NMR solution structure of the precursor for carnobacteriocin B2,
an antimicrobial peptide from
Carnobacterium piscicola
Implications of the a-helical leader section for export and inhibition of type IIa
bacteriocin activity
Tara Sprules
1
, Karen E. Kawulka
1
, Alan C. Gibbs
2
, David S. Wishart
2
and John C. Vederas
1
1
Department of Chemistry and
2
Faculty of Pharmacy, University of Alberta, Edmonton, AB, Canada
Type IIa bacteriocins, which are isolated from lactic acid
bacteria that are useful for food preservation, are potent
antimicrobial peptides with considerable potential as
therapeutic agents for gastrointestinal infections in mam-
mals. They are ribosomally synthesized as precursors with
an N-terminal leader, typically 18–24 amino acid residues
in length, which is cleaved during export from the produ-
cing cell. We have chemically synthesized the full precursor
of carnobacteriocin B2, precarnobacteriocin (preCbnB2),
which has a C-terminal amide rather than a carboxyl, and
also produced preCbnB2(1–64), which is missing two
amino acid residues at the C-terminus (Arg65 and Pro66),

tide synthesis; precarnobacteriocin B2.
Bacteriocins are potent antimicrobial peptides secreted by
bacteria. Those produced by lactic acid bacteria are the
focus of extensive studies because of their potential
application as nontoxic food preservatives, as well as their
possible therapeutic uses in both human and veterinary
medicine [1–5]. Nisin is approved in over 80 countries as
a food additive [6]. Most bacteriocins from lactic acid
bacteria are synthesized as prepeptides which undergo a
variety of post-translational modifications, ranging from
extensive formation of dehydro residues and lanthionine
bridges in the case of lantibiotics (e.g. nisin A) to simple
cleavage of a leader peptide and export across the cell
membrane. These antimicrobial compounds are divided
into classes according to their structural characteristics.
Type IIa bacteriocins are single peptides characterized by
a conserved YGNGVXC motif in the N-terminus, with
the cysteine involved in a disulfide bridge, and are
otherwise unmodified except for cleavage of the leader
from their precursor (Table 1). They show potent activity
against a number of potential Gram-positive food
spoilage and pathogenic bacteria, e.g. Listeria monocyto-
genes, but display no toxicity toward humans or other
eukaryotes. We reported purification and the primary
structure of the first member of this class, leucocin A [7],
and in the meantime over 20 such compounds have been
identified. These heat-stable, cationic peptides typically
have 37–48 amino acid residues. The solution structures
of three type IIa bacteriocins have been determined by
NMR methods: leucocin A [8], carnobacteriocin B2

encode bacteriocin and immunity proteins, which protect
the producer strain from attack by its own antimicrobial
peptides, genes that encode ATP-binding cassette (ABC)
transporter proteins are usually found in these bacteriocin
operons [5]. ABC transporters are a family of large
transmembrane proteins responsible for the ATP-driven
transport of a variety of compounds ranging from ions to
oligosaccharides and proteins. The ABC proteins associ-
ated with type IIa bacteriocin production have an extra
N-terminal cysteine protease domain, which is responsible
for cleavage of the leader peptide after the double glycine
motif [16,17]. This cleavage occurs on the cytoplasmic side
of the membrane during export of the bioactive peptide
[18]. The leader peptide is undoubtedly recognized by the
ABC protein responsible for export and processing. In the
case of carnobacteriocin B2 (CbnB2), a cationic thermo-
stable type IIa bacteriocin produced by Carnobacterium
piscicola LV17B [19], the additional 18 amino acids in the
leader peptide of precarnobacteriocin B2 (preCbnB2) also
render it  125 times less active than the mature peptide
[14]. To help determine the structural basis of the
inhibition of the antimicrobial activity of CbnB2 by the
leader and to assist future analysis of the interaction of
preCbnB2 with its ABC transporter protease, it is
essential to establish the preferred geometry of the
precursor. We now report the chemical synthesis, bio-
chemical production, and solution structure of preCbnB2,
and compare it with the structure of the mature
bacteriocin, CbnB2.
Materials and methods

