Plectasin, a fungal defensin, targets the bacterial cell wall precursor Lipid II.

Abstract

Host defense peptides such as defensins are components of innate immunity and have retained antibiotic activity throughout evolution. Their activity is thought to be due to amphipathic structures, which enable binding and disruption of microbial cytoplasmic membranes. Contrary to this, we show that plectasin, a fungal defensin, acts by directly binding the bacterial cell-wall precursor Lipid II. A wide range of genetic and biochemical approaches identify cell-wall biosynthesis as the pathway targeted by plectasin. In vitro assays for cell-wall synthesis identified Lipid II as the specific cellular target. Consistently, binding studies confirmed the formation of an equimolar stoichiometric complex between Lipid II and plectasin. Furthermore, key residues in plectasin involved in complex formation were identified using nuclear magnetic resonance spectroscopy and computational modeling.

Full text

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Plectasin, a fungal defensin, targets the bacterial cell wall
precursor Lipid II

Metazoan and fungal host defence peptides act as specific inhibitors of bacterial
peptidoglycan biosynthesis


Tanja Schneider1, Thomas Kruse5, Reinhard Wimmer3, Imke Wiedemann1, Vera Sass1, Ulrike
Pag1, Andrea Jansen1, Allan K Nielsen2, Per H Mygind2, Dorotea S Raventós2, Søren Neve2,
Birthe Ravn2, Alexandre MJJ Bonvin4, Leonardo De Maria2, Anders S Andersen2,5, Lora K
Gammelgaard2, Hans-Georg Sahl1 & Hans-Henrik Kristensen2*


1Institute for Medical Microbiology, Immunology and Parasitology – Pharmaceutical
Microbiology Section, University of Bonn, D-53115 Bonn, Germany

2Novozymes AS, DK-2880 Bagsvaerd, Denmark

3Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg
University, DK-9000 Aalborg, Denmark

4Department of Chemistry, Faculty of Science, Utrecht University, 3584 CH Utrecht, the
Netherlands

5Statens Serum Institut, 2300 Copenhagen S, Denmark

*To whom correspondence should be addressed: hahk@novozymes.com (HHK)

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Host defence peptides such as defensins are components of innate immunity and have
retained antibiotic activity throughout evolution. Their activity is thought to be due to
amphipathic structures which enable binding and disruption of microbial cytoplasmic
membranes. Contrary to this, we show here that plectasin, a fungal defensin, acts by
directly binding the bacterial cell wall precursor Lipid II. A wide range of genetic and
biochemical approaches identify cell wall biosynthesis as the pathway targeted by
plectasin. In vitro assays for cell wall synthesis identified Lipid II as the specific cellular
target. Consistently, binding studies confirmed the formation of an equimolar
stoichiometric complex between Lipid II and plectasin. Furthermore, key residues in
plectasin involved in complex formation were identified using NMR spectroscopy and
computational modelling.

