Host defence peptide plectasin targets bacterial cell wall precursor lipid II by a calcium-sensitive supramolecular mechanism

Antimicrobial resistance is a leading cause of mortality, calling for the development of new antibiotics. The fungal antibiotic plectasin is a eukaryotic host defence peptide that blocks bacterial cell wall synthesis. Here, using a combination of solid-state nuclear magnetic resonance, atomic force microscopy and activity assays, we show that plectasin uses a calcium-sensitive supramolecular killing mechanism. Efficient and selective binding of the target lipid II, a cell wall precursor with an irreplaceable pyrophosphate, is achieved by the oligomerization of plectasin into dense supra-structures that only form on bacterial membranes that comprise lipid II. Oligomerization and target binding of plectasin are interdependent and are enhanced by the coordination of calcium ions to plectasin’s prominent anionic patch, causing allosteric changes that markedly improve the activity of the antibiotic. Structural knowledge of how host defence peptides impair cell wall synthesis will likely enable the development of superior drug candidates.


Supplementary Figure 1. ssNMR chemical shift assignments of lipid II in complex with plectasin
A) Chemical shift assignments of Lipid II in complex with plectasin.Overlay of 2D 13 C 13 C PARIS-xy (red) and TOBSY 1 (blue) spectra, sensitive to rigid and mobile regions respectively.The PARIS spectrum was recorded at a magnetic field of 800 MHz ( 1 H frequency), 42 kHz MAS and a sample temperature of 280 K.The TOBSY spectrum was recorded at a magnetic field of 950 MHz ( 1 H frequency), 8 kHz MAS and a sample temperature of 305 K. B) Strip plots of 3D CONH (teal) and CaNH (orange) spectra used for the backbone assignments of the pentapeptide of Lipid II.The CONH was recorded at 700 MHz ( 1 H frequency), 60 kHz MAS and a sample temperature of 305 K.The CaNH was recorded with 50% NUS at 800 MHz ( 1 H frequency), 60 kHz MAS and a sample temperature of 305 K.In contrast to the 13 C detected experiments in A, both A4 and A5 were detectable in these dipolar coupling-based experiments due to the higher heteronuclear dipolar couplings with protons and the increased sensitivity of proton detection.

Supplementary Figure 2. 'FRET-like' DNP-ssNMR confirms that plectasin oligomerizes upon Lipid II binding in membranes.
A,B) Plectasin oligomerizes upon Lipid II binding in membranes.The plectasin-plectasin intermolecular interface was investigated using a FRET-like ssNMR experiment 2 .For this experimental setup, equimolar quantities of 13 C-and 15 N-labelled plectasin where mixed and then added to Lipid-II doped DOPC liposomes.Magnetization transferred from 15 N to 13 C can then only be established via intermolecular contacts between 13 C-and 15 N-labelled molecules.Since this transfer is short-ranged (approximately 0.5 nm), plectasin molecules need to tightly interact to detect 15 N to 13 C magnetization transfer.In order to exclude that intermolecular transfer relates to spurious aggregation due to crowding on the membrane surface, we acquired N(HH)C-spectra 3 using low (0.5 mol%) Lipid II concentration (see methods section for more details).As the low Lipid II concentration very strongly reduce the spectral sensitivity, we used DNP-signal enhancement 2,4,5 to compensate.AMUPol 6 was used as polarization agent.A) Enhancement by DNP of plectasin at low (0.5 mol%) lipid II concentration.Spectra were acquired at 400 MHz ( 1 H frequency), 8 kHz MAS and 100 K sample temperature with the microwave source turned off (red) or on (blue).Spectrum without DNP enhancement was scaled vertically by a factor of 51.53.B) 2D NHHC experiment acquired under DNP.Spectrum was acquired at 400 MHz ( 1 H frequency), 8 kHz MAS and 100 K sample temperature using a 1 H-1 H mixing time of 200 µs.C,D) While no experimental data on the histidine-sidechains were reported, previous studies in micelles 7 conjectured that the H18 would be protonated and would interact with the anionic pentapeptide of Lipid II.This is clearly refuted by our NMR studies in membranes, in which we can directly and clearly determine the protonation states.C) ssNMR 15 N-cp spectrum of 15 N-labeled plectasin in complex with lipid II in DOPG membranes.Spectrum was acquired at 800 MHz ( 1 H frequency), 60 kHz MAS and a sample temperature of 305 K.A long 1 H-15 N cross-polarization contact time of 4.5 ms was used in order to observe the unprotonated imidazole nitrogens.Literature chemical shifts of cationic bi-protonated histidine sidechains are shown in red 8 .The data unambiguously show that both H16 and H18 sidechains are neutral.D) ssNMR 1 H-detected 2D NH spectrum of plectasin in complex with Lipid II in DOPC membranes using a wide spectral width in the 15 N dimension displaying the unfolded peaks of the histidine sidechains.Spectrum was acquired at 1200 MHz ( 1 H frequency), 60 kHz MAS and a sample temperature of 290 K.

Supplementary Figure 3. Influence of membrane composition and bivalent cations on the plectasin-Lipid II complex.
ssNMR amide fingerprints and corresponding CSPs of plectasin in complex with Lipid II in either DOPC or anionic DOPG membranes and with or without the addition of Ca 2+ or Mg 2+ .Spectra were recorded at a magnetic field of 28.2 T (1200 MHz 1 H frequency) at 60 kHz MAS and a sample temperature of 305 K, with the exception of DOPG without calcium, which was recorded at 16.4 T (700 MHz 1 H frequency). A) Example overlay of NH fingerprint of 15 Nplectasin in complex with Lipid II in zwitterionic (DOPC, green) and anionic (DOPG, yellow) lipid vesicles.Note that H16e2 and H18e2 similar 15 N and 1 H chemical shifts in both spectra, but signals are shifted due to different spectral widths in the indirect ( 15 N) dimension and resulting differential spectral backfolding.B) CSPs of DOPC and DOPG membranes both without Ca 2+ .C) CSPs of addition of Ca 2+ in DOPG membranes.D) CSPs of DOPC and DOPG membranes both in the presence of Ca 2+ E) CSPs between addition of Mg 2+ or Ca 2+ in anionic DOPG membranes.F) CSPs between DOPC and TOCL membranes both in the presence of Ca 2+

Supplementary Figure 4. Controls for the growth of S. simulans 22 in bivalent cationdeprived medium
Experimental procedure is described in the methods section.Growth was followed by monitoring the optical density at 600 nm for 18 h in a plate reader.S. simulans grew well at all used Ca 2+ concentrations.

Table 2 .
Solution NMR chemical shifts of free plectasin (in ppm).

Table 3 .
ssNMR chemical shifts of plectasin bound to Lipid II in membranes (in ppm).