title: Orientation and Location of the Cyclotide Kalata B1 in Lipid Bilayers Revealed by Solid-State NMR.
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-state19F-NMR of oriented bilayers in conjunction with the conformationally restricted amino acid L-3-(trifluoromethyl)bicyclopent-[1.1.1]-1-ylglycine as an orientation-sensitive19F-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-sensitive19F-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 by15N-NMR of uniformly labeled protein, and confirmed using solid-state31P- and2H-NMR.31P-NMR relaxation data indicated a change in lipid headgroup dynamics induced by kalata B1. Changes in the2H-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 the19F-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.
authors.affiliation: Institute of Biological Interfaces (IBG-2), Karlsruhe Institute of Technology, Karlsruhe, Germany.
authors.forename: Stephan L
authors.initials: SL
authors.lastname: Grage
authors.affiliation: School of Chemistry, Bio21 Institute, The University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Marc-Antoine
authors.initials: MA
authors.lastname: Sani
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Olivier
authors.initials: O
authors.lastname: Cheneval
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Sónia Troeira
authors.initials: ST
authors.lastname: Henriques
authors.affiliation: Institute of Biological Interfaces (IBG-2), Karlsruhe Institute of Technology, Karlsruhe, Germany.
authors.forename: Constantin
authors.initials: C
authors.lastname: Schalck
authors.affiliation: Institute of Biological Interfaces (IBG-2), Karlsruhe Institute of Technology, Karlsruhe, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Heinzmann
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Joshua S
authors.initials: JS
authors.lastname: Mylne
authors.affiliation: Chemistry Department, Taras Shevchenko National University of Kyiv, Kyiv, Ukraine; Enamine Ltd., Kyiv, Ukraine.
authors.forename: Pavel K
authors.initials: PK
authors.lastname: Mykhailiuk
authors.affiliation: Institute of Biological Interfaces (IBG-2), Karlsruhe Institute of Technology, Karlsruhe, Germany.
authors.forename: Sergii
authors.initials: S
authors.lastname: Afonin
authors.affiliation: Institute of High Technologies, Taras Shevchenko National University of Kyiv, Kyiv, Ukraine.
authors.forename: Igor V
authors.initials: IV
authors.lastname: Komarov
authors.affiliation: School of Chemistry, Bio21 Institute, The University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Frances
authors.initials: F
authors.lastname: Separovic
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: David J
authors.initials: DJ
authors.lastname: Craik
authors.affiliation: Institute of Biological Interfaces (IBG-2), Karlsruhe Institute of Technology, Karlsruhe, Germany; Institute of Organic Chemistry, Karlsruhe Institute of Technology, Karlsruhe, Germany. Electronic address: anne.ulrich@kit.edu.
authors.forename: Anne S
authors.initials: AS
authors.lastname: Ulrich
mesh_terms: D000595:Amino Acid Sequence; D047169:Cyclotides / Q000737:chemistry* / Q000378:metabolism*; D008051:Lipid Bilayers / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D032067:Oldenlandia / Q000378:metabolism; D011487:Protein Conformation
chemical_list: D047169:Cyclotides; D008051:Lipid Bilayers; C091930:kalata B1