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Plectasin is a 40 amino acid residue fungal defensin produced by the saprophytic ascomycete
Pseudoplectania nigrella (1). Plectasin shares primary structural features with defensins from
spiders, scorpions, dragonflies and mussels and folds into a cystine-stabilized alpha-beta-
structure (CSαβ). In vitro and in animal models of infection, plectasin is potently active
against drug-resistant gram-positive bacteria such as streptococci, while the antibacterial
spectrum of an improved derivative, NZ2114 (2), also includes staphylococci such as
methicillin-resistant Staphylococcus aureus (MRSA).
Here we set out to determine the molecular target and specific mechanism by which
plectasin kills bacteria. While many host defence peptides (HDPs) act on and disintegrate the
bacterial membrane several observations suggested that this is not the case for plectasin.
Growth kinetic measurements of the gram-positive bacterium Bacillus subtilis exposed
to plectasin clearly demonstrated that plectasin exhibited kinetic behaviour similar to cell
wall-interfering agents (e.g. vancomycin, penicillin and bacitracin) and not to the rapidly-lytic
membrane-active agents (e.g. polymyxin and novispirin) or non-lytic antibiotics with
replication (ciprofloxacin), transcription (rifampicin) or protein translation (kanamycin,
tetracycline) as their primary target (Fig. 1A). Consistent with this, killing kinetics indicated
that over a period of approximately one generation time (0.5 hours), treated cells were unable
to multiply, but remained viable (Fig. 1B insert), before the number of colony-forming units
decreased (Fig. 1B). Next, the effect of plectasin on macromolecular biosynthesis pathways
was investigated. The incorporation of radiolabelled isoleucine into protein and of thymidine
into nucleic acids was not affected whereas glucosamine, an essential precursor of bacterial
peptidoglycan, was no longer incorporated (Fig. 1C). Finally, treatment of B. subtilis with
plectasin induced severe cell shape deformations as visualized by phase contrast microscopy
(Fig. S1). These characteristics are all typical for compounds interfering with cell-wall
biosynthesis rather than for membrane disintegration (3, 4). Consistently, neither pore
formation as measured by K+ efflux (Fig. 1E) nor changes in membrane potential using TPP+
or DiBAC4 (Fig. S2AB), nor carboxy-fluorescein efflux from liposomes were detected (Fig.
S2C). Thus, despite its amphipathic nature, plectasin does not compromise membrane
integrity reducing the risk of unspecific toxicity.
We obtained further support for the cell-wall interfering activity using DNA
microarrays to compare the transcriptional responses of plectasin-treated cells with response
patterns obtained for a range of reference antibiotics. For both B. subtilis 168 and S. aureus
SG511, we found that the transcriptional profiles overlapped those of established cell wall

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biosynthesis inhibitors such as vancomycin and bacitracin (5, 6, 7, 8) (Fig. S3; Tables S1 and
S2).
The biosynthesis of bacterial cell walls requires a number of steps (9). Initially the N-
acetylmuramic acid-pentapeptide (MurNAc-pentapeptide), a major constituent of the cell wall
building block is produced in the cytoplasm as an UDP-activated precursor before it is
transferred onto a membrane carrier, bactoprenolphosphate (reaction I, Fig 2B). The resulting
membrane-anchored precursor Lipid I is then further modified to the structural cell wall
subunit, Lipid II (reaction II). In some gram-positive bacteria, Lipid II (Fig. 2A) is further
decorated by an interpeptide bridge (a pentaglycine peptide in case of S. aureus (10), reaction
III), before it gets translocated across the cytoplasmic membrane to the outside, where it is
incorporated into the peptidoglycan polymer through the activity of transglycosylases and
transpeptidases (reactions IV). We analysed the intracellular pool of cell wall precursors by
reverse HPLC and mass spectrometry and found accumulation of the soluble molecule, UDP-
MurNAc-pentapeptide in plectasin-treated cells (Fig. 1D), suggesting that one of the later,
membrane-associated or extracellular processes may be targeted by plectasin.
We then analysed the effect of plectasin on the membrane-bound steps of cell wall
biosynthesis in vitro. Cytoplasmic membranes with associated CW biosynthesis apparatus
were isolated and incubated with plectasin and radiolabelled substrates necessary for Lipid II
formation. Using thin layer chromatography and subsequent scintillation counting we found
the overall synthesis reaction to be strongly inhibited (Fig. 2C). For a more detailed analysis,
we cloned the individual CW biosynthesis genes from S. aureus, expressed them in
Escherichia coli and analysed the activity of the purified enzymes in the presence of plectasin
by measuring the amount of product formed. These enzymes included MraY (Fig. 2B,
reaction I); MurG (Fig. 2B, reaction II); FemXAB (Fig. 2B, reaction III); and PBP2 (Fig. 2B,
reaction IV). Whereas the MraY reaction was not affected by plectasin, we found the MurG,
FemX and PBP2 reactions to be inhibited in a dose-dependent fashion (Fig.2C). For these
three enzymes, Lipid I (MurG) or Lipid II (FemX and PBP2) are substrates and significant
inhibition of the reactions was only observed when plectasin was added in equimolar
concentrations with respect to Lipid I or Lipid II (Fig. 2C). Thus plectasin, similar to
glycopeptide antibiotics (e.g. vancomycin (11, 12)) and lantibiotics (13, 14), may form a
stoichiometric complex with the substrate rather than inhibiting the enzyme. To further
validate this we incubated either Lipid I or II with plectasin in various molar ratios and used
thin layer chromatography to analyse the migration behaviour. Free Lipid I and II as well as
free peptide were found to migrate to defined positions in the chromatogram, while the Lipid

