iBet uBet web content aggregator. Adding the entire web to your favor.
iBet uBet web content aggregator. Adding the entire web to your favor.



Link to original content: https://pubmed.ncbi.nlm.nih.gov/18042681
Chemical synthesis and 1H-NMR 3D structure determination of AgTx2-MTX chimera, a new potential blocker for Kv1.2 channel, derived from MTX and AgTx2 scorpion toxins - PubMed Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Jan;17(1):107-18.
doi: 10.1110/ps.073122908. Epub 2007 Nov 27.

Chemical synthesis and 1H-NMR 3D structure determination of AgTx2-MTX chimera, a new potential blocker for Kv1.2 channel, derived from MTX and AgTx2 scorpion toxins

Cyril Pimentel  1 , Sarrah M'Barek, Violetta Visan, Stephan Grissmer, François Sampieri, Jean-Marc Sabatier, Hervé Darbon, Ziad Fajloun
Affiliations

Chemical synthesis and 1H-NMR 3D structure determination of AgTx2-MTX chimera, a new potential blocker for Kv1.2 channel, derived from MTX and AgTx2 scorpion toxins

Cyril Pimentel et al. Protein Sci. 2008 Jan.

Abstract

Agitoxin 2 (AgTx2) is a 38-residue scorpion toxin, cross-linked by three disulfide bridges, which acts on voltage-gated K(+) (Kv) channels. Maurotoxin (MTX) is a 34-residue scorpion toxin with an uncommon four-disulfide bridge reticulation, acting on both Ca(2+)-activated and Kv channels. A 39-mer chimeric peptide, named AgTx2-MTX, was designed from the sequence of the two toxins and chemically synthesized. It encompasses residues 1-5 of AgTx2, followed by the complete sequence of MTX. As established by enzyme cleavage, the new AgTx2-MTX molecule displays half-cystine pairings of the type C1-C5, C2-C6, C3-C7, and C4-C8, which is different from that of MTX. The 3D structure of AgTx2-MTX solved by (1)H-NMR, revealed both alpha-helical and beta-sheet structures, consistent with a common alpha/beta scaffold of scorpion toxins. Pharmacological assays of AgTx2-MTX revealed that this new molecule is more potent than both original toxins in blocking rat Kv1.2 channel. Docking simulations, performed with the 3D structure of AgTx2-MTX, confirmed this result and demonstrated the participation of the N-terminal domain of AgTx2 in its increased affinity for Kv1.2 through additional molecular contacts. Altogether, the data indicated that replacement of the N-terminal domain of MTX by the one of AgTx2 in the AgTx2-MTX chimera results in a reorganization of the disulfide bridge arrangement and an increase of affinity to the Kv1.2 channel.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Sequence comparison between MTX, AgTx2, and AgTx2-MTX. Chemical synthesis and disulfide bridged organization of AgTx2-MTX chimera. (A) Amino acid sequences (one-letter code) and half-cystine pairings of MTX and AgTx2. Half-cystine residues are numbered by order of appearance from the N to the C terminus. The relative positioning of secondary structures (helix and β strands of the β-sheet structure) is indicated for each peptide. Disulfide bridges are depicted by solid lines. For AgTx2-MTX, the amino acid sequence of the 39-mer is shown. The amino acid sequence of this chimera derived from AgTx2 and MTX are shaded in dark and light gray, respectively. (B) AgTx2-MTX at different stages of its chemical synthesis. HPLC profiles of the crude reduced peptide (left), crude peptide after oxidative folding (middle), and purified AgTx2-MTX (right). (C) Half-cystine pairings of the AgTx2-MTX chimera. Assignment of the half-cystine pairings was achieved by analysis of the peptides yielded by enzyme cleavage (trypsin and chymotrypsin) of AgTx2-MTX.
Figure 2.
Figure 2.
Pharmacological activity of AgTx2-MTX, AgTx2, and MTX. (A) Concentration-dependent inhibition curves of IKCa1 currents by AgTx2-MTX (□), AgTX2 (■), and MTX (●). Fits of the data yield IC50 values of 7.3 ± 0.6 nM (AgTx2-MTX), 2.2 ± 0.2 nM (MTX), and 1152 ± 156 nM (AgTx2) for IKCa1. (B) Concentration-dependent inhibition curves of Kv1.2 currents by AgTx2-MTX (□), AgTx2 (■), and MTX (●). Fits of the data yield IC50 values of 0.14 ± 0.01 nM (AgTx2-MTX), 0.51 ± 0.04 nM (MTX), and 26.8 ± 5.2 nM (AgTx2) for Kv1.2.
Figure 3.
Figure 3.
Statistical properties of AgTx2-MTX structure calculation. (A) Sequence of AgTx2-MTX and sequential assignments. Filled circles (●) represent 3JHN-Hα coupling constants ≥8 Hz and open circles (○) those ≤6 Hz. Collected sequential nOe are classified into strong, medium, and weak nOe, and are indicated by thick, medium, and thin lines, respectively. The last line indicates the secondary elements (extended regions). (B) nOe (left) and RMSD (right) distribution vs. sequence of AgTx2-MTX. Intraresidue nOe are in black, sequential nOe in dark gray, medium nOe in light gray, and long-range nOe in white. RMSD values for backbone and all heavy atoms are in black and gray, respectively.
Figure 4.
Figure 4.
Structural properties of AgTx2-MTX. (A) Stereo pair view of the best fit of 20 structures of AgTx2-MTX. (B) PyMOL ribbon drawing of the averaged minimized AgTx2-MTX structure (DeLano Scientific). The eight half-cysteine residues are numbered according to their positions in the AgTx2-MTX amino acid sequence.
Figure 5.
Figure 5.
Structural alignment of AgTx2 with AgTx2-MTX (left) and MTX with AgTx2-MTX (right). Only amino acids conserved between the toxins and in interaction with the channel are shown.
Figure 6.
Figure 6.
Important contacts (strong interaction) between AgTx2-MTX and Kv1.2 channel. Map detailing major molecular contacts between AgTx2-MTX and Kv1.2 channel (for clarity, not all the contacts are shown). The docking of AgTx2-MTX onto the Kv1.2 channel can be imagined by a 180° vertical rotation of AgTx2-MTX from right to left.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bartels, C., Xia, T.H., Billeter, M., Guntert, P., Wüthrich, K. The program XEASY for computer-supported NMR spectral analysis of biological macromolecules. J. Biomol. NMR. 1995;5:110–118. - PubMed
    1. Blanc, E., Sabatier, J.M., Kharrat, R., Meunier, S., El Ayeb, M., Van Reitschoten, J., Darbon, H. Solution structure of maurotoxin, a scorpion toxin from Scorpio maurus, with high affinity for voltage-gated potassium channels. Proteins. 1997;29:321–333. - PubMed
    1. Bontems, F., Roumestand, C., Gilquin, B., Ménez, A., Toma, F. Refined structure of charybdotoxin: Common motifs in scorpion toxins and insect defensins. Science. 1991;254:1521–1523. - PubMed
    1. Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 1998;54:905–921. - PubMed
    1. Darbon, H., Blanc, E., Sabatier, J.M. Animal toxins and potassium channels. In: Darbon H., Sabatier J.M., editors. Perspectives in drug discovery and design. Vol. 15. Kluwer/Dordrecht; The Netherlands: 1999. pp. 40–60.

Publication types