Insights into the Mechanism of Action of the Two-Peptide Lantibiotic Lacticin 3147

Article abstract of DOI:10.1021/jacs.7b04728

Lacticin 3147 is a two peptide lantibiotc (LtnA1 and LtnA2) that displays nanomolar activity against many Gram-positive bacteria. Lacticin 3147 may exert its antimicrobial effect by several mechanisms. Isothermal titration calorimetry experiments show that only LtnA1 binds to the peptidoglycan precursor lipid II, which could inhibit peptidoglycan biosynthesis. An experimentally supported model of the resulting complex suggests that the key binding partners are the C-terminus of LtnA1 and pyrophosphate of lipid II. A combination of in vivo and in vitro assays indicates that LtnA1 and LtnA2 can induce rapid membrane lysis without the need for lipid II binding. However, the presence of lipid II substantially increases the activity of lacticin 3147. Furthermore, studies with synthetic LtnA2 analogues containing either desmethyl- or oxa-lanthionine rings confirm that the precise geometry of these rings is essential for this synergistic activity.

Full text of DOI:10.1021/jacs.7b04728

Article
pubs.acs.org/JACS
Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX-XXX
Insights into the Mechanism of Action of the Two-Peptide Lantibiotic
Lacticin 3147
Alireza Bakhtiary,† Stephen A. Cochrane,§ Pascal Mercier,‡ Ryan T. McKay,† Mark Miskolzie,†
Clarissa S. Sit,¶ and John C. Vederas*,†
†Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada
§School of Chemistry and Chemical Engineering, Queens University Belfast, Belfast BT9 5AG, United Kingdom
‡National High Field NMR Centre, University of Alberta, Edmonton, Alberta T6G 2E1, Canada
¶Department of Chemistry, Saint Mary’s University, Halifax, Nova Scotia B3H 3C3, Canada
S
* Supporting Information
ABSTRACT: Lacticin 3147 is a two peptide lantibiotc
(LtnA1 and LtnA2) that displays nanomolar activity against
many Gram-positive bacteria. Lacticin 3147 may exert its
antimicrobial effect by several mechanisms. Isothermal
titration calorimetry experiments show that only LtnA1
binds to the peptidoglycan precursor lipid II, which could
inhibit peptidoglycan biosynthesis. An experimentally sup-
ported model of the resulting complex suggests that the key
binding partners are the C-terminus of LtnA1 and
pyrophosphate of lipid II. A combination of in vivo and in
vitro assays indicates that LtnA1 and LtnA2 can induce rapid
membrane lysis without the need for lipid II binding. However,
the presence of lipid II substantially increases the activity of lacticin 3147. Furthermore, studies with synthetic LtnA2 analogues
containing either desmethyl- or oxa-lanthionine rings confirm that the precise geometry of these rings is essential for this
synergistic activity.
Lantibiotics are grouped into Type-A and -B peptides, based
on their functional and structural features.12 Type-A lantibiotics
are elongated, with similar arrangements of their thioether
bridges. This group is subdivided into Type-A(I) and Type-
A(II). Type-A(I) lantibiotics contain several charged residues
and have a net negative or slightly positive charge, whereas
Type-A(II) lantibiotics are highly negatively charged. Type-B
lantibiotics are typically shorter than Type-A and adopt a
globular structure. Although these types differ considerably,
examples from all types have been shown to bind lipid II.
