Structure, folding and stability of a minimal homologue from Anemonia sulcata of the sea anemone potassium channel blocker ShK

Bankala Krishnarjuna, Christopher A. MacRaild, Punnepalli Sunanda, Rodrigo A.V. Morales, Steve Peigneur, Jason Macrander, Heidi H. Yu, Marymegan Daly, Srinivasarao Raghothama, Vikas Dhawan, Satendra Chauhan, Jan Tytgat, Michael W. Pennington, Raymond S. Norton

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Peptide toxins elaborated by sea anemones target various ion-channel sub-types. Recent transcriptomic studies of sea anemones have identified several novel candidate peptides, some of which have cysteine frameworks identical to those of previously reported sequences. One such peptide is AsK132958, which was identified in a transcriptomic study of Anemonia sulcata and has a cysteine framework similar to that of ShK from Stichodactyla helianthus, but is six amino acid residues shorter. We have determined the solution structure of this novel peptide using NMR spectroscopy. The disulfide connectivities and structural scaffold of AsK132958 are very similar to those of ShK but the structure is more constrained. Toxicity assays were performed using grass shrimp (Palaemonetes sp) and Artemia nauplii, and patch-clamp electrophysiology assays were performed to assess the activity of AsK132958 against a range of voltage-gated potassium (KV) channels. AsK132958 showed no activity against grass shrimp, Artemia nauplii, or any of the KV channels tested, owing partly to the absence of a functional Lys-Tyr dyad. Three AsK132958 analogues, each containing a Tyr in the vicinity of Lys19, were therefore generated in an effort to restore binding, but none showed activity against any of KV channels tested. However, AsK132958 and its analogues are less susceptible to proteolysis than that of ShK. Our structure suggests that Lys19, which might be expected to occupy the pore of the channel, is not sufficiently accessible for binding, and therefore that AsK132958 must have a distinct functional role that does not involve KV channels.

Original languageEnglish
Pages (from-to)169–178
Number of pages10
JournalPeptides
Volume99
DOIs
Publication statusPublished - Jan 2018

Keywords

  • Cysteine-rich peptide
  • K channel
  • NMR spectroscopy
  • Sea anemone
  • Structure

Cite this

Krishnarjuna, Bankala ; MacRaild, Christopher A. ; Sunanda, Punnepalli ; Morales, Rodrigo A.V. ; Peigneur, Steve ; Macrander, Jason ; Yu, Heidi H. ; Daly, Marymegan ; Raghothama, Srinivasarao ; Dhawan, Vikas ; Chauhan, Satendra ; Tytgat, Jan ; Pennington, Michael W. ; Norton, Raymond S. / Structure, folding and stability of a minimal homologue from Anemonia sulcata of the sea anemone potassium channel blocker ShK. In: Peptides. 2018 ; Vol. 99. pp. 169–178.
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abstract = "Peptide toxins elaborated by sea anemones target various ion-channel sub-types. Recent transcriptomic studies of sea anemones have identified several novel candidate peptides, some of which have cysteine frameworks identical to those of previously reported sequences. One such peptide is AsK132958, which was identified in a transcriptomic study of Anemonia sulcata and has a cysteine framework similar to that of ShK from Stichodactyla helianthus, but is six amino acid residues shorter. We have determined the solution structure of this novel peptide using NMR spectroscopy. The disulfide connectivities and structural scaffold of AsK132958 are very similar to those of ShK but the structure is more constrained. Toxicity assays were performed using grass shrimp (Palaemonetes sp) and Artemia nauplii, and patch-clamp electrophysiology assays were performed to assess the activity of AsK132958 against a range of voltage-gated potassium (KV) channels. AsK132958 showed no activity against grass shrimp, Artemia nauplii, or any of the KV channels tested, owing partly to the absence of a functional Lys-Tyr dyad. Three AsK132958 analogues, each containing a Tyr in the vicinity of Lys19, were therefore generated in an effort to restore binding, but none showed activity against any of KV channels tested. However, AsK132958 and its analogues are less susceptible to proteolysis than that of ShK. Our structure suggests that Lys19, which might be expected to occupy the pore of the channel, is not sufficiently accessible for binding, and therefore that AsK132958 must have a distinct functional role that does not involve KV channels.",
keywords = "Cysteine-rich peptide, K channel, NMR spectroscopy, Sea anemone, Structure",
author = "Bankala Krishnarjuna and MacRaild, {Christopher A.} and Punnepalli Sunanda and Morales, {Rodrigo A.V.} and Steve Peigneur and Jason Macrander and Yu, {Heidi H.} and Marymegan Daly and Srinivasarao Raghothama and Vikas Dhawan and Satendra Chauhan and Jan Tytgat and Pennington, {Michael W.} and Norton, {Raymond S.}",
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Krishnarjuna, B, MacRaild, CA, Sunanda, P, Morales, RAV, Peigneur, S, Macrander, J, Yu, HH, Daly, M, Raghothama, S, Dhawan, V, Chauhan, S, Tytgat, J, Pennington, MW & Norton, RS 2018, 'Structure, folding and stability of a minimal homologue from Anemonia sulcata of the sea anemone potassium channel blocker ShK' Peptides, vol. 99, pp. 169–178. https://doi.org/10.1016/j.peptides.2017.10.001

