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Acta Biochim Biophys
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doi:10.1111/j.1745-7270.2005.00078.x |
Sequence-specific Assignment of 1H-NMR Resonance and Determination of the Secondary Structure of Jingzhaotoxin-I
Xiong-Zhi ZENG, Qi ZHU, and Song-Ping LIANG*
College of Life Sciences,
Hunan Normal University, Changsha 410081, China
Received: January
14, 2005
Accepted: May 9,
2005
This work was
supported by grants from the National Natural Science Foundation of China (No.
30170193 and No. 30430170)
*Corresponding author:
Tel, 86-731-8872556; Fax, 86-21-8861304; E-mail, [email protected]
Abstract Jingzhaotoxin-I (JZTX-I) purified from the venom of the spider Chilobrachys jingzhao is a novel neurotoxin preferentially inhibiting cardiac sodium channel inactivation by binding to receptor site 3. The structure of this toxin in aqueous solution was investigated using 2-D 1H-NMR techniques. The complete sequence-specific assignments of proton resonance in the 1H-NMR spectra of JZTX-I were obtained by analyzing a series of 2-D spectra, including DQF-COSY, TOCSY and NOESY spectra, in H2O and D2O. All the backbone protons except for Gln4 and more than 95% of the side-chain protons were identified by daN, dad, dbN and dNN connectivities in the NOESY spectrum. These studies provide a basis for the further determination of the solution conformation of JZTX-I. Furthermore, the secondary structure of JZTX-I was identified from NMR data. It consists mainly of a short triple-stranded antiparallel b-sheet with Trp7-Cys9, Phe20-Lys23 and Leu28-Trp31. The characteristics of the secondary structure of JZTX-I are similar to those of huwentoxin-I (HWTX-I) and hainantoxin-IV (HNTX-IV), whose structures in solution have previously been reported.
Key words Jingzhaotoxin-I (JZTX-I); 2-D nuclear magnetic resonance (NMR); sequence-specific assignment; secondary structure
Spiders are remarkable for their reliance on predation as a trophic strategy. Their evolutionary success is largely a result of the production of a complex venom that is designed to quickly subdue or kill their prey [1]. Spider venom glands are extraordinary special organs that have evolved through hundreds of millions of years, and their secreted toxin components have special structural diversities and great specificity in terms of biological activities [2,3]. Many spider toxins have been used as invaluable tools for studying receptors, ion channels, nerve cell communication and immunology, and as potential lead structures in the design and creation of new highly specific and effective insecticides and pharmaceuticals [4,5]. Furthermore, antibodies raised against the critical toxin components have the potential to block the toxic effects and reduce the pain caused by spider envenomation [6].
Recently, a new peptide neurotoxin, Jingzhaotoxin-I (JZTX-I), purified from the venom of the spider Chilobrachys jingzhao, has been identified [7]. JZTX-I is a 33-residue peptide toxin containing three disulfide bridges Cys2-Cys17, Cys9-Cys22 and Cys16-Cys29, determined by partial reduction, sequencing and multi-enzymatic digestion. Moreover, JZTX-I is also an a-like sodium channel toxin first reported in spider venoms, inhibiting channel fast-inactivation kinetics of both TTX-resistant (TTX-R) voltage-gated sodium channels (VGSCs) on rat cardiac myocytes and TTX-sensitive (TTX-S) VGSCs expressed on rat dorsal root ganglion (DRG) neurons as well as cotton bollworm central nerve ganglia. It may contain important ligands for distinguishing cardiac VGSC subtypes [8].
In order to study the structure-function relationship of JZTX-I, we determined the structure of JZTX-I in solution using two-dimensional proton nuclear magnetic resonance (2-D 1H-NMR) spectroscopy. The complete sequence-specific assignments of proton resonances and the secondary structure of JZTX-I are reported in this paper.
Experimental Procedures
JZTX-I was isolated from the venom of the spider Chilobrachys jingzhao and purified by ion exchange and reverse phase high performance liquid chromatography (RP-HPLC) as described previously [8]. The purity of the peptide was confirmed by N-terminal sequencing, RP-HPLC and mass spectrometry analysis. The sample was prepared by dissolving the lyophilized powder of JZTX-I in 550 ml of buffer (H2O:D2O=9:1, V/V) containing 0.02% NaN3 and 0.1 mM EDTA, with the final concentration of JZTX-I being 3.5 mM at pH 5.0. Sodium 3-(trimethyl-silyl)propionate-2,2,3,3-D4 (TSP) was added to the mixture at a final concentration of 200 mM as an internal chemical shift reference. For experiments in D2O, the sample used in H2O experiments was lyophilized, redissolved in 99.8% D2O, and then allowed to stand at room temperature for 24 h. After lyophilization, the peptide powder was redissolved in 550 ml of 99.96% D2O (Cambridge Isotope Laboratories) [9].