Nisin (1) is a Type-A(I) lantibiotic routinely used in the
food industry to protect against pathogenic Gram-positive
bacteria (Figure 1).13 Nisin exerts its antimicrobial effect by
pore formation on bacterial membranes, which is enhanced by
the presence of lipid II,14 and binds to lipid II through its N-
terminal lanthionine rings, forming a “pyrophosphate cage”.14d
Mersacidin (2) is a Type-B lantibiotic that also binds to lipid
II.15 2D-NMR experiments have shown that when lipid II is
added to mersacidin, it undergoes several chemical shift
perturbations in its C-terminus.15b There are also two
component lantibiotic systems wherein one peptide binds to
lipid II. These two component lantibiotics work synergistically
INTRODUCTION
■
The continued emergence of antibiotic-resistant bacteria
necessitates the discovery of novel antimicrobial compounds.1
In recent years, antimicrobial peptides (AMPs) have been the
vanguard of new antimicrobial agents.2 Bacteriocins are a class
of AMPs produced by bacteria that often display strong activity
against pathogenic bacterial strains.3 The lantibiotics are a
subclass of bacteriocins defined by their sulfur-containing rings,
which result from the 1,4-conjugate addition of cysteine to
dehydroalanine (lanthionine) or dehydrobutyrine (methyllan-
thionine) during their biosynthesis.4 Understanding the
mechanism of action of antimicrobial compounds is important,
as it can enable the rational design of more active and/or
synthetically more accessible analogues.5 For lantibiotics, this
can be achieved by chemical synthesis or genetic engineer-
ing.4−6 A significant number of known AMPs bind to the
peptidoglycan precursor lipid II. Peptidoglycan is structurally
essential and unique to bacteria, and many antibiotics target this
pathway.7 Lipid II binding AMPs can exert their antimicrobial
effect through a variety of mechanisms. This includes inhibition
of peptidoglycan biosynthesis (vancomycin, plectasin, and
teixobactin),8,9,2a disruption of the proton-motive-force (tride-
captin A1),10 and membrane lysis/pore formation (lanti-
biotics).11
Received: May 8, 2017
© XXXX American Chemical Society
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Figure 1. Bead structures of some lipid II-binding lantibiotics. Residues known or suspected to be involved in lipid II binding are light green.
Lanthionine, methyl lanthionine, and amino-vinyl-cysteine bridges are red. Type-A(I) = nisin (1), and Type-B = mersacidin (2). Haloduracin and
lacticin 3147 are two-component lantibiotics, in which Halα (3) and LtnA1 (5) are Type-B-like and bind to lipid II. Halβ (4) and LtnA2 (6) are
type-A(I) like but do not bind to lipid II. Synthetic oxa-LtnA2 (7) and desMe-LtnA2 (8) are also shown.
to kill bacteria and include the haloduracins16 (Halα (3) and β
(4)) and lacticin 3147 (LtnA1 (5) and LtnA2 (6)).17 Halα and
LtnA1 are Type-B-like lantibiotics with lanthionine bridging
patterns (B, C, and D rings) very similar to mersacidin.
Mutations in these areas have been shown to reduce
antimicrobial activity.18,19 Halβ and LtnA2 are both Type-
A(I)-like lantibiotics with overall charges of +1. The similarities
between mersacidin, Halα, and LtnA1 are suggestive of a
conserved lipid II binding motif.15,16
LtnA1 and LtnA2 operate in a 1:1 stoichiometry.17d Our group
has previously synthesized oxa- and desmethyl-lanthionine
analogues of LtnA2 (oxa-LtnA2 (7) and desMe-LtnA2 (8)),
wherein lanthionine sulfurs are replaced with oxygen or methyls
are missing from methyllanthionine residues, respectively.21,22
We also completed the first total syntheses of LtnA1 and
LtnA2.23 Mechanistic studies on other lipid II binding peptides
inspired us to study the mechanism of action of lacticin 3147
and the structure of the LtnA1 interaction with lipid II.10
Lacticin 3147 is produced by Lactococcus lactis subspecies
lactis DPC3147 and has broad-spectrum activity against Gram-
positive bacteria.17 Previous mechanistic studies revealed that
LtnA1 binds to lipid II on the surface of the cell membrane.20 It
has been proposed that this complex then recruits LtnA2, and
the resulting ternary complex inhibits cell wall synthesis and
causes pore formation. Previous activity assays have shown that
RESULTS AND DISCUSSION
■
Isothermal Titration Calorimetry. Isothermal titration
calorimetry (ITC) is commonly used to assess the lipid II
binding properties of antimicrobial peptides.24,25,10 We used
ITC to study the interactions between natural and synthetic
analogues of lacticin 3147 peptides, as well as synergistic
B
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Figure 2. ITC binding experiments with natural and synthetic lacticin 3147 analogues and phospholipid LUVs containing 1 mol % Gram-positive
lipid II. (A) Lipid II added to LtnA1. (B) LtnA1 added to LtnA2. (C) Lipid II added to 1:1 LtnA1:LtnA2. (D) LtnA1 added to desMe-LtnA2. (E)
LtnA1 added to oxa-LtnA2. (F) Lipid II added to 1:1 LtnA1:desMe-LtnA2. (G) Lipid II added to 1:1 LtnA1:oxa-LtnA2. Binding affinity (Kd) and
number of binding sites (N) are shown.
