Orientation and Location of the Cyclotide Kalata B1 in Lipid Bilayers Revealed by Solid-State NMR

Stephan L. Grage, Marc-Antoine Sani, Olivier Cheneval, Sonia Troeira Henriques, Constantin Schalck, Ralf Heinzmann, Joshua S. Mylne, Pavel K. Mykhailiuk, Sergii Afonin, Igor V. Komarov, Frances Separovic, David J. Craik, Anne S. Ulrich

Research output: Contribution to journalArticle

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Abstract

Cyclotides are ultra-stable cyclic disulfide-rich peptides from plants. Their biophysical effects and medically interesting activities are related to their membrane-binding properties, with particularly high affinity for phosphatidylethanolamine lipids. In this study we were interested in understanding the molecular details of cyclotide-membrane interactions, specifically with regard to the spatial orientation of the cyclotide kalata B1 from Oldenlandia affinis when embedded in a lipid bilayer. Our experimental approach was based on the use of solid-state F-19-NMR of oriented bilayers in conjunction with the conformationally restricted amino acid L-3-(trifluoromethyl)bicyclopent-[1.1.1]-1-ylglycine as an orientation-sensitive F-19-NMR probe. Its rigid connection to the kalata B1 backbone scaffold, together with the well-defined structure of the cyclotide, allowed us to calculate the protein alignment in the membrane directly from the orientation-sensitive F-19-NMR signal. The hydrophobic and polar residues on the surface of kalata B1 form well-separated patches, endowing this cyclotide with a pronounced amphipathicity. The peptide orientation, as determined by NMR, showed that this amphipathic structure matches the polar/apolar interface of the lipid bilayer very well. A location in the amphiphilic headgroup region of the bilayer was supported by N-15-NMR of uniformly labeled protein, and confirmed using solid-state P-31- and H-2-NMR. P-31-NMR relaxation data indicated a change in lipid headgroup dynamics induced by kalata B1. Changes in the H-2-NMR order parameter profile of the acyl chains suggest membrane thinning, as typically observed for amphiphilic peptides embedded near the polar/apolar bilayer interface. Furthermore, from the F-19-NMR analysis two important charged residues, E7 and R28, were found to be positioned equatorially. The observed location thus would be favorable for the postulated binding of E7 to phosphatidylethanolamine lipid headgroups. Furthermore, it may be speculated that this pair of side chains could promote oligomerization of kalata B1 through electrostatic intermolecular contacts via their complementary charges.

Original languageEnglish
Pages (from-to)630-642
Number of pages13
JournalBiophysical Journal
Volume112
Issue number4
DOIs
Publication statusPublished - 28 Feb 2017
Externally publishedYes

