Reinvestigation of the biological activity of D-allo-ShK protein

Bobo Dang, Sandeep Chhabra, Michael William Pennington, Raymond S. Norton, Stephen B.H. Kent

Research output: Contribution to journalArticleResearchpeer-review

Abstract

ShK toxin from the sea anemone Stichodactyla helianthus is a 35-residue protein that binds to the Kv1.3 ion channel with high affinity. Recently we determined the X-ray structure of ShK toxin by racemic crystallography, in the course of which we discovered that D-ShK has a near-background IC50 value ~50,000 times lower than that of the L-ShK toxin. This lack of activity was at odds with previously reported results for an ShK diastereomer designated D-allo-ShK, for which significant biological activity had been observed in a similar receptor-blocking assay. As reported, D-allo-ShK was made up of D-amino acids, but with retention of the natural stereochemistry of the chiral side chains of the Ile and Thr residues, i.e. containing D-allo-Ile and D-allo-Thr along with D-amino acids and glycine. To understand its apparent biological activity, we set out to chemically synthesize D-allo-ShK and determine its X-ray structure by racemic crystallography. Using validated allo-Thr and allo-Ile, both L-allo-ShK and D-allo-ShK polypeptide chains were prepared by total chemical synthesis. Neither the L-allo-ShK nor the D-allo-ShK polypeptides folded, whereas both L-ShK and D-ShK folded smoothly under the same conditions. Re-examination of NMR spectra of the previously reported D-allo-ShK protein revealed that diagnostic Thr and Ile signals were the same as for authentic D-ShK. On the basis of these results, we conclude that the previously reported D-allo-ShK was in fact D-ShK, the true enantiomer of natural L-ShK toxin, and that the apparent biological activity may have arisen from inadvertent contamination with trace amounts of L-ShK toxin.

Original languageEnglish
Pages (from-to)12599-12605
Number of pages7
JournalJournal of Biological Chemistry
Volume292
Issue number30
DOIs
Publication statusPublished - 2017

Cite this

Dang, Bobo ; Chhabra, Sandeep ; Pennington, Michael William ; Norton, Raymond S. ; Kent, Stephen B.H. / Reinvestigation of the biological activity of D-allo-ShK protein. In: Journal of Biological Chemistry. 2017 ; Vol. 292, No. 30. pp. 12599-12605.
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abstract = "ShK toxin from the sea anemone Stichodactyla helianthus is a 35-residue protein that binds to the Kv1.3 ion channel with high affinity. Recently we determined the X-ray structure of ShK toxin by racemic crystallography, in the course of which we discovered that D-ShK has a near-background IC50 value ~50,000 times lower than that of the L-ShK toxin. This lack of activity was at odds with previously reported results for an ShK diastereomer designated D-allo-ShK, for which significant biological activity had been observed in a similar receptor-blocking assay. As reported, D-allo-ShK was made up of D-amino acids, but with retention of the natural stereochemistry of the chiral side chains of the Ile and Thr residues, i.e. containing D-allo-Ile and D-allo-Thr along with D-amino acids and glycine. To understand its apparent biological activity, we set out to chemically synthesize D-allo-ShK and determine its X-ray structure by racemic crystallography. Using validated allo-Thr and allo-Ile, both L-allo-ShK and D-allo-ShK polypeptide chains were prepared by total chemical synthesis. Neither the L-allo-ShK nor the D-allo-ShK polypeptides folded, whereas both L-ShK and D-ShK folded smoothly under the same conditions. Re-examination of NMR spectra of the previously reported D-allo-ShK protein revealed that diagnostic Thr and Ile signals were the same as for authentic D-ShK. On the basis of these results, we conclude that the previously reported D-allo-ShK was in fact D-ShK, the true enantiomer of natural L-ShK toxin, and that the apparent biological activity may have arisen from inadvertent contamination with trace amounts of L-ShK toxin.",
author = "Bobo Dang and Sandeep Chhabra and Pennington, {Michael William} and Norton, {Raymond S.} and Kent, {Stephen B.H.}",
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Reinvestigation of the biological activity of D-allo-ShK protein. / Dang, Bobo; Chhabra, Sandeep; Pennington, Michael William; Norton, Raymond S.; Kent, Stephen B.H.

In: Journal of Biological Chemistry, Vol. 292, No. 30, 2017, p. 12599-12605.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Reinvestigation of the biological activity of D-allo-ShK protein

AU - Dang, Bobo

AU - Chhabra, Sandeep

AU - Pennington, Michael William

AU - Norton, Raymond S.

AU - Kent, Stephen B.H.

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AB - ShK toxin from the sea anemone Stichodactyla helianthus is a 35-residue protein that binds to the Kv1.3 ion channel with high affinity. Recently we determined the X-ray structure of ShK toxin by racemic crystallography, in the course of which we discovered that D-ShK has a near-background IC50 value ~50,000 times lower than that of the L-ShK toxin. This lack of activity was at odds with previously reported results for an ShK diastereomer designated D-allo-ShK, for which significant biological activity had been observed in a similar receptor-blocking assay. As reported, D-allo-ShK was made up of D-amino acids, but with retention of the natural stereochemistry of the chiral side chains of the Ile and Thr residues, i.e. containing D-allo-Ile and D-allo-Thr along with D-amino acids and glycine. To understand its apparent biological activity, we set out to chemically synthesize D-allo-ShK and determine its X-ray structure by racemic crystallography. Using validated allo-Thr and allo-Ile, both L-allo-ShK and D-allo-ShK polypeptide chains were prepared by total chemical synthesis. Neither the L-allo-ShK nor the D-allo-ShK polypeptides folded, whereas both L-ShK and D-ShK folded smoothly under the same conditions. Re-examination of NMR spectra of the previously reported D-allo-ShK protein revealed that diagnostic Thr and Ile signals were the same as for authentic D-ShK. On the basis of these results, we conclude that the previously reported D-allo-ShK was in fact D-ShK, the true enantiomer of natural L-ShK toxin, and that the apparent biological activity may have arisen from inadvertent contamination with trace amounts of L-ShK toxin.

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DO - 10.1074/jbc.M117.793943

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