Figure 3. (A) Cartoon schematic of the assay used to assess membrane lysis of the lacticin 3147 analogues. (B) Relative fluorescence vs time graph
showing the effects of adding lacticin 3147 analogues to LUVs containing BCECF. Peptide concentrations are 5 μM. (C) Relative fluorescence vs
time graph showing the effects of adding lacticin 3147 analogues to LUVs doped with 1 mol % lipid II and containing BCECF. Peptide
concentrations are 100 nM.
mixtures, and Gram-positive lipid II. Gram-positive lipid II
often contains lysine in its pentapeptide, whereas Gram-
negative lipid II usually contains diaminopimelic acid. To better
mimic a Gram-positive bacterial membrane, 1 mol % lipid II
(containing a lysine residue) was incorporated into large
unilamellar vesicles (LUVs) for the ITC experiments. In these
LUVs, lipid II is symmetrically distributed between the inner
and outer leaflets and cannot translocate the artificial
membrane without a flippase. Therefore, the actual concen-
tration of lipid II present on the outer leaflet is 0.5 mol %.26
LtnA1 binds to membrane-embedded lipid II in a 2:1 ratio with
a binding affinity (Kd) of 0.9 μM, which is consistent with its
minimum inhibitory concentration (1 μM) (Figure 2A).
However, none of the LtnA2 analogues bind to lipid II (Figure
S1). Given the synergistic relationship between LtnA1 and
LtnA2, the binding interaction between these peptides was also
assessed by ITC. LtnA1 binds strongly to LtnA2 in a 1:1 ratio
with a Kd of 1.2 μM (Figure 2B). When lipid II LUVs were
added to a 1:1 mixture of LtnA1:LtnA2, the binding isotherm
changed (Figure 2C). The resulting isotherm is characteristic of
a heterotropic interaction, in which cooperative binding occurs
between a macromolecule (LtnA1) and two different ligands
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(LtnA2 and lipid II).27 Therefore, it is not accurate to measure
the number of binding sites or binding affinity by this method
as it only gives an average of the various possible binding states
between LtnA1, LtnA2, and lipid II embedded in the LUVs.
The binding of LtnA1 to synthetic LtnA2 analogues was also
tested, and it was found that LtnA1 binds weakly to desMe-
LtnA2 (Figure 2D) and does not bind to oxa-LtnA2 at all
(Figure 2E). These measurements are consistent with our
previous observations that desMe-LtnA2 has reduced syner-
gistic activity with LtnA1, but oxa-LtnA2 is not synergistic with
LtnA1.21,22 The binding of LtnA1 + des-LtnA2 to lipid II
(Figure 2F) and LtnA1 + oxa-LtnA2 to lipid II (Figure 2G)
were then analyzed. The resulting curves are very similar to
LtnA1 + LtnA2 + lipid II (Figure 2C). In the case of oxa-
LtnA2, this is surprising, given that it does not bind to LtnA1. It
is possible that upon formation of the LtnA1:lipid II complex,
oxa-LtnA2 is then able to bind to this structure. However, at
this stage, further experiments were required to determine how
these binding studies relate to antimicrobial activity.
on an agar plate, and grown overnight. The number of colony
forming units (CFUs) per mL were then counted and plotted
over the course of the 8 h experiment (Figure 4A and B). In a
In Vitro Membrane Lysis Assays. To link the ITC results
to the effect on cell membranes, a series of pore-formation
assays were performed using LUVs containing the pH-sensitive
dye 2′,7′-bis(2-carboxyethyl)-5-carboxy-fluorescein
(BCECF).28 In this assay, LUVs are constructed with an
internal pH of 8 and then placed in buffer at pH 6. If pore
formation occurs, there is a decrease in intra-vesicle pH, which
is observed as a decrease in BCECF fluorescence (Figure
3A).10,28 To assess the effect of membrane bound lipid II on
membrane lysis, LUVs with and without lipid II were
constructed. In LUVs lacking lipid II, no membrane lysis was
observed for any of the lacticin 3147 analogues at
concentrations up to 5 μM (Figure 3B). However, a 1:1
mixture of LtnA1:LtnA2 caused rapid membrane lysis. The
synergistic mixture of LtnA1:desMe-LtnA2 caused a smaller
degree of membrane lysis in vesicles lacking lipid II, however
no effect was observed for LtnA1:oxa-LtnA2. These results are
consistent with the ITC studies, which showed that oxa-LtnA2
does not bind to LtnA1. When lipid II was incorporated into
the LUVs, the concentration of 1:1 LtnA1:LtnA2 or 1:1
LtnA1:desMe-LtnA2 required for membrane lysis dropped
significantly to 100 nM (Figure 3C). Consistent with the
absence of lipid II binding found during the ITC studies, none
of the LtnA2 analogues caused membrane lysis. In contrast,
LtnA1, which was shown by ITC to bind to lipid II, causes
moderate membrane lysis at 100 nM. These results show that
although lacticin 3147 can cause membrane lysis without lipid
II, the presence of lipid II significantly enhances activity. An
anomaly observed during the ITC studies was the binding curve
for 1:1 LtnA1:oxa-LtnA2 + lipid II. Even though oxa-LtnA2
does not bind to LtnA1, the binding curve was nearly identical
to the synergistic mixture of the natural peptides + lipid II,
suggesting a similar interaction. However, the degree of
membrane lysis observed for 1:1 LtnA1:oxa-LtnA2 is identical
to LtnA1 alone, showing that the major interaction resulting in
the ITC binding curve is LtnA1 binding to lipid II. Having
linked lipid II binding to membrane lysis, we next sought to
relate this to antimicrobial activity.