The NMR spectra were collected on a Varian Inova 600 (Varian Inc,
California, America) or a Bruker DRX-500 (Bruker BioSpin Corporation,
Switzerland) spectrometer with a sample temperature of 300 K and 310 K,
respectively. Two-dimensional DQF-COSY, TOCSY and NOESY measurements were
recorded in a phase-sensitive mode by the time-proportional phase
incrementation (TPPI) method following standard pulse sequences and phase
cycling. TOCSY spectra were obtained with a mixing time of 85 ms and 100 ms.
NOESY spectra were recorded in D2O with a mixing time of 200 ms and in H2O
with a mixing time of 100 ms, 200 ms and 400 ms. Solvent suppression was
achieved by the presaturation method. All 2-D measurements were recorded with
1024´512 frequency data points and were zero-filled to yield 2048´1024 data
matrices except for the DQF-COSY spectrum. The DQF-COSY spectrum was recorded
with 2048´512 data points in two dimensions, respectively, and zero-filled to
yield 4096´1024 points to measure the coupling constants. All spectra were
processed and analyzed using Felix 98.0 software (Biosym Technologies) running
on a Silicon Graphics O2 workstation. The signal was multiplied by a sine bell
square window function with a 90° phase shift in both dimensions prior to
Fourier transformation.
As for the experiment involving the slow exchange of backbone amide protons, the sample lyophilized from H2O was redissolved in D2O and was identified by analyzing a series of 1-D spectra recorded at time points of 8 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h and 24 h. A TOCSY spectrum was recorded after 6 h of exchange.
Results and Discussion
The sequence-specific proton resonance assignment of JZTX-I was
performed using standard 2-D homonuclear NMR experiments [10]. It was performed
in two steps: (1) identification of the spin systems; and (2) sequential
assignment of resonances.
The 33 residues of JZTX-I were divided into four groups according to their spin systems and structural characteristics: (1) four Gly containing AX spin systems, each has two a protons; (2) seven residues containing methyl, including Ala1, Ala18, Ala21, Leu26, Leu28, Ile24 and Ile30; (3) fourteen AMX spin systems, including six Cys, three Trp, two Phe, Ser32, Asn19 and Tyr27; and (4) eight residues containing long side-chain spin systems, including three Lys, three Pro, Glu11 and Gln4. The proton resonances of JZTX-I were assigned to the spin systems of specific residue types by analyzing scalar coupling patterns observed in TOCSY and DQF-COSY spectra. The residue types that were immediately identified were Gly, Ala, Lys, Leu and Ile.
On the basis of the amino acid sequence of JZTX-I, 33 NHi-CaHi cross-peaks were expected in the fingerprint region of the DQF-COSY
spectrum because Ala1, Pro14, Pro15 and Pro33 do not exhibit cross-peaks, while
four Gly exhibit eight cross-peaks. Fig. 1 shows 26 NHi-CaHi cross-peaks. Among them, the cross-peaks of Cys2, Gln4, Trp7, Lys8,
Cys9, Lys13 and Cys22, can not be found. These peaks, except for Gln4, were
overlapped by H2O, but they were observed in NOESY and TOCSY spectra in D2O.
In the TOCSY spectrum with a mixing time of 85 ms, all the spin systems of
JZTX-I, except for Ala1, Gln4, Pro14, Pro15 and Pro33, are shown in Fig. 2.
Sequence-specific assignments were carried out by looking for daN, dbN, dad and dNN connectivities in the NOESY spectrum with a mixing time of 200 ms.
