Three-Dimensional Structure in Solution of the Calcium Channel Blocker .omega.-Conotoxin MVIIA

Kohno, Toshiyuki; Kim, Jae Il; Kobayashi, Kozue; Kodera, Yasuhiro; Maeda, Tadakazu; Sato, Kazuki

doi:10.1021/bi00032a020

Cited by 111 publications

(119 citation statements)

References 35 publications

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“…Based on analyses of the determined secondary and tertiary structure elements, the backbone conformation of ␦-CTX TxVIA is similar to those of -CTX GVIA (14 -17) and -CTX MVIIA (18,19) reported previously. However, novel charge distribution and hydrophobic characteristics are present in ␦-CTX TxVIA.…”

supporting

confidence: 77%

“…As shown in Fig. 3, the global conformation of ␦-CTX TxVIA is similar to those of the structurally well characterized -CTX GVIA (14 -17) and -CTX MVIIA (18,19). Despite the relatively low amino acid sequence homology among the many conotoxins, the toxin structures exhibit similar locations and orientations of the secondary structure elements in their three-dimensional coordinates.…”

Section: Resultsmentioning

confidence: 52%

“…The amino acid sequences of ␦-CTX PVIA and NgVIA are similar. Especially, both peptides have the same sequence between residues Hyp 6 and Ser 19 . In these sequences a hydrophobic residue, Leu 12 , in ␦-CTX TxVIA is replaced by a positively charged residue, Lys, in both ␦-CTX PVIA and NgVIA.…”

Section: Structure-function Relationships Of ␦-Ctxmentioning

confidence: 99%

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Three-dimensional Solution Structure of the Sodium Channel Agonist/Antagonist δ-Conotoxin TxVIA

Kohno

Sasaki

Kobayashi

et al. 2002

Journal of Biological Chemistry

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The three-dimensional solution structure of ␦-conotoxin TxVIA, a 27-mer peptide agonist/antagonist of sodium channels, was determined by two-dimensional 1 H NMR spectroscopy with simulated annealing calculations. A total of 20 converged structures of ␦-conotoxin TxVIA were obtained on the basis of 360 distance constraints obtained from nuclear Overhauser effect connectivities, 28 torsion angle constraints, and 27 constraints associated with hydrogen bonds and disulfide bonds. The atomic root mean square difference about the averaged coordinate positions is 0.35 ؎ 0.07 Å for the backbone atoms (N, C ␣ , C) and 0.98 ؎ 0.14 Å for all heavy atoms of the entire peptide. The molecular structure of ␦-conotoxin TxVIA is composed of a short triplestranded antiparallel ␤-sheet. The overall ␤-sheet topology is ؉2x, ؊1, which is the same as those for other conotoxins. However, the three-dimensional structure of ␦-conotoxin TxVIA has an unusual hydrophobic patch on one side of the molecule, which may play an important role in the sodium channel binding. These results provide a molecular basis for understanding the mechanism of sodium channel modulation through the toxinchannel interaction and insight into the discrimination of different ion channels.

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supporting

confidence: 77%

Section: Resultsmentioning

confidence: 52%

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Three-dimensional Solution Structure of the Sodium Channel Agonist/Antagonist δ-Conotoxin TxVIA

Kohno

Sasaki

Kobayashi

et al. 2002

Journal of Biological Chemistry

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“…The MVIIA molecule could conceivably adopt two orientations in the outer vestibule of the N-type channel pore. Alternatively, it has been shown by NMR analysis that MVIIA can dynamically swap between two conformations by rearranging the disulfide backbone (20,21). Future work examining MVIIA mutants with a single defined structure, or other related peptides such as -conotoxin CVID (22) might serve to test such a hypothesis.…”

Section: Discussionmentioning

confidence: 99%

Determinants of Inhibition of Transiently Expressed Voltage-gated Calcium Channels by ω-Conotoxins GVIA and MVIIA

Feng

Doering

Winkfein

et al. 2003

Journal of Biological Chemistry

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The Conus magus peptide toxin -conotoxin MVIIA is considered an irreversible, specific blocker of N-type calcium channels, and is now in clinical trials as an intrathecal analgesic. Here, we have examined the action of MVIIA on mutant and wild type calcium channels transiently expressed in tsA-201 cells. Although we have shown previously that mutations in a putative external EF-hand motif in the domain IIIS5-H5 region alters block by both -conotoxin GVIA and MVIIA (Feng, Z. P., Hamid, J., Doering, C., Bosey, G. M., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 15728 -15735), the introduction of five point mutations known to affect GVIA blocking (and located downstream of the EFhand) affected MVIIA block to a smaller degree compared with GVIA. These data suggest that despite some overlap, MVIIA and GVIA block does not share identical channel structural determinants. At higher concentrations (ϳ3 M), MVIIA reversibly blocked L-, P/Q-, and R-type, but not T-type channels, indicating that the overall architecture of the MVIIA site is conserved in all types of high voltage-activated calcium channels. A kinetic analysis of the MVIIA effects on the N-type channel showed that MVIIA blocked resting, open, and inactivated channels. Although the development of MVIIA block did not appear to be voltage-, nor frequency-dependent, the degree of recovery from block strongly depended on the potential applied during washout. Interestingly, the degree of washout was highly variable and appeared to weakly depend on the holding potential applied during toxin application. We propose a model in which N-type calcium channels can form both reversible and irreversible complexes with MVIIA.A number of predatory species such as spiders, scorpions, and fish hunting mollusks have developed venoms to rapidly stun and kill their prey. Their venoms typically contain a mixture of potent peptide toxins that have evolved to specifically bind to voltage-and ligand-gated ion channels (1). Among calcium channel blocking peptides, -conotoxin GVIA (isolated from Conus geographus) and -conotoxin MVIIA (isolated from Conus magus) are perhaps the most widely known. Both of these toxins are thought to specifically block the pore of N-type calcium channels from a variety of species. Block by both toxins is considered to be virtually irreversible at normal membrane potentials (i.e. Refs. 2 and 3), but complete reversibility can be obtained upon strong hyperpolarization (4), or by removal of extracellular divalent carrier ions (5). Our current understanding of the molecular basis of N-type channel block came initially from a study by Ellinor et al. (6) who showed that block of transiently expressed N-type channels by -conotoxin GVIA was dramatically attenuated by sequence substitution in the domain III S5-S6 region of the Ca v 2.2 ␣ 1 subunit. More recently, Feng et al. (7) showed that additional point mutations in an EF-hand consensus motif in this region affected the development of, and recovery from, block by both GVIA and MVIIA, consistent...