Figure 4. Time-kill assays. Lactococcus lactis subsp. cremoris HP cells
treated with 250 nM (A) or 1 μM (B) of peptide and the number of
viable cells determined at different time points.
time-kill assay, bacteriostatic agents (e.g., chloramphenicol) are
easily identified, as the number of CFUs will remain constant
over the time-course of the experiment. Bactericidal agents kill
cells, therefore the number of CFUs will decrease over time if
cells are exposed to a bactericidal compound. Furthermore,
membrane lysing agents cause rapid cell death, with the number
of CFUs typically being reduced to 0 within 30 min. At both
250 nM and 1 μM, LtnA1 halted cell growth but did not
significantly reduce the number of CFUs over an 8 h period.
This shows that LtnA1 binding to lipid II exerts a bacteriostatic
effect. In contrast, LtnA2 is bactericidal. At 250 nM all cells are
killed over the 8 h time-course, however at 1 μM the time taken
to kill all cells is reduced to 30 min. This suggests that at higher
concentrations, LtnA2 alone can kill Gram-positive bacteria by
lysis of the cell membrane. In the in vitro membrane lysis assay,
LtnA2 showed no lytic activity against LUVs with or without
lipid II. Therefore, these artificial membranes either lack the
lipid composition required for LtnA2-induced membrane lysis
or LtnA2 binds to a membrane embedded biomolecule that is
not present in the LUVs. To confirm this, an in vivo membrane
lysis assay was required. The synergistic mixture of LtnA1 and
LtnA2 kills all cells within 10 min at 250 nM and 5 min at 1
μM, which is highly suggestive of rapid membrane lysis. These
results corroborate that the synergistic mixture of LtnA1 and
LtnA2 kills Gram-positive bacteria by rapid membrane lysis. To
confirm these findings in vivo, SYTOX Green membrane lysis
assays were performed.
Time-Kill Assays. Time-kill assays are often used to
determine if an antibiotic is bacteriostatic or bactericidal.10 In
these experiments, Lactococcus lactis subsp. cremoris HP cells
were incubated in the presence of set concentrations of lacticin
3147 peptides (1 μM or 250 nM) for 8 h. At certain time
points, an aliquot of bacterial suspension was removed, spread
In Vivo Membrane Lysis Assays. To assess the effect of
lacticin 3147 peptides on whole-cell membranes, in vivo
membrane lysis assays were performed using SYTOX Green
(Figure 5). SYTOX Green is a membrane-impermeable reagent
D
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a
Scheme 1. Total Synthesis of (E,E)-Farnesyl Lipid II (9)
Figure 5. Lactococcus lactis subsp. cremoris HP cells pretreated with
SYTOX Green were exposed to antimicrobial peptides (100 nM) and
the extent of membrane lysis visualized as an increase in fluorescence.
a
that penetrates cells with compromised cell membranes,
binding to nucleic acids and emitting a fluorescent signal.29
Lactococcus lactis subsp. cremoris HP cells were incubated with
SYTOX Green, and peptides were then added to a final
concentration of 100 nM. Nisin (1) is known to cause rapid
pore formation at this concentration and indeed showed rapid
membrane lysis in the SYTOX Green assay. LtnA1 did not
cause membrane lysis, which is consistent with the previous
experiments in this study. The synergistic mixture of LtnA1 and
LtnA2 caused immediate membrane lysis, although not to the
same extent as nisin. Gratifyingly, LtnA2, which did not cause
membrane lysis in the in vitro assay but showed bactericidal
kinetics consistent with a lytic peptide, caused membrane lysis
in this whole-cell experiment. This provides further evidence
that either the artificial membranes lack the lipid content
required for LtnA2 lysis or they lack a membrane embedded
biomolecule that LtnA2 binds to induce membrane lysis.