hexafluorophosphate as activation agent, and N-methyl-
pyrrolidinone as solvent. Test cleavages were performed
after every five-residue coupling, and the desired product
was confirmed by MALDI-TOF MS. Peptide was cleaved
from the resin with a mixture of 87.5% trifluoroacetic acid,
5% phenol, 5% water, 2.5% dithiothreitol, and 2.5%
anisole for  90 min at 20 °C. The filtrate from the cleavage
reactions was collected, combined with trifluoroacetic acid
washes (3 · 2 min, 1 mL), and concentrated in vacuo.Cold
diethyl ether ( 15 mL) was added to precipitate the crude
cleaved peptide. Disulfide bond formation was achieved by
Table 1. Comparison of the amino acid sequence of selected type IIa bacteriocins. Conserved hydrophobic residues within the leader peptide are
italicized, and hydrophilic residues are underlined. The YGNGVXC motif is highlighted in bold text.
Bacteriocin Leader peptide Mature peptide Reference
PreCbnB2
MNSVKELNVKEMKQLHGG VNYGNGVSCSKTKCSVNWGQAFQERYTAGINSFVSGVASGAGS
IGRRP
[19]
Sakacin G
MKNAKSLTIQEMKSITGG KYYGNGVSCNSHGCSVNWGQAWTCGVNHLANGGHGVC [20]
Plantaricin 423
MMKKIEKLTEKEMANIIGG KYYGNGVTCGKHSCSVNWGQAFSCSVSHLANFGHGKC [21]
Piscicolin 126
MKTVKELSVKEMQLTTGG KYYGNGVSCNKNGCTVDWSKAIGIIGNNAAANLTTGGAAGWNKG [22]
CbnBM1 MKSVKELNKKEMQQINGG AISYGNGVYCNKEKCWVNKAENKQAITGIVIGGWASSLAGMGH [19]
Leucocin A
MMNMKPTESYEQLDNSALEQVVGG KYYGNGVHCTKSGCSVNWGEAFSAGVHRLANGGNGFW [7]
Pediocin
MKKIEKLTEKEMANIIGG KYYGNGVTCGKHSCSVDWGKATTCIINNGAMAWATGGHQGNHKC [23,24]
Mesentericin

]
2
SO
4
, or alternatively
[
15
NH
4
]
2
SO
4
and
D
-[U-
13
C]glucose (99% isotopic purity;
Cambridge Isotope Laboratories, Woburn, MA, USA),
were used as sole nitrogen and carbon sources. Recom-
binant protein production was induced with 0.3 m
M
isopropyl thio-b-
D
-galactoside when A
600
of the cell culture
reached 0.5. The culture was incubated for a further 3 h at
37 °C, and the cells were harvested by centrifugation. The
cell pellet was resuspended in column buffer (20 m

culture. The target peptide was cleaved from MBP with
Factor Xa (Borean Biologics Aps, Aarhus, Denmark) in
20 m
M
Tris/HCl/100 m
M
NaCl/1 m
M
CaCl
2
[0.01 mgÆ(mg
fusion protein)
)1
] overnight at room temperature. The
resulting peptide was separated from MBP on a C18
column (Waters PrepPak), with a 20 minute gradient of
20–60% acetonitrile in water with 0.1% trifluoroacetic acid.
The target peptide was eluted at 28% acetonitrile. Fractions
containing this peptide were combined and lyophilized.
Typical yield is 4–6 mg per litre of initial fermentation. MS
analysis (see below) of the MBP fusion protein was in
agreement with that expected (mass 48 300 Da) for unlabe-
led protein. However, the MS of the target peptide showed
that the two C-terminal amino acid residues (Arg65 and
Pro66) were absent [observed 6739.2 ± 0.8 for unlabeled
peptide; calculated 6738.5 for preCbnB2(1–64) missing the
two C-terminal amino acids; calculated 6991.8 for preC-
bnB2 having all 66 residues]. The mass spectra of
15
N-labeled preCbnB2(1–64) derived from [