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“…The three-dimensional structures of ω-conotoxin MVIIA, MVIIC have been determined by two-dimensional NMR, revealing a triple-stranded antiparallel β-sheet stabilized by three disulfide bonds. [7][8][9] Although IpTx a and ω-conotoxins exhibit high structural similarity, their biological functions are very different. IpTx a activates ligand-gated Ca 2+ channels.…”

Section: Resultsmentioning

confidence: 99%

Determination of Disulfide Bond Connectivity of Cysteine-rich Peptide IpTx_a

Lee¹,

Sato²,

Kim³

2013

Bulletin of the Korean Chemical Society

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Disulfide bonds play a crucial role in the folding and structural stabilization of many important extracellular peptides and proteins including hormones, enzymes, growth factors, toxins, and immunoglobulins.1,2 Cysteine-rich peptides stabilized by intramolecular disulfide bonds have often been isolated from venoms of microbes, animals and plants. These peptides typically have much higher stability and improved biopharmaceutical properties compared to their linear counterparts. Therefore the correct disulfide bond formation of small proteins and peptides has been extensively studied for a better understanding of their folding mechanism and achieving efficient generation of the naturally occurring biologically active product. Imperatoxin A (IpTx a ), a peptide toxin containing 6 cysteine residues, was isolated from the venom of scorpion Pandinus imperator, selectively binds the ryanodine receptors and activates Ca 2+ release from sarcoplasmic reticulum (SR).3,4 IpTx a increases the binding of ryanodine to ryanodine receptors (RyRs) 4,5 and encourages reconstituted single channel to induce subconductance states. 5We previously reported the three-dimensional structure of IpTx a determined by solution NMR spectroscopy.6 The molecular structure of IpTx a consists of two antiparallel β-strands connected by four chain reversals. The overall structure of IpTx a is stabilized by three disulfide bonds. This topology and disulfide bond pattern is classified as an 'inhibitor cysteine knot' fold found in numerous toxic and inhibitory peptides, 7-11 including ω-conotoxins. Figure 1 shows the sequence alignment of IpTx a with ω-conotoxin GVIA, MVIIA, and MVIIC demonstrating the same number and position of cysteines, although they have very low Dali Z-scores and sequence identity. IpTx a exhibits a large functional surface area identified by alanine-scanning mutagenesis, and the hydrophobic core of IpTx a is entirely composed of the four out of six cysteines that form intramolecular disulfide bonds to stabilize the overall toxin structure. Without cysteine residues, IpTx a completely lost its molecular structure and biological activity.6 These results indicate that the information of the authentic disulfide bond connectivity of cysteine-rich peptides or proteins might help not only to understand their three-dimensional conformation but to prepare biologically active products. Here we report the disulfide bond connectivity of IpTx a determined by a combined approach of enzymatic fragmentation and chemical synthesis. ExperimentalPeptide Synthesis and Oxidative Folding. Solid phase peptide synthesis was conducted on an Applied Biosystems 433A peptide synthesizer. The linear precursor of IpTx a was synthesized by solid phase methodology of Fmoc chemistry starting from Fmoc-Arg(Pmc)-Alko resin using a variety of blocking groups for the protection of amino acids. MALDI-TOF-MS was measured on a PerSeptive Biosystems Voyager Linear mass spectrometer by using α-cyano-4-hydroxy-cinnamic acid as a matrix. High performance liquid chromatog...

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Three-Dimensional Structure in Solution of the Calcium Channel Blocker .omega.-Conotoxin MVIIA

Cited by 111 publications

References 35 publications

Three-dimensional Solution Structure of the Sodium Channel Agonist/Antagonist δ-Conotoxin TxVIA

Three-dimensional Solution Structure of the Sodium Channel Agonist/Antagonist δ-Conotoxin TxVIA

Determinants of Inhibition of Transiently Expressed Voltage-gated Calcium Channels by ω-Conotoxins GVIA and MVIIA

Determination of Disulfide Bond Connectivity of Cysteine-rich Peptide IpTx_a

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Three-Dimensional Structure in Solution of the Calcium Channel Blocker .omega.-Conotoxin MVIIA

Cited by 111 publications

References 35 publications

Three-dimensional Solution Structure of the Sodium Channel Agonist/Antagonist δ-Conotoxin TxVIA

Three-dimensional Solution Structure of the Sodium Channel Agonist/Antagonist δ-Conotoxin TxVIA

Determinants of Inhibition of Transiently Expressed Voltage-gated Calcium Channels by ω-Conotoxins GVIA and MVIIA

Determination of Disulfide Bond Connectivity of Cysteine-rich Peptide IpTxa

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Determination of Disulfide Bond Connectivity of Cysteine-rich Peptide IpTx_a