Cite this

Grage, S. L., Sani, M-A., Cheneval, O., Henriques, S. T., Schalck, C., Heinzmann, R., ... Ulrich, A. S. (2017). Orientation and Location of the Cyclotide Kalata B1 in Lipid Bilayers Revealed by Solid-State NMR. Biophysical Journal, 112(4), 630-642. https://doi.org/10.1016/j.bpj.2016.12.040
Grage, Stephan L. ; Sani, Marc-Antoine ; Cheneval, Olivier ; Henriques, Sonia Troeira ; Schalck, Constantin ; Heinzmann, Ralf ; Mylne, Joshua S. ; Mykhailiuk, Pavel K. ; Afonin, Sergii ; Komarov, Igor V. ; Separovic, Frances ; Craik, David J. ; Ulrich, Anne S. / Orientation and Location of the Cyclotide Kalata B1 in Lipid Bilayers Revealed by Solid-State NMR. In: Biophysical Journal. 2017 ; Vol. 112, No. 4. pp. 630-642.
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abstract = "Cyclotides are ultra-stable cyclic disulfide-rich peptides from plants. Their biophysical effects and medically interesting activities are related to their membrane-binding properties, with particularly high affinity for phosphatidylethanolamine lipids. In this study we were interested in understanding the molecular details of cyclotide-membrane interactions, specifically with regard to the spatial orientation of the cyclotide kalata B1 from Oldenlandia affinis when embedded in a lipid bilayer. Our experimental approach was based on the use of solid-state F-19-NMR of oriented bilayers in conjunction with the conformationally restricted amino acid L-3-(trifluoromethyl)bicyclopent-[1.1.1]-1-ylglycine as an orientation-sensitive F-19-NMR probe. Its rigid connection to the kalata B1 backbone scaffold, together with the well-defined structure of the cyclotide, allowed us to calculate the protein alignment in the membrane directly from the orientation-sensitive F-19-NMR signal. The hydrophobic and polar residues on the surface of kalata B1 form well-separated patches, endowing this cyclotide with a pronounced amphipathicity. The peptide orientation, as determined by NMR, showed that this amphipathic structure matches the polar/apolar interface of the lipid bilayer very well. A location in the amphiphilic headgroup region of the bilayer was supported by N-15-NMR of uniformly labeled protein, and confirmed using solid-state P-31- and H-2-NMR. P-31-NMR relaxation data indicated a change in lipid headgroup dynamics induced by kalata B1. Changes in the H-2-NMR order parameter profile of the acyl chains suggest membrane thinning, as typically observed for amphiphilic peptides embedded near the polar/apolar bilayer interface. Furthermore, from the F-19-NMR analysis two important charged residues, E7 and R28, were found to be positioned equatorially. The observed location thus would be favorable for the postulated binding of E7 to phosphatidylethanolamine lipid headgroups. Furthermore, it may be speculated that this pair of side chains could promote oligomerization of kalata B1 through electrostatic intermolecular contacts via their complementary charges.",
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Grage, SL, Sani, M-A, Cheneval, O, Henriques, ST, Schalck, C, Heinzmann, R, Mylne, JS, Mykhailiuk, PK, Afonin, S, Komarov, IV, Separovic, F, Craik, DJ & Ulrich, AS 2017, 'Orientation and Location of the Cyclotide Kalata B1 in Lipid Bilayers Revealed by Solid-State NMR' Biophysical Journal, vol. 112, no. 4, pp. 630-642. https://doi.org/10.1016/j.bpj.2016.12.040

Orientation and Location of the Cyclotide Kalata B1 in Lipid Bilayers Revealed by Solid-State NMR. / Grage, Stephan L.; Sani, Marc-Antoine; Cheneval, Olivier; Henriques, Sonia Troeira; Schalck, Constantin; Heinzmann, Ralf; Mylne, Joshua S.; Mykhailiuk, Pavel K.; Afonin, Sergii; Komarov, Igor V.; Separovic, Frances; Craik, David J.; Ulrich, Anne S.

In: Biophysical Journal, Vol. 112, No. 4, 28.02.2017, p. 630-642.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Orientation and Location of the Cyclotide Kalata B1 in Lipid Bilayers Revealed by Solid-State NMR

AU - Grage, Stephan L.

AU - Sani, Marc-Antoine

AU - Cheneval, Olivier

AU - Henriques, Sonia Troeira

AU - Schalck, Constantin

AU - Heinzmann, Ralf

AU - Mylne, Joshua S.

AU - Mykhailiuk, Pavel K.

AU - Afonin, Sergii

AU - Komarov, Igor V.

AU - Separovic, Frances

AU - Craik, David J.

AU - Ulrich, Anne S.