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I/II-plectasin complex remained at the start point (Fig. 2D). Only at an equimolar ratio, was
neither free Lipid I/II nor free peptide detectable, indicating the formation of a 1:1
stoichiometric complex.
We further analysed the interaction of both Lipid I and II with plectasin using a
liposome system with membranes composed of phosphatidylcholine and Lipid II (0.2 or 0.5
mol%) and 14C-labelled plectasin. We found the maximum number of plectasin molecules that
bound to liposomes to approximately match the number of Lipid II molecules available on the
liposome surface (Fig. S4). Using Scatchard plot analysis, we determined an equilibrium
binding constant of 1.8x10-7 mol for Lipid II and 1.1x10-6 mol for Lipid I, suggesting that the
second sugar in Lipid II, the N-acetyl glucosamine contributes to the stability of the complex.

the membrane interface we measured chemical shifts changes for 15N-labelled plectasin.
To gain further insight into the structural nature of the plectasin/Lipid II interaction at
Heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectra
were measured either in solution or upon binding membrane-mimicking
dodecylphosphocholine (DPC) micelles (Fig. S5, Fig. S6). Fitting the binding data to a
Langmuir isotherm yielded a free enthalpy of binding ΔG=-27±1 kJ/mol (Fig. S7). Backbone
HN and N atoms of ten residues (G6, W8, D9, A31, K32, G33, G34, F35, V36 and C37),
which in the tertiary structure all locate to one end of plectasin, exhibited marked changes in
chemical shifts (Δδobs>0.15 ppm) (Fig. 3A; residues labelled yellow), suggesting an
orientation in which one end of plectasin specifically is located in the membrane interface.
To identify the residues on plectasin that bind Lipid II, we then titrated plectasin
bound to DPC with Lipid II. With increasing concentrations of Lipid II, another set of NMR
signals appeared and became stronger, whereas the NMR signals of apo-plectasin bound to
DPC micelles became weaker, until they disappeared at equimolar concentrations of plectasin
and Lipid II, supporting the 1:1 binding stoichiometry found by TLC. Addition of extra
plectasin to the mixture brought the signals of apo-plectasin forward again, further addition of
Lipid II to equimolarity led to the disappearance of the signals again. From a 3D-HNCA
spectrum we could assign backbone HN, N and Cα signals of the plectasin:Lipid II:DPC
complex . The strongest changes in chemical shift (Δδobs>0.22 ppm) were obtained for amino
acids F2, C4, D12, Y29, A31, G33, C37 and K38 (Fig. S5, Fig. S6). Most of these residues
localize in a coherent patch in close proximity to the residues affected by binding to DPC (Fig.
3B, residues labelled magenta). A31, G33 and C37 exert chemical shift-changes both upon
addition of DPC and Lipid II. To further verify this, site-saturated mutagenesis (where a given

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amino acid is changed to each of the other 19 natural amino acids) was carried out at all
positions in plectasin except the 6 cysteines. The mutant libraries were expressed in S.
cerevisiae and 400-600 transformants of each position tested for activity against S. aureus in a
plate overlay assay. No amino acid substitutions at positions D12, Y29 or G33 resulted in
activity against S. aureus, whereas only the very conservative mutations of A31 to G and K38
to R resulted in activity against S. aureus. At other amino acid positions not involved in DPC
or Lipid II binding, a wide range of non-homologous amino acid substitutions gave rise to
plectasin variants retaining antimicrobial activity.
To visualize the complex between Lipid II and plectasin, docking studies using the
GOLD and HADDOCK programs were performed (15, 16). In accordance with the NMR data,
evidence in favour of a primary binding site involving the interaction of the pyro-phosphate
moiety of Lipid II with the amide protons F2, G3, C4 and C37 of plectasin via hydrogen
bonding was obtained (Fig. 3C). Several of the other large chemical shift changes are present
in residues involved in secondary structure interactions (e.g. formation of beta-sheets), which
most likely undergo structural changes upon binding to the target. Taken together, these data
strongly support a model in which plectasin gains affinity and specificity through binding to
the solvent-exposed part of Lipid II while the hydrophobic part of plectasin is located in the
membrane interface. Thus, plectasin shares functional features with the lantibiotic nisin in that
for both peptides the pyrophosphate moiety is most relevant for binding of Lipid II, although
nisin inserts deeply into the membrane bilayer, forming pores and causing major
delocalization of Lipid II (17,18).
To test whether inhibition of CW biosynthesis is restricted to plectasin or represents a
general feature we tested a series of defensin peptides from other fungi, mollusc and
arthropods for Lipid II binding and inhibition of the overall Lipid II synthesis and FemX
reaction (Fig. S8A). Two fungal defensins, oryzeasin (from Aspergillus oryzea) and eurocin
(Eurotium amstelodami) did inhibit the enzymatic reactions and bind to Lipid II in
stoichiometric numbers as did the two defensins from invertebrates, lucifensin from maggots
of the blowfly Lucilia sericata and gallicin from the mussel Mytilus galloprovinciali (Fig.
S8BCD). In contrast, heliomicin from the tobacco budworm Heliothis virescens which share
the conserved cysteine pattern did not show affinity for Lipid II and had no activity in these
assays. These data clearly demonstrate that among the host defence peptides of eukaryotic
organisms specific inhibitors of CW biosynthesis can be found which directly target Lipid II,
“the bacterial Achilles’ heel” for antibiotic attack (19).