Solution NMR Derived Model of LtnA1:Lipid II
Complex. Having identified LtnA1 as a lipid II binding
peptide, we next sought to identify how it binds using NMR
experiments. Natural lipid II contains a long undecaprenyl
chain (C55) that is not very amenable to NMR studies, as it has
poor solubility and its multiple methylene units can cover
important signals from the peptide. Therefore, a shorter chain
version, (E,E)-farnesyl lipid II (9) was synthesized by adapting
previously published methodology.10 Glycosyl acceptor 10 was
prepared in 5 steps and glycosyl donor 11 in 4 steps from
commercially available starting materials.10 A TMSOTf-
catalyzed glycosylation, followed by protecting group manipu-
lation, phosphorylation of the MurNAc anomeric position, and
deprotection of the 2-(phenylsulfonyl)ethyl ester with DBU
gave acid 12 (Scheme 1). Coupling to deprotected Boc-
tetrapeptide 13 (Scheme S1) then produced disaccharidyl
pentapeptide 14 in 54% yield. Finally, deprotection of the
dibenzyl phosphate moiety, followed by coupling to CDI
activated (E,E)-farnesyl phosphate and HPLC purification
yielded (E,E)-farnesyl lipid II (9) in 15% isolated yield.
(a) TMSOTf, 4 Å MS, CH2Cl2, rt, 18 h, 61%. (b) (i) ZnCl2, AcOH/
Ac2O, rt, 24 h; (ii) Zn, THF/AcOH/Ac2O, rt, 24 h, 63% (two steps).
(c) (i) H2, Pd/C, MeOH, rt, 3 h; (ii) (iPr)2NP(OBn)2, tetrazole,
CH2Cl2, rt, 2 h; (iii) 30% H2O2 in THF, −78 °C to rt, 2 h, 84% (three
steps). (d) DBU, CH2Cl2, rt, 0.5 h, quant. (e) Boc-Tetrapeptide 13,
TFA/CH2Cl2, 2 h; NHS, EDCI.HCl, DMF, rt, 18 h, 54%. (f) (i) H2,
Pd/C, MeOH, rt, 2.5 h, (ii) CDI activated (E,E)-farnesyl phosphate,
DMF, rt, 4 d, (iii) NaOH, H2O/dioxane, 37°C, 2 h, 15% (three steps).
and not due to a solvent/dielectric effect from high
concentrations of peptide present, the same experiment was
repeated with LtnA2. In this instance, no change in the 31P
NMR of lipid II occurred (Figure S3). Chemical shift
perturbations also occurred in the amide protons of Ala1 and
D-Glu2 as well as Lys3-Hβ (Figure S4). The largest 1H-
chemical shift changes in LtnA1 were observed in the amide
N−H signals of His23, Glu24, Cys25, Met26, Trp28, Cys29,
and Lys30 (Figure S5). Seven intermolecular NOEs were also
found (Figure S6) between D-Glu2 (lipid II) and Asn15, Cys19
and Abu20 (LtnA1), as well as Lys3 (lipid II) and Asn15
(LtnA1). The chemical shift assignments and NOE peak list
from the LtnA1:lipid II complex were then used to generate a
model of the complex using CYANA (Figures 6 and S7).30
Based on the observed 31P chemical shift perturbations and
intermolecular NOEs, as well as the earlier work of Sahl et
al.,13d four loose artificial NOE restraints were introduced
involving residues Glu24 and Lys30 with both phosphate atoms
for relative anchoring of LtnA1 and lipid II. The resulting
CYANA generated model is consistent with all intermolecular
NOEs and chemical shift perturbations.
LtnA1 binds to the pyrophosphate and pentapeptide
moieties of lipid II (Figure 6A). Closer analysis of the structure
revealed the D-ring (res. 22−30) of LtnA1 forms a hydrophilic
groove that binds to the pyrophosphate on lipid II (Figure 6B).
