Fig. 4: Plectasin oligomerizes in membranes upon lipid II binding.
a, Confocal microscopy of GUVs doped with NBD-tagged lipid II shows that plectasin induces cluster formation. b, Plectasin oligomerization upon lipid II binding was probed in a Förster resonance energy transfer-like ssNMR set-up21,22 with an equimolar mixture of 15N- and 13C-labelled (‘mixed-labelled’) plectasin molecules. Left: a series of 1D DNP-ssNMR NHHC21 experiments with increasing 1H-1H magnetization transfer times applied to mixed-labelled plectasin in lipid II-doped liposomes show the presence of plectasin oligomers. Right (controls) The absence of signals without 15N-labelling or without lipid II show that this experiment specifically detects lipid II-bound oligomers. c, ITC data show that mutation of either H16 or H18 to an alanine strongly reduces lipid II binding affinity. d, A DNP-ssNMR 2D NHHC21 spectrum shows the involvement of histidine side chains in the oligomerization interface. His, histidine. e, The only histidine residues in plectasin, H16 and the conserved H18, are localized on opposite sides. f, Snapshots of a time-lapse HS-AFM video (Supplementary Video 4) following the oligomerization of plectasin in supported DOPC/DOPG lipid bilayers doped with 1% lipid II. The experiment was repeated three times (three independent samples; n = 3) with similar results. g, Same as f but in the presence of 1 mM Ca2+ (Supplementary Video 5). h, AFM height profiles show that plectasin forms a dynamic (Supplementary Video 6), loosely packed supra-structure (left) in the absence of Ca2+, while a uniform, densely packed supra-structure is formed in the presence of Ca2+. Images were taken from AFM measurements (f and g) after full coverage of the membranes by the plectasin supra-structures (Supplementary Video 7). The experiment was repeated three times (three independent samples; n = 3) with similar results. i, Cross-sections across images shown in h illustrate the markedly different behaviour of the supra-structures on the membrane surface with (in blue) and without (in red) Ca2+. Source data are provided.