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Vancomycin, one of the very few remaining drugs for the treatment of multi-resistant
gram-positive infections, has been shown to predominantly bind the D-alanyl-D-alanine (D-
ala-D-ala) part of the pentapeptide in Lipid II (11) (Fig. 2A). However, high-level
vancomycin resistance has been observed in both enterococci (VRE) and staphylococci
(VRSA). Importantly, there is no cross-resistance between vancomycin and plectasin, and in
contrast to vancomycin, plectasin is not competitively inhibited by the presence of the D-ala-
D-ala ligand (Fig. S9). This further demonstrates that the primary interactions to Lipid II
differ between plectasin and vancomycin and taken together these results suggest that future
development of true cross-resistance between vancomycin and plectasin is unlikely.

Plectasin and its improved derivatives such as NZ2114 possess a range of features -
such as potent activity in vitro under physiological conditions and in animal models of
infection, low potential for unwanted toxicities, extended serum stability and in vivo half-life,
and cost-effective large-scale manufacturing – which combined with a validated microbial
target make it a promising lead for further drug development.

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Reference List

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8. T. Mascher, N. G. Margulis, T. Wang, R. W. Ye, J. D. Helmann, Mol. Microbiol. 50,
1591 (2003).

9. H. J. van Heijenoort, Microbiol. Mol. Biol. Rev. 71, 620 (2007).
10. T. Schneider et al., Mol. Microbiol. 53, 675 (2004).
11. P. E. Reynolds, Eur. J. Clin. Microbiol. Infect. Dis. 8, 943 (1989).
12. N. E. Allen, T. I. Nicas, FEMS Microbiol. Rev. 26, 511 (2003).
13. H. Brotz et al., Mol. Microbiol. 30, 317 (1998).
14. J. M. Willey, W. A. van der Donk, Annu. Rev. Microbiol. 61, 477 (2007).
15. G. Jones, P. Willett, R. C. Glen, A. R. Leach, R. Taylor, J. Mol. Biol. 267, 727 (1997).
16. C. Dominguez, R. Boelens, A. M. Bonvin, J. Am. Chem. Soc. 125, 1731 (2003).
17. S. T. Hsu et al., Nat. Struct. Mol. Biol. 11, 963 (2004).
18. H. E. Hasper et al., Science 313, 1636 (2006).
19. E. Breukink, B. de Kruijff, Nat. Rev. Drug Discov. 5, 321 (2006).
20. We thank Michaele Josten, Annette Hansen and Marianne R Markvardsen for expert
technical assistance and acknowledge the Carlsberg Research Center for use of the 800MHz
NMR spectrometer and the Obel Foundation for supporting the NMR laboratory at Aalborg
University. HGS acknowledges financial support by the German Research Foundation (SA
292/10-2 and SA 292/13-1), the BMBF (SkinStaph) and by the BONFOR programme of the
Medical Faculty, University of Bonn. AMJJB acknowledges financial support from the
Netherlands Organization for Scientific Research (VICI grant #700.56.442). ASA
acknowledges financial support from The Danish Research council for Technology and
Production (274-05-0435). Competing interest statement: Allan K Nielsen, Dorothea S
Raventós, Søren Neve, Birthe Ravn, Leonardo De Maria, Anders S Andersen & Hans-Henrik
Kristensen are employees of Novozymes. The authors declare they have no other competing