Structure, folding and stability of a minimal homologue from Anemonia sulcata of the sea anemone potassium channel blocker ShK. / Krishnarjuna, Bankala; MacRaild, Christopher A.; Sunanda, Punnepalli; Morales, Rodrigo A.V.; Peigneur, Steve; Macrander, Jason; Yu, Heidi H.; Daly, Marymegan; Raghothama, Srinivasarao; Dhawan, Vikas; Chauhan, Satendra; Tytgat, Jan; Pennington, Michael W.; Norton, Raymond S.

In: Peptides, Vol. 99, 01.2018, p. 169–178.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Structure, folding and stability of a minimal homologue from Anemonia sulcata of the sea anemone potassium channel blocker ShK

AU - Krishnarjuna, Bankala

AU - MacRaild, Christopher A.

AU - Sunanda, Punnepalli

AU - Morales, Rodrigo A.V.

AU - Peigneur, Steve

AU - Macrander, Jason

AU - Yu, Heidi H.

AU - Daly, Marymegan

AU - Raghothama, Srinivasarao

AU - Dhawan, Vikas

AU - Chauhan, Satendra

AU - Tytgat, Jan

AU - Pennington, Michael W.

AU - Norton, Raymond S.

PY - 2018/1

Y1 - 2018/1

N2 - Peptide toxins elaborated by sea anemones target various ion-channel sub-types. Recent transcriptomic studies of sea anemones have identified several novel candidate peptides, some of which have cysteine frameworks identical to those of previously reported sequences. One such peptide is AsK132958, which was identified in a transcriptomic study of Anemonia sulcata and has a cysteine framework similar to that of ShK from Stichodactyla helianthus, but is six amino acid residues shorter. We have determined the solution structure of this novel peptide using NMR spectroscopy. The disulfide connectivities and structural scaffold of AsK132958 are very similar to those of ShK but the structure is more constrained. Toxicity assays were performed using grass shrimp (Palaemonetes sp) and Artemia nauplii, and patch-clamp electrophysiology assays were performed to assess the activity of AsK132958 against a range of voltage-gated potassium (KV) channels. AsK132958 showed no activity against grass shrimp, Artemia nauplii, or any of the KV channels tested, owing partly to the absence of a functional Lys-Tyr dyad. Three AsK132958 analogues, each containing a Tyr in the vicinity of Lys19, were therefore generated in an effort to restore binding, but none showed activity against any of KV channels tested. However, AsK132958 and its analogues are less susceptible to proteolysis than that of ShK. Our structure suggests that Lys19, which might be expected to occupy the pore of the channel, is not sufficiently accessible for binding, and therefore that AsK132958 must have a distinct functional role that does not involve KV channels.

AB - Peptide toxins elaborated by sea anemones target various ion-channel sub-types. Recent transcriptomic studies of sea anemones have identified several novel candidate peptides, some of which have cysteine frameworks identical to those of previously reported sequences. One such peptide is AsK132958, which was identified in a transcriptomic study of Anemonia sulcata and has a cysteine framework similar to that of ShK from Stichodactyla helianthus, but is six amino acid residues shorter. We have determined the solution structure of this novel peptide using NMR spectroscopy. The disulfide connectivities and structural scaffold of AsK132958 are very similar to those of ShK but the structure is more constrained. Toxicity assays were performed using grass shrimp (Palaemonetes sp) and Artemia nauplii, and patch-clamp electrophysiology assays were performed to assess the activity of AsK132958 against a range of voltage-gated potassium (KV) channels. AsK132958 showed no activity against grass shrimp, Artemia nauplii, or any of the KV channels tested, owing partly to the absence of a functional Lys-Tyr dyad. Three AsK132958 analogues, each containing a Tyr in the vicinity of Lys19, were therefore generated in an effort to restore binding, but none showed activity against any of KV channels tested. However, AsK132958 and its analogues are less susceptible to proteolysis than that of ShK. Our structure suggests that Lys19, which might be expected to occupy the pore of the channel, is not sufficiently accessible for binding, and therefore that AsK132958 must have a distinct functional role that does not involve KV channels.

KW - Cysteine-rich peptide

KW - K channel

KW - NMR spectroscopy

KW - Sea anemone

KW - Structure

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U2 - 10.1016/j.peptides.2017.10.001

DO - 10.1016/j.peptides.2017.10.001

M3 - Article

VL - 99

SP - 169

EP - 178

JO - Peptides

JF - Peptides

SN - 0196-9781

ER -