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

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.

A kinked antimicrobial peptide from Bombina maxima. II. Behavior in phospholipid bilayers.

The preceding contribution by Toke et al. has studied the structure of the cationic antimicrobial peptide maximin-4 in detergent micelles and in organic solvent, revealing a different kink angle and side-chain interactions in the two different environments. Here, we have examined the same peptide in lipid bilayers using oriented circular dichroism (OCD) and solid-state (15)N nuclear magnetic resonance (NMR) in aligned samples. OCD showed that maximin-4 is helical and adopts an oblique alignment in the membrane, and lacks the characteristic realignment response that is often observed for amphipathic α-helical peptides at a peptide:lipid ratio between 1:100 and 1:20. Solid-state (15)N-NMR experiments suggest that maximin-4 also remains unaffected by lipid charge and temperature. Analyzing (15)N labels in positions Ala12, Ala13, and Leu14, an oblique tilt angle of the N-terminal helix of ~130° relative to the membrane normal was found, in good agreement with the amphiphilic profile of this segment. An additional constraint at Ala22 in the C-terminal segment is found to be compatible with a continuous α-helix, but unfavorable side-chain interactions make this solution unlikely. Instead, a kink at Gly16 seems fully compatible with all known constraints and with the biophysical expectations in the membrane-bound state, given the liquid-state NMR structures. It thus seems that the flexible kink in maximin-4 allows the two helical segments to adjust to the local environment. The irregular amphiphilic profile and the resulting versatility in shape might explain why maximin-4 lacks the realignment response that has been characteristically observed for many related frog peptides forming straight amphipathic α-helices.