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Biosci Rep
2013 Jun 27;333:. doi: 10.1042/BSR20130052.
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Functional evolution of scorpion venom peptides with an inhibitor cystine knot fold.
Gao B
,
Harvey PJ
,
Craik DJ
,
Ronjat M
,
De Waard M
,
Zhu S
.
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The ICK (inhibitor cystine knot) defines a large superfamily of polypeptides with high structural stability and functional diversity. Here, we describe a new scorpion venom-derived K+ channel toxin (named λ-MeuKTx-1) with an ICK fold through gene cloning, chemical synthesis, nuclear magnetic resonance spectroscopy, Ca2+ release measurements and electrophysiological recordings. λ-MeuKTx-1 was found to adopt an ICK fold that contains a three-strand anti-parallel β-sheet and a 310-helix. Functionally, this peptide selectively inhibits the Drosophila Shaker K+ channel but is not capable of activating skeletal-type Ca2+ release channels/ryanodine receptors, which is remarkably different from the previously known scorpion venom ICK peptides. The removal of two C-terminal residues of λ-MeuKTx-1 led to the loss of the inhibitory activity on the channel, whereas the C-terminal amidation resulted in the emergence of activity on four mammalian K+ channels accompanied by the loss of activity on the Shaker channel. A combination of structural and pharmacological data allows the recognition of three putative functional sites involved in channel blockade of λ-MeuKTx-1. The presence of a functional dyad in λ-MeuKTx-1 supports functional convergence among scorpion venom peptides with different folds. Furthermore, similarities in precursor organization, exon-intron structure, 3D-fold and function suggest that scorpion venom ICK-type K+ channel inhibitors and Ca2+ release channel activators share a common ancestor and their divergence occurs after speciation between buthidae and non-buthids. The structural and functional characterizations of the first scorpion venom ICK toxin with K+ channel-blocking activity sheds light on functionally divergent and convergent evolution of this conserved scaffold of ancient origin.
Figure 1. Molecular cloning of λ-MK1(A) PCR strategy for isolating λ-MK1 cDNA from the M. eupeus venom gland. UTR, untranslated region; SP, signal peptide; PP, propeptide; MP, mature peptide; (B) Comparison of nucleotide and deduced amino acid sequences of λ-MK1 and its orthologous gene BmCa1. Mm, Mesobuthus martensii; Me, Mesobuthus eupeus. Exons and introns are shown by upper and lower letters, respectively. SP-, PP- and MP-coding regions and the corresponding amino acid sequences are shown in different colours. Identical nucleotide and non-identical amino acid sequences are shadowed in yellow and italicized/underlined, respectively. Three insertions in the intron regions are shadowed in grey and short repeats are shown in brown. The poly(A) signal (ATTAAA) is underlined once. BmCa-1 was cloned and sequenced in this work, whose sequence shows nine polymorphic sites different from the previously reported sequence (GenBank® accession number DQ206446), indicated in pink. Arrows indicate positions of primers and their sequences are provided in Supplementary Table S1 (available at http://www.bioscirep.org/bsr/033/bsr033e047add.htm).
Figure 2. Scorpion venom ICK peptides(A) Amino acid sequences of precursors. Hydrophobic residues in SPs and acidic and basic residues in PPs and MPs are shown in green, red and blue, respectively. Amino acids disrupted by one intron are underlined once. Six conserved cysteines are shadowed in yellow and identical residues to λ-MK1 in grey. JZTX-15 is a spider ICK peptide from Chilobrachys guangxiensis. Sequence source: JZTX-15 (ABY71684); BmCa-1 (AF419253; BoTx758 (ACJ23131); Hj1a (ADY39527); Opicalcine-1 (AAP73822); Opicalcin-2 (P60253); Hadrucalcin (ACC99422); ImKTx1 [21]; MCa (P60254); Imperatoxin A (P59868); (B) An NJ tree rooted with JZTX-15, a spider ICK peptide (accession No. EU233865), which was reconstructed by using the mature amino acid sequences in Figure 2(A). Two distinct clades are shown in different colours. Numbers above branches are bootstrap values and the scale bar indicates total amino acid divergence; (C) Gene structure similarity between λ-MK1 and opicalcine-1 [23]. 1 and 2 represent intron phases.
