Mapping hydrophobic residues of the interaction surface of charybdotoxin

Stampe, Per; Kolmakova-Partensky, Ludmilla; Miller, Christopher

doi:10.1016/s0006-3495(92)81761-7

Cited by 25 publications

(25 citation statements)

References 8 publications

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“…By site-directed mutagenesis studies they identified in the residues located in the / l strand 25-29 and 32-36 of the p sheet of charybdotoxin the surface of interaction of the toxin for the channel. In addition, their mutations of Glpl and Phe2 residues [60], present on the adjacent p strand I, produced results identical to those we reported here for the analogues lacking Glpl [i.e. charybdotoxin-(2-37)] and Phe2 [i.e.…”

Section: Discussionsupporting

confidence: 87%

“…A recent 'H-NMR analysis has revealed the same motif in iberiotoxin [23]. The same alp motif is also found in other longer toxins (60)(61)(62)(63)(64)(65) residues) present in scorpion venoms and active on Na' channels (20,21,24,251. Thus, all scorpion toxins, whether they are activc on Na' or K ' channels, secm to possess a similar basic fold rorrncd by a triple-stranded p sheet.…”

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confidence: 54%

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Synthesis of charybdotoxin and of two N‐terminal truncated analogues

Vita

Bontems

Bouet

et al. 1993

European Journal of Biochemistry

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Charybdotoxin and two N-terminal truncated peptides, corresponding to the 2-37 and 7 -37 sequences, were obtained by stepwise solid-phase synthesis using W-t-butyloxycarbonyl and benzyltype side-chain protection. While this strategy was generally useful, the S-acetamidomethyl protecting group used for the six cysteines was not completely stable under HF treatment and its subsequent removal by mercury(I1) treatment was neither complete nor devoid of side reactions. The completely deprotected native and truncated sequences were folded efficiently in the presence of glutathione and were finally purified by high-pressure liquid chromatography with overall yields of 4.0-5.096. Each protein was characterised chemically. structurally and functionally. 'H-NMR spectroscopy was used and a complete assignment of all the protons of the three synthetic proteins was achieved. NMR data show that synthetic charybdotoxin is indistinguishable from the natural protein. The two truncated proteins contain the same elements of secondary structure and a similar overall threedimensional structure, in agreement with circular dichroic measurements. The shortest analogue, however, may have local structural perturbations and/or higher flexibility. Biological activity on dog epithelial Ca'+-activated K' channels and on rat brain synaptosomal voltage-dependent K' channels show that synthetic charybdotoxin was as potent as the natural toxin on both channels. For both channels, deletion of the first amino acid, 5-oxoproline (pyroglutamic acid) decreased only slightly the potency of the inhibitor, while deletion of the entire 1-6 segment reduced potency much more. We conclude that the N-terminal region of charybdotoxin plays a functional role in tuning the toxin's biological activity but is not essential for the folding and stability of the structure. The structure of the shortest analogue represents an interesting example of how a well organised and stable alp fold can be engineered with only 31 amino acid residues.Protein toxins are used by venomous animals to subdue prey. These molecules, which generally have a molecular mass ranging between 2-12 kDa, are active towards different molecular targets [1-41 and are characterised by the presence of several disulfide bridges. Scorpion venoms contain a variety of proteins known to inhibit Na' and K' channels. Charybdotoxin, present as a minor component (= 0.2%) in the venom of the Israeli Lpiurus quinquestriatus scorpion, was the first discovered high-affinity inhibitor of high-conductance Ca'+-activated K' channels present in skelctal muscles [5, 61. proteins 11, 3, 4, 20, 211 with subtly different specificities and which therefore represent valuable tools in order to study diffcrcnt K' channels.We have recently reported the three-dimensional solution structure of charybdotoxin solved by two-dimensional NMR [20, 211 and the refined and detailed description of the sidechain organisation [22]. This analysis revealed the presence of a well ordered structure containing three antiparallel strands fo...