PY - 2017/2/28

Y1 - 2017/2/28

N2 - Cyclotides are ultra-stable cyclic disulfide-rich peptides from plants. Their biophysical effects and medically interesting activities are related to their membrane-binding properties, with particularly high affinity for phosphatidylethanolamine lipids. In this study we were interested in understanding the molecular details of cyclotide-membrane interactions, specifically with regard to the spatial orientation of the cyclotide kalata B1 from Oldenlandia affinis when embedded in a lipid bilayer. Our experimental approach was based on the use of solid-state F-19-NMR of oriented bilayers in conjunction with the conformationally restricted amino acid L-3-(trifluoromethyl)bicyclopent-[1.1.1]-1-ylglycine as an orientation-sensitive F-19-NMR probe. Its rigid connection to the kalata B1 backbone scaffold, together with the well-defined structure of the cyclotide, allowed us to calculate the protein alignment in the membrane directly from the orientation-sensitive F-19-NMR signal. The hydrophobic and polar residues on the surface of kalata B1 form well-separated patches, endowing this cyclotide with a pronounced amphipathicity. The peptide orientation, as determined by NMR, showed that this amphipathic structure matches the polar/apolar interface of the lipid bilayer very well. A location in the amphiphilic headgroup region of the bilayer was supported by N-15-NMR of uniformly labeled protein, and confirmed using solid-state P-31- and H-2-NMR. P-31-NMR relaxation data indicated a change in lipid headgroup dynamics induced by kalata B1. Changes in the H-2-NMR order parameter profile of the acyl chains suggest membrane thinning, as typically observed for amphiphilic peptides embedded near the polar/apolar bilayer interface. Furthermore, from the F-19-NMR analysis two important charged residues, E7 and R28, were found to be positioned equatorially. The observed location thus would be favorable for the postulated binding of E7 to phosphatidylethanolamine lipid headgroups. Furthermore, it may be speculated that this pair of side chains could promote oligomerization of kalata B1 through electrostatic intermolecular contacts via their complementary charges.

AB - Cyclotides are ultra-stable cyclic disulfide-rich peptides from plants. Their biophysical effects and medically interesting activities are related to their membrane-binding properties, with particularly high affinity for phosphatidylethanolamine lipids. In this study we were interested in understanding the molecular details of cyclotide-membrane interactions, specifically with regard to the spatial orientation of the cyclotide kalata B1 from Oldenlandia affinis when embedded in a lipid bilayer. Our experimental approach was based on the use of solid-state F-19-NMR of oriented bilayers in conjunction with the conformationally restricted amino acid L-3-(trifluoromethyl)bicyclopent-[1.1.1]-1-ylglycine as an orientation-sensitive F-19-NMR probe. Its rigid connection to the kalata B1 backbone scaffold, together with the well-defined structure of the cyclotide, allowed us to calculate the protein alignment in the membrane directly from the orientation-sensitive F-19-NMR signal. The hydrophobic and polar residues on the surface of kalata B1 form well-separated patches, endowing this cyclotide with a pronounced amphipathicity. The peptide orientation, as determined by NMR, showed that this amphipathic structure matches the polar/apolar interface of the lipid bilayer very well. A location in the amphiphilic headgroup region of the bilayer was supported by N-15-NMR of uniformly labeled protein, and confirmed using solid-state P-31- and H-2-NMR. P-31-NMR relaxation data indicated a change in lipid headgroup dynamics induced by kalata B1. Changes in the H-2-NMR order parameter profile of the acyl chains suggest membrane thinning, as typically observed for amphiphilic peptides embedded near the polar/apolar bilayer interface. Furthermore, from the F-19-NMR analysis two important charged residues, E7 and R28, were found to be positioned equatorially. The observed location thus would be favorable for the postulated binding of E7 to phosphatidylethanolamine lipid headgroups. Furthermore, it may be speculated that this pair of side chains could promote oligomerization of kalata B1 through electrostatic intermolecular contacts via their complementary charges.

KW - ANTIMICROBIAL PEPTIDE PGLA

KW - CYCLIC CYSTINE KNOT

KW - PLANT CYCLOTIDES

KW - MEMBRANES

KW - SPECTROSCOPY

KW - F-19-NMR

KW - POLYPEPTIDE

KW - PHOSPHATIDYLCHOLINES

KW - OPTIMIZATION

KW - RESONANCE

U2 - 10.1016/j.bpj.2016.12.040

DO - 10.1016/j.bpj.2016.12.040

M3 - Article

VL - 112

SP - 630

EP - 642

JO - Biophysical Journal

JF - Biophysical Journal

SN - 0006-3495

IS - 4

ER -

Grage SL, Sani M-A, Cheneval O, Henriques ST, Schalck C, Heinzmann R et al. Orientation and Location of the Cyclotide Kalata B1 in Lipid Bilayers Revealed by Solid-State NMR. Biophysical Journal. 2017 Feb 28;112(4):630-642. https://doi.org/10.1016/j.bpj.2016.12.040