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. and Ulrich, Anne S. (2017) Orientation and location of the cyclotide kalata B1 in lipid bilayers revealed by solid-state NMR. Biophysical Journal, 112 4: 630-642. doi:10.1016/j.bpj.2016.12.040


Author 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.
Title Orientation and location of the cyclotide kalata B1 in lipid bilayers revealed by solid-state NMR
Journal name Biophysical Journal   Check publisher's open access policy
ISSN 1542-0086
0006-3495
Publication date 2017-02-28
Year available 2017
Sub-type Article (original research)
DOI 10.1016/j.bpj.2016.12.040
Open Access Status Not yet assessed
Volume 112
Issue 4
Start page 630
End page 642
Total pages 13
Place of publication St. Louis, MO, United States
Publisher Cell Press
Language eng
Subject 1304 Biophysics
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-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-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-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-NMR of uniformly labeled protein, and confirmed using solid-state P- and H-NMR. P-NMR relaxation data indicated a change in lipid headgroup dynamics induced by kalata B1. Changes in the H-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-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.
Formatted 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 19F-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 19F-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 19F-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 15N-NMR of uniformly labeled protein, and confirmed using solid-state 31P- and 2H-NMR. 31P-NMR relaxation data indicated a change in lipid headgroup dynamics induced by kalata B1. Changes in the 2H-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 19F-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.
Keyword Biophysics
Biophysics
Q-Index Code C1
Q-Index Status Provisional Code
Grant ID APP1084965
DE120103152
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: HERDC Pre-Audit
Institute for Molecular Bioscience - Publications
 
Versions
Version Filter Type
Citation counts: TR Web of Science Citation Count  Cited 1 times in Thomson Reuters Web of Science Article | Citations
Scopus Citation Count Cited 1 times in Scopus Article | Citations
Google Scholar Search Google Scholar
Created: Tue, 14 Mar 2017, 00:24:17 EST by System User on behalf of Learning and Research Services (UQ Library)