Residue types that were previously assigned were used as starting points for
the sequential assignment process. When a daN-type
NOE was observed, a sequential connectivity was established only if an
additional dbN or dNN NOE was also
observed. The spin systems of residues Pro14, Pro15 and Pro33 were identified
by the observation of strong sequential NOE cross-peaks between the a proton of the
residue prior to the proline and the d protons of the proline, which also indicate
that residues Pro14, Pro15 and Pro33 in JZTX-I all take the trans
configuration. Gly3, Gln4, Phe5, Lys8, Cys9, Cys22 and Lys23 were confirmed by
the observed dbN and dNN
connectivities, although no sequential daN(i,i+1) connectivities were found. At the end of the sequential assignment procedure,
all the backbone protons (except for Gln4) and more than 95% of the side-chain
protons had been assigned. Although no NH-CaH and NH-CbH proton resonances of
Gln4 were observed over the pH range of 4.0-6.5 and temperature range of 288-310 K, this complete lack of signals is possibly indicative of a
chemical exchange which resulted in the undetectable 1H
resonances. Fig. 3 shows the sequential daN(i,i+1) connectivities in the NH-CaH fingerprint region of the NOESY spectrum with a mixing time of 200
ms. Table 1 shows the summary of the chemical shifts of proton
resonances of JZTX-I. All the amide protons of JZTX-I resonate at conventional
frequencies with the exception of Gly25, which shows an unusual chemical shift
for its amide proton (5.376 ppm at 300 K). During the structural calculations
for JZTX-I, an explanation of this unusual chemical shift was obtained, when
the Gly25 amide proton was placed in the neighborhood of the aromatic ring of
Tyr27. The ring current of Tyr27 creates an electromagnetic shield that
dramatically affects the chemical shifts of the Gly25 amide proton. This was
also observed for the chemical shifts of Gln4, which is similarly affected by
the ring current of Trp7.
The regular secondary structure elements of the JZTX-I molecule were
characterized according to the criteria described by Wüthrich [10]. The extent
and relative orientation of b-strands were based on strong sequential daN,
interstrand dNN and NH-CaH connectivities,
slow-exchange amide protons, and large 3JNH-CaH
coupling constants, which distinguished the periphery and strands in the b-sheet. The NMR
data summarized in Fig. 4 show that there are three short b-strands from
Trp7 to Cys9, Phe20 to Lys23, and Leu28 to Trp31. They are arranged in an
antiparallel fashion with coils and turns. The analysis of the CaH chemical shifts was in accordance with the three-strand
antiparallel b-sheet, in which most of the residues showed downfield shifts [11].
Fig. 5 shows the b-sheet region, which is in agreement with the standard criteria.
According to up-to-date records from the protein data bank, the 3-D solution structures of 32 spider toxins have been determined by using 1H-NMR spectroscopy. Some of these toxins include the P-type calcium channel antagonist w-agatoxin-IVA [12] and insect sodium channel inhibitor m-agatoxins from the venom of the American funnel web spider [13]; the N-type calcium channel inhibitor HWTX-I [14], tetrodotoxin-sensitive sodium channel antagonist HWTX-IV [15] and SHL-I [16] from the Chinese bird spider Selenocosmia huwena; the potassium channel inhibitor Patx1 [17] from the venom of the spider Phrixotrichus auratus; the proton-gated cation channel blocker psalmotoxin 1 from the South American tarantula [18]; and the mechanosensitive ion channel inhibitor Gsmtx-4 [19] from the tarantula Grammostola spatulata. These toxins display low sequence homology and diverse bioactivity, but they all share the same structural scaffold known as the “inhibitor cystine knot” (ICK) architectural motif [20]. The ICK motif, which consists of several loops that emerge from a double-stranded or triple-stranded antiparallel b-sheet structure, is reticulated by at least three disulfide bridges. Two of the disulfide bridges, together with the amino acid backbone, form a ring, which is penetrated by the third disulfide bridge. However, the diverse bioactivities of those spider toxins derive from the local structural differences.
The structure of JZTX-I is characterized by a cystine knot and a small triple-stranded (Trp7 to Cys9, Phe20 to Lys23, and Leu28 to Trp31) antiparallel b-sheet. It is now evident that JZTX-I shares the same cystine knot motif as the spider toxins mentioned above on the basis of the secondary structure analysis.
In summary, the complete sequence-specific assignment of proton
resonance in the 1H-NMR spectra has been made and the secondary structure elements of
JZTX-I have been obtained. These results will provide a basis for the
structural calculation and detail analysis of JZTX-I.
Acknowledgements
We thank Mr. Xian-Zhong YAN of the National Center of Biomedical
Analysis (China) and Guan-Zhong TU of the Beijing Institute of Microchemistry
(Beijing, China) for collecting the 1H-NMR spectra.
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