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Section: Discussionsupporting

confidence: 87%

mentioning

confidence: 54%

Synthesis of charybdotoxin and of two N‐terminal truncated analogues

Vita

Bontems

Bouet

et al. 1993

European Journal of Biochemistry

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“…It suffices to recall that changing only amino acid residue in certain types of scorpion toxins can modify the affinity by three orders of magnitude, as shown for the case of charybdotoxin [43]. Also the fact that CeErg4, reported here, and CllErg1 [42] have the same amino acid sequence is not unusual.…”

Section: Discussionsupporting

confidence: 51%

Two Novel Ergtoxins, Blockers of K+-channels, Purified from the Mexican Scorpion Centruroides elegans elegans

et al. 2008

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Voltage-gated potassium channels of the ether-a-go-go related gene (ERG) family are implicated in many important cellular processes. Three such genes have been cloned (erg1, erg2 and erg3) and shown to be expressed in the central nervous system (CNS) of mammalians. This communication describes the isolation and characterization of two isoforms of scorpion toxin (CeErg4 and CeErg5, systematic nomenclature gamma-KTx1.7 and gamma-KTx1.8, respectively) that can discriminate the various subtypes of ERG channels of human and rat. These peptides were purified from the venom of the Mexican scorpion Centruroides elegans elegans. They contain 42 amino acid residues, tightly folded by four disulfide bridges. Both peptides block in a reversible manner human and rat ERG1 channels, but have no effect on human ERG2. They also block completely and irreversibly the rat ERG2 and the human ERG3 channels hence are excellent tools for the discrimination of the various sub-types of ion-channels studied.

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“…Combining the structure–activity profiles of ChTx [33,34], IbTx [5,8,19], IbTx-chimeras [12], and our previous IbTx derivative studies [2], a suitable thiol-ester ligation construct was designed, containing an orthogonally protected ‘spinster thiol’. Native chemical ligation requires a C-terminal peptide segment (Peptide B) to contain an N-terminal Cys (see Fig.…”

Section: Discussionmentioning

confidence: 99%

“…However, a single amino acid substitution may also have significant consequences on a toxin’s specificity, targeting and overall pharmacological behavior, in some cases rendering a toxin candidate biologically inactive (see Refs. [33,34]). Here, careful design based on comparative structural and pharmacological identity of similar peptide toxin sequences is an important aspect of successful peptide toxin bioengineering.…”

Section: Introductionmentioning

confidence: 99%

Synthesis of an iberiotoxin derivative by chemical ligation: A method for improved yields of cysteine-rich scorpion toxin peptides

et al. 2009

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Automated and manual solid phase peptide synthesis techniques were combined with chemical ligation to produce a 37-residue peptide toxin derivative of iberiotoxin which contained: (i) substitution of Val 16 to Ala, to facilitate kinetic feasibility of native chemical ligation, and; (ii) substitution of Asp 19 to orthogonally protected Cys-4-MeOBzl for chemical conjugate derivatization following peptide folding and oxidation. This peptide ligation approach increased synthetic yields approximately 12-fold compared to standard linear peptide synthesis. In a functional inhibition assay, the ligated scorpion toxin derivative, iberiotoxin V16A/D19-Cys-4-MeOBzl, exhibited 'native-like' affinity (K d = 1.9 nM) and specificity towards the BK Ca 2+ -activated K + Channel (K Ca 1.1). This was characterized by the rapid association and slow dissociation rates (k on = 4.59 × 10 5 M −1 s −1 ; k off = 8.65 × 10 −4 s −1 ) as determined by inhibition of macroscopic whole-cell currents of cloned human K Ca 1.1 channel. These results illustrate the successful application of peptide chemical ligation to improve yield of cysteine-rich peptide toxins over traditional solid phase peptide synthesis. Native chemical ligation is a promising method for improving production of biologically active disulfide containing peptide toxins, which have diverse applications in studies of ion-channel function.

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Mapping hydrophobic residues of the interaction surface of charybdotoxin

Cited by 25 publications

References 8 publications

Synthesis of charybdotoxin and of two N‐terminal truncated analogues

Synthesis of charybdotoxin and of two N‐terminal truncated analogues

Two Novel Ergtoxins, Blockers of K+-channels, Purified from the Mexican Scorpion Centruroides elegans elegans

Synthesis of an iberiotoxin derivative by chemical ligation: A method for improved yields of cysteine-rich scorpion toxin peptides

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