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financial interest. DNA microarray data can be accessed through ArrayExpress, accession
number: E-MTAB-60. NMR assignment of 1H, 15N and 13C atoms of plectasin have been
deposited in the BioMagResBank (accession no. 16739)


Supporting Online Material
Materials and Methods
Figs S1 to S9
Tables S1 to S2
References

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FIGURE LEGENDS
Fig. 1.
Effect of Plectasin on intact cells. (A) Classification of antimicrobial compounds using optical
density measurements. Growth kinetic measurements of B. subtilis exposed to plectasin or
various antibiotics with known cellular targets. 2-4 times the minimal inhibitory concentration
(MIC) of the respective compounds were used. Plectasin (black) falls into the cluster of cell
wall biosynthesis inhibiting antibiotics (red colours). (B) Killing kinetics of plectasin;
Staphylococcus simulans 22 treated with plectasin at 2 × MIC (open diamonds) and 4 x MIC
(squares); control without peptide (triangles). Insert shows a similar experiment with more
time points within the first 60 minutes demonstrating the absence of killing in the first 30 min
of treatment (C) Impact of plectasin on macromolecular biosynthesis in B. subtilis 168.
Incorporation of [14C]-thymidine into nucleic acids, of L-[14C]-isoleucine into protein and of
[3H]–glucosamine in cell wall was measured in untreated controls (squares) and plectasin
treated cells (open circles); glucosamine incorporation into cell wall material was selectively
inhibited. (D) Intracellular accumulation of the ultimate soluble cell wall precursor UDP-
MurNAc-pentapeptide in vancomycin-treated (dotted line) and plectasin-treated (dashed line)
cells of S. simulans 22. Cells were treated for 30 min with plectasin or vancomycin, which is
known to form a complex with Lipid II1. Treated cells were extracted with boiling water and
the intracellular nucleotide pool analyzed by reversed HPLC. UDP-MurNAc-pentapeptide
was identified by mass spectrometry using the negative mode and 1 mg/ml 6-aza-2-
thiothymine (in 50% (v/v) ethanol/20 mM ammonium citrate) as matrix; the calculated
monoisotopic mass is 1149.35; in addition to the singly-charged ion, the mono- and di-sodium
salts are detected. (E) Plectasin is unable to form pores in the cytoplasmic membrane of S.
simulans 22. Potassium efflux from living cells was monitored with a potassium-sensitive
electrode. Ion leakage is expressed relative to the total amount of potassium released after
addition of 1 µM of the pore forming lantibiotic nisin (100%, open diamonds). Plectasin was
added at 0.2 µM (triangles) and 1 µM (open triangles); controls without peptide antibiotics
(squares).

Fig. 2.
Inhibition of membrane associated cell wall biosynthesis steps. (A) Structure of the cell wall
precursor Lipid II. (B) The membrane-bound steps of cell wall precursor biosynthesis and

