2005) 1116–1126

site selectivity on the sodium channel, J. Mol. Biol. 292 (1999) 125–135. X.L He, J.P. Deng, M. Wang, Y. Zhang, D.C. Wang, Structure of a new neurotoxin from the scorpion Buthus martensi Karsch at ˚ , Acta Crystallogr. Sect. D 56 (2000) 25–33. 1.76 A N.X. Zhang, G. Wu, Z.H. Wang, H.M. Wu, Purification, characterization and 1H NMR resonance assignment of an a-like neurotoxin BmK 16 from the venom of Chinese scorpion Buthus martensi Karsch, Chin. J. Chem. 21 (2003) 1378–1384. G. Wu, Y.M. Li, D.S. Wei, F.H. He, S.K. Jiang, G.Y. Hu, H.M. Wu, Solution structure of BmP01 from the venom of scorpion Buthus martensi Karsch, Biochem. Biophys. Res. Commun. 276 (2000) 1148–1154. Y.Q. Xu, J.H. Wu, J.M. Pei, Y.Y. Shi, Y.H. Ji, Q.H. Tong, Solution structure of BmP02, a new potassium channel blocker from the venom of the Chinese scorpion Buthus martensi Karsch, Biochemistry 39 (2000) 13669–13675. F.H. He, Y.M. Li, G. Wu, C.Y. Cao, H.M. Wu, Threedimensional structure of BmP03 from venom of scorpion Buthus martensi Karsch, Acta Chim. Sin. 58 (2000) 850–855. E. Blanc, R. Romi-Lebrun, O. Bornet, T. Nakajima, H. Darbon, Solution structure of two new toxins from the venom of the Chinese scorpion Buthus martensi Karsch blockers of potassium channels, Biochemistry 37 (1998) 12412–12418. J.G. Renisio, R. Romi-Lebrun, E. Blanc, O. Bornet, T. Nakajima, H. Darbon, Solution structure of BmKTX, a K+ blocker toxin from the Chinese scorpion Buthus Martensi, Proteins 38 (2000) 70–78. Z. Cai, C.Q. Xu, Y.Q. Xu, W.Y. Lu, C.W. Chi, Y.Y. Shi, J.H. Wu, Solution structure of BmBKTx1, a new BKCa channel blocker from the Chinese scorpion Buthus martensi Karsch, Biochemistry 43 (2004) 3764–3771. Y.F. Wang, X. Chen, N.X. Zhang, G. Wu, H.M. Wu, The solution structure of BmTx3B, a member of the scorpion toxin sub-family a-KTx 16, Proteins 58 (2005) 489–497. N.X. Zhang, M.H. Li, X. Chen, Y.F. Wang, G. Wu, G.Y. Hu, H.M. Wu, Solution structure of BmKK2, a new potassium channel blocker from the venom of Chinese scorpion Buthus martensi Karsch, Proteins 55 (2004) 835–845. N.X. Zhang, X. Chen, M.H. Li, C.Y. Cao, Y.F. Wang, G. Wu, G.Y. Hu, H.M. Wu, Solution structure of BmKK4, the first member of sub-family a-KTx 17 of scorpion toxins, Biochemisry 43 (2004) 12469–12476. H.M. Wu, G. Wu, X.L. Huang, F.H. He, S.K. Jiang, Purification, characterization and structural study of the neuro-peptides from scorpion Buthus martensi Karsch, Pure Appl. Chem. 71 (1999) 1157–1162. U. Piantini, O.W. Sorensen, R.R. Ernst, Multiple quantum filters for elucidating NMR coupling networks, J. Am. Chem. Soc. 104 (1982) 6800–6801. C. Griesinger, G. Otting, K. Wu¨thrich, R.R. Ernst, Clean TOCSY for proton spin system identification in macromolecules, J. Am. Chem. Soc. 110 (1988) 7870–7872.

[21] J. Jeener, B.H. Meier, P. Bachmann, R.R. Ernst, Investigation of exchange process by two-dimensional NMR spectroscopy, J. Chem. Phys. 71 (1979) 4546–4553. [22] A.D. Bax, D.G. Davis, MLEV-17-based two-dimensional homonuclear magnetization transfer spectroscopy, J. Magn. Reson. 65 (1985) 355–366. [23] K. Wu¨thrich, in: K. Wu¨thrich (Ed.), NMR of Proteins and Nucleic Acid, Wiley, New York, 1986, p. 292. [24] P. Gu¨ntert, C. Mumenthaler, K. Wu¨thrich, Torsion angle dynamics for NMR structure calculation with the new program DYANA, J. Mol. Biol. 273 (1997) 283–298. [25] S.J. Weiner, P.A. Kollman, AMBER: assisted model building with energy refinement. A general program for modeling molecules and their interactions, J. Comput. Chem. 2 (1981) 287–309. [26] S.J. Weiner, P.A. Kollman, D.T. Nguyen, D.A. Case, An all atom force field for simulations of proteins and nucleic acids, J. Comput. Chem. 2 (1986) 230–252. [27] R.A. Laskowski, M.W. MacArthur, D.S. Moss, J.M. Thornton, PROCHECK: a program to check the stereochemical quality of protein structures, J. Appl. Crstallorgr. 26 (1993) 283–291. [28] R.A. Laskowski, J.A.C. Rullmann, M.W. MacArthur, R. Kaptein, J.M. Thornton, AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR, J. Biomol. NMR 8 (1996) 477–486. [29] R. Koradi, M. Billeter, K. Wu¨thrich, MOLMOL: a program for display and analysis of macromolecular structures, J. Mol. Graph. 14 (1996) 51–55. [30] Y.M. Li, G. Wu, N.X. Zhang, H.M. Wu, Purification and primary structure of a novel peptide from the Chinese scorpion Buthus martensi Karsch, Acta Biochim. Biophys. Sin. 32 (2000) 351–355. [31] P. Gu¨ntert, W. Braun, K. Wu¨thrich, Efficient computation of three-dimensional protein structures in solution from nuclear magnetic resonance data using the program DIANA and the supporting programs CALIBA, HABAS and GLOMSA, J. Mol. Biol. 217 (1991) 517–530. [32] F. Bontems, C. Roumestand, B. Gilquin, F. Toma, Refined structure of charybdotoxin: common motifs in scorpion toxins and insect defensins, Science 254 (1991) 1521–1523. [33] E. Blanc, J.M. Sabatier, R. Kharrat, S. Meunier, M. el Ayeb, J. Van Rietschoten, H. Darbon, Solution structure of maurotoxin, a scorpion toxin from Scorpio maurus, with high affinity for voltagegated potassium channels, Proteins 29 (1997) 321–333. [34] R.C. Rodriguez de la Vega, E. Merino, B. Becerril, L.D. Possani, Novel interactions between K+ channels and scorpion toxins, Trends Pharmacol. Sci. 24 (2003) 222–227. [35] C.Q. Xu, S.Y. Zhu, C.W. Chi, J. Tytgat, Turret and pore block of K+ channels: what is the difference?, Trends Pharmacol. Sci. 24 (2003) 446–448. [36] C.G. Wang, Z. Cai, W. Lu, J. Wu, Y. Xu, Y. Shi, C.W. Chi, A novel short-chain peptide BmKX from the chinese scorpion Buthus martensi karsch, sequencing, gene cloning and structure determination, Toxicon 45 (2005) 309–319.