Voyager Elite instrument in the positive ion mode with an
acceleration voltage of 20 kV using a nitrogen laser
(k ¼ 337 nm). Samples were prepared using a-cyano-4-
hydroxycinnamic acid (Aldrich) or sinapinic acid (Aldrich)
as a matrix, and fixed to a gold or stainless-steel target
before analysis. The instrument was calibrated daily before
each experiment using apomyoglobin [MH
+
¼ 16 952.56]
and trypsinogen [MH
+
¼ 23 981.9] as standards for MBP
fusion proteins and insulin [MH
+
¼ 5734.59] and insulin
chain B [MH
+
¼ 3496.96] for peptides.
CD spectroscopy
All CD measurements were performed by R. Luty (Depart-
ment of Biochemistry, University of Alberta) on a Jasco
J-720 spectrophotometer equipped with
JASCO J
700 soft-
ware. A thermally controlled quartz cell with a 0.02 cm path
length over 180–250 nm was used. CD spectra of preCbnB2
at different concentrations and varying the solvent from 0%
to 70% trifluoroethanol in water were collected at 25 °C.
Data were collected every 0.05 nm and were the average of
eight scans. The bandwidth was set at 1.0 nm and the

N HSQC-TOCSY [30],
15
NHSQC-NOESY
[30],
13
CHSQC,
13
C HSQC-NOESY [31] and a 2D
NOESY spectrum were recorded at 35 °C.
15
NHSQC,
15
N HSQC-NOESY, HNHA [32–34],
13
CHSQCand
13
C HSQC-NOESY spectra were recorded at 20 °C.
15
N
HSQC-NOESY mixing times were 200 ms; the
13
CHSQC-
NOESY experiments were recorded with 150 ms mixing
1750 T. Sprules et al.(Eur. J. Biochem. 271) Ó FEBS 2004
times. The TOCSY experiment was recorded with a 60-ms
spinlock. Chemical shifts were referenced to an internal
standard of 2,2-dimethyl-2-silapentane-5-sulfonic acid [35].
Data were processed with NMRpipe [36], and data analysis
was performed with NMRView [37]. Data were multiplied
by a 90°-shifted sine-bell-squared function in all dimensions.

aN
ratios less than 1, and to 120° ±100° for d
Na
/d
aN
ratios greater than 1. Initially 100 structures were calculated
with CNS 1.1 [39] using 327 intraresidue, 201 sequential, 115
medium-range and six long-range NOEs. The resulting
structures were subjected to a second round of simulated
annealing with the addition of 22 hydrogen bonds and 91
dihedral angles. The 20 lowest energy accepted structures
(no NOE violations >0.5 A
˚
, no dihedral angle violations
>5°)withnoresiduesinthedisallowedregionofthe
Ramachandran plot (
PROCHECK
[40] analysis) were chosen
to represent the structure. The co-ordinates of preCbnB2(1–
64) have been deposited in the Protein Data Bank as 1RY3.
Results and discussion
Production of target peptides
Like other bacteriocins, CbnB2 is ribosomally synthesized
as a prepeptide, PreCbnB2, by C. piscicola [19]. PreCbnB2
consists of the mature CbnB2 sequence (48 amino acids)
preceded at the N-terminus by an 18-amino acid leader
peptide, which is cleaved at the Gly–Gly site during the
maturation process to liberate the active peptide. The goal
of this study was determination of the 3D solution structure
of preCbnB2 to assist in obtaining a molecular level

4
)
2
SO
4
, or alternatively (
15
NH
4
)
2
SO
4
and
D
-[U-
13
C]glucose, as sole nitrogen and carbon sources.
Purification of substantial quantities (70–100 mg per litre of
fermentation) of fusion protein is easy, but large-scale
proteolytic cleavage of the MBP portion by Factor Xa also
results in removal of two C-terminal amino acids of
preCbnB2, namely Arg65 and Pro66, to yield the truncated
preCbnB2(1–64) as the major product. However, this
material displays CD spectra and antimicrobial properties
indistinguishable from the complete preCbnB2. The two
missing hydrophilic residues are at the C-terminus of the
peptide, which is unstructured in the mature CbnB2 [9], and
distant from the N-terminal leader portion. The C-terminal
section is also random coil in preCbnB2(1–64) (see below).