The LtnA1:lipid II complex was analyzed in MacPyMol to
identify intermolecular interactions between LtnA1 and lipid II.
The amide protons of Glu24, Cys25, Met26, and Lys30 form
hydrogen bonds with the phosphate oxygen atoms in lipid II
(Figure 6C). Furthermore, the electron-deficient imidazole ring
on His23 forms a π-anion bond31 (π-clamp) with a P−O
oxygen anion in lipid II as well as a H-bond. Chemical shift
perturbations were found in all of these LtnA1 residues, further
supporting the CYANA generated LtnA1:lipid II complex.
Comparison to Other Lipid II Binding Lantibiotics.
Having identified that LtnA1 binds to the pyrophosphate of
lipid II, we next sought to compare this binding mechanism to
other lipid II-binding lantibiotics (Figure 7). The NMR
Extensive solvent screening identified 1:1:1
CD3OH:CD3CN:H2O as a suitable solvent for the NMR
studies. LtnA1 and (E,E)-farnesyl lipid II were both
1
characterized separately in this solvent system by H NMR,
1
1H−1H COSY, 1H−1H TOCSY, H−1H NOESY, and 31P
NMR (Tables S1−S2). The chemical shifts were then assigned
in a 1:1 mixture of LtnA1 and (E,E)-farnesyl lipid II (Tables
S3−S4). A significant chemical shift perturbation (∼1.0 ppm)
was observed in the pyrophosphate signals of lipid II when
LtnA1 was added (Figure S2). To ensure this was a real effect
E
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Figure 6. (A) Model of the complex formed between LtnA1 and (E,E)-farnesyl lipid II. LtnA1 is shown as a surface representation with hydrophobic
residues green and hydrophilic residues yellow. Lipid II is represented as a stick model. LtnA1 binds mainly to the pyrophosphate and pentapeptide
regions of lipid II. (B) The pyrophosphate binding pocket of LtnA1 as a surface representation. (C) Key binding interactions between the C-terminal
residues of LtnA1 and pyrophosphate of lipid II. H-bonds between the lipid II pyrophosphate and the amide protons of Glu24, Cys25, Met26, and
Lys30 were identified (yellow dash) as well as a possible π-anion interaction (turquoise dash) and H-bond between the lipid II pyrophosphate and
the imidazole ring of His23.
Figure 7. (A) NMR solution structure of the nisin-lipid II complex (1WCO). (B) Model of the LtnA1-lipid II complex and (C) lipid II docked into
the NMR solution structure of mersacidin bound to lipid II in DPC micelles (1MQZ). In (A−C) peptides are shown as a surface representation with
hydrophobic residues green and hydrophilic residues yellow, and lipid II is represented as a stick model. Nisin adopts an elongated structure and
binds to lipid II with its N-terminal residues, whereas both LtnA1 and mersacidin adopt a globular structure and bind to lipid II through their C-
terminal residues. (D) The nisin-lipid II pyrophosphate cage, (E) the LtnA1-lipid II cage, and (F) a model of the proposed mersacidin-lipid II
intermolecular bonds. In (D−F) the peptide backbone is shown a stick model wherein carbon = green, oxygen = red, nitrogen = blue, and sulfur =
yellow, and some side-chain residues have been included. The pyrophosphate moiety within lipid II is represented by spheres with the rest of lipid II
omitted for clarity, unless needed to show key intermolecular bonds. Nisin forms a tight pyrophosphate cage with several intermolecular hydrogen
bonds, whereas the LtnA1 pyrophosphate cage is looser, with less intermolecular H-bonds but with a π-clamping interaction. The mersacidin-lipid II
model suggests H-bonding between Glu-17 and both the pyrophosphate and GlcNAc moieties of lipid II and is supported by all experimental
evidence of mersacidin-lipid II binding to date.