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bactoprenol (C55P) carrier cycling in staphylococci. Cell wall biosynthesis starts in the
cytoplasm with the formation of the soluble precursor UDP-MurNAc-pentapeptide (UDP-
MurNAc-pp). This precursor is linked to the membrane carrier bactoprenolphosphate (C55P)
by MraY yielding Lipid I (step I). Lipid II is formed by MurG which adds N-acetyl-
glucosamine (GlcNAc) (step II). When the interpeptide bridge, which only occurs in some
Gram-positive bacteria, is accomplished (step III), the monomeric peptidoglycan unit is
translocated across the cytoplasmic membrane to the outside and incorporated into the cell
wall (step IV). (C) Inhibition of membrane associated steps of cell wall biosynthesis by
plectasin. In all tests, plectasin was added in molar ratios of 0.1 to 1 with respect to the
amount of the appropriate lipid substrate C55P, Lipid I or Lipid II used in the individual test
system. The amount of reaction products synthesized in the absence of plectasin was taken as
100%. Product analysis was done by thin layer chromatography (see D) and subsequent
scintillation counting of stained and excised product-containing bands; radiolabelling was
based on [3H]-labelled C55P (for Lipid I), [14C]-GlcNAc for Lipid II and [14C]-glycine for
Lipid II-Gly1. Error bars represents +/- SD and the experiments were repeated at least three
times. Technical details on the assays, the cloning and purification of the enzymes are given
in Materials and Methods. (D) Estimation of the stoichiometry of plectasin:Lipid II binding.
Lipid II was incubated in the presence of plectasin at the molar concentration ratios indicated.
The stable complex of plectasin with the Lipid II remains at the application spot whereas both
components migrate to the sites indicated. At a molar ratio of 1:1 neither free Lipid II nor free
plectasin were observed. Data obtained with Lipid I were comparable (not shown).
Fig. 3.
NMR-based model of the plectasin/Lipid II-complex. (A) Surface representation of plectasin
with the residues showing significant chemical shift perturbations upon binding to DPC
micelles indicated in yellow. (B) Surface representation of plectasin with the residues
showing significant chemical shift perturbations upon Lipid II titration shown in magenta. (C)
Detailed view of the pyrophosphate binding pocket. In this proposed HADDOCK-generated
model the pyrophosphate moiety forms hydrogen bonds to F2, G3, C4 and C27 and the D-γ-
Glutamate of Lipid II forms a salt bridge with the N-terminus of plectasin and the side-chain
of His18.

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Figure 1. Effect of plectasin on intact cells
B
E
C
0.2 µM plectasin
contro
l
0
20
40
60
80
100
050100150200250300 [sec]
% potassium release
addition of
peptide
no peptide antibiotic
nisin [1 µM]
plectasin [1 x MIC]
plectasin [5 x MIC]
A
00
102030[min]
14C-isoleucine
0
0.5
1.0
1.5
2.0
2.5
CPM x 103
0
0.5
1.0
1.5
2.0
2.5
0102030[min]
CPM x 103
14C-thymidine
0
5
10
15
20
25
01020 30[min]
3H-glucosamine
CPM x 103
010 203040
[min]
101
102
103
104
105
106
107
108
109
CFU/ml
120 240
plectasin-
treated
untreated
D
UDP-MurNAc-
pentapeptide
[min]
0
10
2030
vancomycin-
treated
0
0.1
0.2
A260
m/z
1148.723
1170.624
1192.444
0
1000
2000
3000
4000
intensity [a.u.]
1100
1200
0123456
2x MIC
4x MIC
control
[h]
log cfu/ mL
9
8
4
5
6
7

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A
B
O
O
O
PO
O
O
P
O
O
O
2
8
L-Ala
D-Glu
L-Lys
D-Ala
D-Ala
O
O
HO
CH2
HO
NH
CO
CH3
HO
CH
CO
CH2
OH
NH
CO
CH3
H3C
bactoprenol
(C55)
pentapeptide-
sidechain
MurNAc
GlcNAc
Gly-Gly-Gly-Gly-Gly-
interpeptide-
bridge
plectasin
Lipid II
lipid II : plectasin ratio
1:0
1:0.1
1:0.251:0.5
1:1
1:2
0:1
C
D
Figure 2. Inhibition of membrane associated cell wall biosynthesis steps
UDP
UMP
UDP
II
MraY
MurG
FemXAB
PBP
I
I
III
IV
cytoplasm
peptidoglycan
P
P
P
P
P
P
P
P
P
P
P
UDP
MurNAc
GlcNAc
C55-P
Ppentaglycine-
interpeptidebridge
pentapeptide-
sidechain
Pi
10
20
30
40
50
60
70
80
90
100
product formed (%)
substrate
used
product
formed
overall
reaction
(I-II)
MraY
(I)
MurG
(II)
FemX
(III)
PBP2
(IV)
C55P C55P
lipid Ilipid IIlipid II
lipid IIlipid Ilipid IIlipid II-Gly1C55PP
plectasin :
substrate ratio
0.1:1
0.25:10.5:1 1:1 nisin
…
Lipid II/ plectasin
complex

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Figure 3. NMR-based model of the plectasin/Lipid II-complex