3D structure of the section corresponding to the mature
peptide. Complete proton, nitrogen and carbon assignments
were obtained for PreCbnB2(1–64) using a combination of
15
N HSQC-TOCSY,
15
N HSQC-NOESY and
13
CHSQC
spectra. Sequential assignments were made by following the
pattern of d
N,N±1
NOEs. Analysis of
3
J
HNHa
coupling
constants, Ha,Ca and Cb chemical shifts, and NOEs
Ó FEBS 2004 Solution structure of precarnobacteriocin B2 (Eur. J. Biochem. 271) 1751
indicated the presence of two a-helices: one running from
residues )15 to )5 in the leader peptide, and a second from
residues 20–38 in the C-terminal portion of the molecule. No
other regular secondary-structure elements were identified.
The solution structure of PreCbnB2(1–64) was calculated
based on 649 NOEs, 91 dihedral angles, and 22 hydrogen
bonds. The two a-helices are separated by a stretch of
relatively unstructured peptide (Fig. 2). The rmsd for helix
1, from residues )15 to )5 is 0.6 ± 0.2 A
˚
, and for helix 2

for previous experiments with mature CbnB2 [9]) may also
Fig. 1.
15
N HSQC of preCbnB2. Spectra
recorded in 70 : 30 trifluoroethanol/H
2
Oat
35 °C and 600 MHz.
Fig. 2. Solution structure of preCbnB2 in 70% trifluoroethanol. The
positions of the N-terminus and C-terminus are indicated, as are
the residues at the start and finish of each a-helix. The position of the
double glycine motif where cleavage occurs during maturation of
the bacteriocin is indicated by an arrow.
1752 T. Sprules et al.(Eur. J. Biochem. 271) Ó FEBS 2004
account for this very minor difference. The side chain
chemical shifts are very similar across the protein (within ±
0.1 p.p.m.), with the most variance observed at the
N-terminus, next to the junction with the leader peptide.
Similar patterns of NOEs are observed as well. Thus, the
critical a-helix in the Ômature portionÕ of the bacteriocin is
conserved in lipophilic environments, not only in all type IIa
bacteriocins examined to date [8–10], but also in the
precursor, preCbnB2.
Mutations that disrupt the helix of mature CbnB2, for
example replacement of Phe33 by serine, render the
bacteriocin completely inactive and greatly alter its retention
time on RP-HPLC [14]. Many additional experiments
support the importance of this helix for membrane-bound
receptor recognition and consequent antimicrobial activity
[2,10]. For example, Fimland et al. [42] have shown that

(Fig. 4), thereby making contact between the hydrophobic
faces of the two helices possible to give a closed ÔjackknifeÕ
structure. This could interfere to some extent with recog-
nition of the mature bacteriocin a-helix by the putative
receptor in membranes of target bacteria and somewhat
diminish the activity of the prebacteriocin. Although no
Fig. 3. Amphipathic a-helical structure in PreCbnB2. (A) Leader pep-
tide a-helix. (B) C-terminal a-helix. The charged residues are coloured
blue, hydrophilic residues white, and hydrophobic residues purple.
Fig. 4. Superimposition of preCbnB2 structures. (A) Superimposition
of the backbone of the leader peptide a-helix for 20 structures. (B)
Superimposition of the backbone of helix 2. Both helix domains are
highly conserved in each case, but the flexible linker region displaces
their relative positions.
Ó FEBS 2004 Solution structure of precarnobacteriocin B2 (Eur. J. Biochem. 271) 1753
long-range NOEs were observed between side chains in the
two a-helices under the conditions used in this study, this
may reflect a relatively weak or short-lived interaction. The
hydrophilic positively charged face of the leader helix, which
contains three lysines, could potentially also interact with
the negatively charged membrane of Gram-positive bac-
teria, thereby hindering the ability of the prebacteriocin to
fully interact with the target receptor. These observations
are consistent with the maintenance of significant antibac-
terial activity for the prebacteriocin, but at a greatly reduced
level ( 125 fold).
The amphipathic nature of the leader peptide is likely to
be critical for its export and processing. Analysis of the
sequences of leader peptides for type IIa bacteriocins
(Table 1) indicates that an a-helix as seen in this section

are gratefully acknowledged for assistance with NMR experiments. We
thank Mark Williams (Department of Medical Microbiology &
Immunology, University of Alberta) for helpful discussions. These
investigations were supported by the Natural Sciences and Engineering
Research Council of Canada, the Alberta Heritage Foundation for
Medical Research, CanBiocin Ltd (Edmonton, AB), and the Canada
Research Chair in Bioorganic and Medicinal Chemistry.
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