Figure 3. Oxidative refolding and identification of chemically synthetic λ-MK1 and λ-MK1a(A) RP-HPLC showing retention time (TR) difference between the reduced (R) and oxidized (O) peptides. C18 column was equilibrated with 0.1% (w/v) TFA (trifluoroacetic acid) and purified proteins were eluted from the column with a linear gradient from 0 to 60% acetonitrile in 0.1% (w/v) TFA within 40 min; (B) MALDI-TOF MS of the oxidized peptides. The two main peaks in each spectrum correspond to the singly and doubly protonated forms of these peptides.
Figure 4. CD spectra of λ-MK1, λ-MK1-GP and λ-MK1aPeptides are dissolved in H2O at a concentration of 0.3 mg/ml. Data are expressed as mean residue molar ellipticity (θ).
Figure 5. Evaluation of the activity of λ-MK1 on Ca2+ release channels/ryanodine receptors(A) [3H]-ryanodine binding assay. Specific [3H]-ryanodine binding on SR vesicles was measured at pCa 7 (left panel) and pCa 5 (right panel) in the presence of 5 nM [3H]-ryanodine. Control corresponds to the specific binding measured in the absence of toxin. In order to ascertain the functional state of the SR membrane preparation, we tested the effect of MCa on ryanodine binding in identical conditions; (B) Ca2+ release measurements. Heavy SR vesicles were actively loaded with Ca2+ by three additions of 20 μM (final concentration) of CaCl2 in the monitoring chamber. In these conditions, addition of λ-MK1 (1 μM final concentration) does not produce any Ca2+ release. In contrast, addition of 50 nM MCa induces strong Ca2+ release.
Figure 6. λ-MK1 and λ-MK1a differentially inhibits Kv channels expressed in Xenopus oocytes(A) Representative whole-cell current traces in control and peptide conditions are shown. The dotted line indicates the zero-current level. The asterisk marks steady-state current traces in the presence of 10 μM peptides. Traces shown are representative traces of 3 independent experiments (n=3); (B) Blocking effects of λ-MK1 and its mutants with carboxyl terminal modifications on five cloned Kv channels expressed in Xenopus oocytes. The peptide concentration used here is 10 μM and data are presented as mean±S.E. (n=3); (C) The dose-response curve of λ-MK1 on the Shaker channel obtained by plotting the percentage blocked current as a function of increasing toxin concentrations. Each point represents mean±S.E. (n=3). These data points were fitted according to the Hill equation; (D) Kinetics of inhibition and reversibility of λ-MK1 on the Shaker channel. Fast inhibition and reversibility of the inhibition upon washout is shown by open circles and black circles, respectively.
Figure 7. The dose–response curve of MCa on rKv1.1Each point represents mean±S.E. (n=3). The representative whole-cell current traces in control and different concentrations of peptide conditions are shown in the inset. The dotted line indicates the zero-current level.
Figure 8. 3D structure of λ-MK1a(A) Structure-based sequence alignment between λ-MK1a and MCa. Disulfide bridge connectivities and secondary structure elements (α-helix: cylinder; β-strand: arrow) were extracted from the coordinates of λ-MK1a (this work) and MCa (pdb entry 1C6W); (B) Superimposition of backbone heavy atoms (N, Cα and C) for a family of 20 lowest energy NMR structures; a ribbon representation with disulfide connectivities shown in a stick model. The two termini are labelled with N-ter and C-ter and the strands (β) and helix (3.10α) are labelled in red. For comparison, the structure of MCa is also shown.
Figure 9. The putative functional surface of λ-MK1(A) A possible interaction between λ-MK1 and the Kv channel, where Arg17, Tyr18 and Gly36/Pro37 are assumed to be implicated in direct binding to the channel pore; (B) Sequence alignment of the pore region of Drosophila Shaker and Kv1.1–Kv1.4 channels. Shaker-specific residues in the turret are italicized underlined once.
Figure 10. Bacterial ICK peptides(A) Schematic domain organization. Signal peptides were predicted by SignalP 3.0 HMM (http://www.cbs.dtu.dk/services/SignalP/). GenBank® accession numbers: HoHP (H. ochraceum hypothetical protein): YP_003267557; KrICK (K. racemifer ICK): ZP_06968211. The glycine-rich hinge region is boxed in red; (B) Multiple sequence alignment. Conserved amino acids between bacterial and scorpion ICK peptides are highlighted in yellow; (C), Structural models. Ribbon representations showing overall folds of each peptide with disulfide connectivities in ball and stick format. The models were obtained by comparative modeling and the target-template alignment is shown in Supplementary Figure S1 (available at http://www.bioscirep.org/bsr/033/bsr033e047add.htm).
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