solution structure of the nisin:lipid II complex was previously
determined in DMSO.13d This Type-A(I) lantibiotic adopts an
elongated structure in which the N-terminal residues bind to
lipid II (Figure 7A). In contrast, as a Type-B-like lantibiotic,
LtnA1 adopts a globular structure, with lipid II binding
occurring at the C-terminus (Figure 7B). The pyrophosphate
cage adopted by nisin (Figure 7D) is shallower than that
adopted by LtnA1 (Figure 7E), with binding occurring
exclusively through amide intermolecular hydrogen bonds. As
well as intermolecular hydrogen bonding, LtnA1 also uses an
interesting π-clamp interaction to bind to the lipid II
pyrophosphate. For both nisin and LtnA1, the major
interaction is with the lipid II pyrophosphate. The NMR
solution structure of mersacidin (2) was previously determined
in DPC micelles containing Gram-positive lipid II.14b However,
the NMR solution structure adopted by lipid II in this complex
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was not determinable. Mersacidin has significantly higher
affinity for lipid II than lipid I (which lacks GlcNAc)32 and has
increased activity in calcium enriched media.33 These findings
suggest that mersacidin may bind to both the pyrophosphate
and GlcNAc moieties in lipid II. In the absence of a structure of
the mersacidin:lipid II complex, lipid II analogue 9 was docked
into the NMR solution structure of mersacidin using AutoDock
Vina (Figure 7C and F).34 This Type-B lantibiotic adopts a
globular structure similar to LtnA1. In the resulting model, the
C-terminal residues of mersacidin that have previously been
shown to interact with lipid II are involved in lipid II binding.
There are predicted intermolecular H-bonds between the
amides of Dha16 and Glu17 and the pyrophosphate as well as
the carboxylate group of Glu17 and GlcNAc. Glu17 was
previously shown be very important in the antimicrobial activity
of mersacidin, with mutation of this residue to alanine
abolishing activity.18 Care must be taken when comparing the
structures of nisin, LtnA1, and mersacidin bound to lipid II, as
they were calculated in different solvents. Ideally these would
be calculated in the same solvent system for comparison,
however this is not possible due to the differing solubility’s of
the respective peptide:lipid II complexes.
rapid membrane lysis by pore formation. LtnA1 and Halα both
bind to lipid II on the surface of the cell membrane and then
direct their partner peptide to the membrane to kill bacteria.
This may allow for the delivery of different antimicrobial
compounds across the cell membrane, as has been shown for
other antimicrobial peptides.37 Although we now have a
detailed understanding of how lacticin 3147 interacts with
lipid II, the synergistic interaction between LtnA1 and LtnA2 at
the molecular level is less well understood. Further studies are
ongoing in this area.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.7b04728.
■
S
Additional ITC data, pore-formation assay results, time-
kill assays, SYTOX Green assays, NMR data, CYANA
data, and experimental procedures (PDF)
AUTHOR INFORMATION
Corresponding Author
*john.vederas@ualberta.ca
■
Haloduracin is another two-component lantibiotic, com-
posed of Type-B-like (Halα, (3)) and Type-A(I)-like (Halβ
(4)) peptides.16a Analogues to the lacticin 3147 peptides, Halα,
and Halβ operate synergistically in a 1:1 ratio to kill Gram-
positive bacteria. Previous studies have also shown that Halα
inhibits the transglycosylation reaction between lipid II and
penicillin-binding protein 1b (PBP1b) by binding to lipid II in a
2:1 ratio.35 These findings suggest a 2:2:1 Halα:Halβ:lipid II
complex is formed, which inhibits cell wall biosynthesis and
mediates pore formation. Previous studies on the nisin:lipid II
binding interaction by ITC showed a 1:1 binding ratio, however
further in vivo studies using fluorescently labeled lipid II
suggested a 2:1 nisin:lipid II ratio in the resulting complex
formed.36 Like Halα and nisin, our studies suggest that LtnA1
binds to lipid II in a 2:1 ratio. Furthermore, given the 1:1
synergistic activity of LtnA1 and LtnA2, our results suggest that
lacticin 3147 exerts its antimicrobial effect by a similar
mechanism to haloduracin by forming a 2:2:1 LtnA1:LtnA2:-
lipid II complex, that results in rapid membrane lysis by pore
formation.
ORCID
Stephen A. Cochrane: 0000-0002-6239-6915
John C. Vederas: 0000-0002-2996-0326
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Wayne Moffat, Brandon Findlay, and Gareth
Lambkin for assistance with NMR analyses, fluorescence
experiments, and biochemical assays, respectively. Alberta
Innovates Health Solutions and the Natural Sciences and
Engineering Research Council of Canada provided funding. We
thank The Natural Sciences and Engineering Research Council
of Canada, Alberta Innovates Health Solutions, and the
Wellcome Trust (110270/A/15/Z) for funding.
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