Thermolysin-linearized microcin J25 retains the structured core
of the native macrocyclic peptide and displays antimicrobial activity
Alain Blond
1
, Michel Cheminant
1
, Delphine Destoumieux-Garzo
´
n
1
, Isabelle Se
´
galas-Milazzo
2
,
Jean Peduzzi
1
, Christophe Goulard
1
and Sylvie Rebuffat
1
1
Laboratory of Chemistry and Biochemistry of Natural Substances, Department of Regulation, Development and
Molecular Diversity, National Museum of Natural History, Paris, France;
2
IRCOF, ECOBS, UMR 6014 CNRS,
IFRMP 23, University of Rouen, France
Microcin J25 (MccJ25) is the single macrocyclic antimicro-
bial peptide belonging to the ribosomally synthesized class of
microcins that are secreted by Enterobacteriaceae.Itshowed
potent antibacterial activity against several Salmonella and

, determined
by two-dimensional NMR, consists of a boot-shaped hair-
pin-like well-defined 8–19 region flanked by disordered N
and C termini. This structure is remarkably similar to that of
cyclic MccJ25, and includes a short double-stranded anti-
parallel b-sheet (8–10/17–19) perpendicular to a loop
(Gly11–His16). The thermolysin-linearized MccJ25-L
1)21
had antibacterial activity against E. coli and S. enteritidis
strains, while both synthetic analogues lacked activity and
organized structure. We show that the 8–10/17–19 b-sheet,
as well as the Gly11–His16 loop are required for moderate
antibacterial activity and that the Phe21–Pro6 loop and the
MccJ25 macrocyclic backbone are necessary for complete
antibacterial activity. We also reveal a highly stable 8–19
structured core present in both the native MccJ25 and the
thermolysin-linearized peptide, which is maintained under
thermolysin treatment and resists highly denaturing condi-
tions.
Keywords: antimicrobial peptide; conformational stability;
microcin; molecular modeling; solution structure.
Since the pioneering works of the 1980s, which led to the
discovery of the insect cecropins [1], the mammalian
defensins [2,3] and the amphibian magainins [4], numerous
antimicrobial peptides have been isolated from a wide
variety of species. Many bacteria produce antimicrobial
peptides and proteins, including bacteriocins [5] and colicins
[6], as a method of defence against other microorganisms.
Among them, microcins are antimicrobial peptides that are
synthesized ribosomally by Enterobacteriaceae [7,8]. These

as the concentrations needed for these membrane activities
are, in some cases, much higher than those required for
the antibiotic action. In addition, a recent study showed
that an E. coli strain displaying a mutation in the gene
encoding the RNA polymerase b¢ subunit is resistant
to MccJ25, which suggests that RNA polymerase could
Correspondence to S. Rebuffat, Laboratoire de Chimie et Biochimie
des Substances Naturelles, Muse
´
um National d’Histoire Naturelle,
63 rue Buffon, 75231 Paris, Cedex 05, France.
Fax:+33140793135,Tel.:+33140793118,
E-mail: rebuff[email protected]
Abbreviations: Mcc, microcin; MIC, minimum inhibitory concentra-
tion; PB, poor broth; CSD, chemical shift deviations; RTD-1,
rhesus theta-defensin-1; SFTI-1, sunflower trypsin inhibitor; TMS,
tetramethylsilane.
(Received 22 August 2002, revised 21 October 2002,
accepted 30 October 2002)
Eur. J. Biochem. 269, 6212–6222 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03340.x
be the intracellular target for the microcin [18]. To date,
the precise mechanism of action of MccJ25 remains
unknown.
The 21-residue primary structure [19], and the three-
dimensional NMR solution structure [20] of cyclic MccJ25
have been determined. The peptide forms a distorted
antiparallel b-sheet, which twists and folds back on itself.
Residues 7–10 and 17–20 form the more regular part of the
b-sheet between the Phe21–Pro6 and Gly11–His16 loops. A
cavity delimited by two crab pincer-like regions that

Despite identical sequences, the folding and activity of the
enzymatically generated MccJ25-L
1)21
and chemically syn-
thesized sMccJ25-L
1)21
were completely different. This
finding was used as a basis to discuss the high stability of the
MccJ25 structured core and its involvement in the antibac-
terial activity.
EXPERIMENTAL PROCEDURES
MccJ25 and MccJ25-L
1)21
sample preparation
Native MccJ25 was purified according to the procedure
described previously [19]. Briefly, E. coli J02
Mcc+
(a
generous gift from A M. Pons, Universite
´
de La
Rochelle, France) was grown in 2 L M63 minimal
medium and the culture supernatant was applied onto a
C8 Sep-Pak cartridge (Waters, France). Two successive
elution steps were performed with (50 : 50, v/v) and
(80 : 20, v/v) methanol/water mixtures. MccJ25, found in
the (80 : 20) methanol/water Sep-Pak fraction, was further
purified on an RP-HPLC semipreparative column (Cap-
cell C18, 5 lm, 7.5 · 250 cm; Interchim, France) under
isocratic conditions in a (61 : 39) methanol/water mixture

Inertsil ODS2 column (5 lm, 4.6 · 250 mm; Interchim,
France) under isocratic elution in a (31 : 69, v/v) acetonit-
rile/water solution containing 0.1% CF
3
COOH (flow rate:
1mLÆmin
)1
). Absorbance was monitored at 226 nm. Purity
of MccJ25-L
1)21
was ascertained by MALDI-TOF MS on
an Applied Biosystem Applera (USA) Voyager De-Pro
system used in a positive linear mode, with sinapinic acid as
a matrix. Calibration was performed with a mixture of
standards including bovine insulin (MH
+
at m/z 5734.59),
thioredoxin (MH
+
at m/z 11674.48) and apomyoglobin
(MH
+
at m/z 16952.56) (Applied Biosystems).
Peptide synthesis and purification
sMccJ25-L
1)21
(VGIGTPISFY
10
GGGAGHVPEY
20

.ThesMccJ25-L
12)11
sample was
purified in two steps on the same column as sMccJ25-L
1)21
at a flow-rate of 2 mLÆ min
)1
. The first RP-HPLC separation
was performed with a biphasic gradient composed of a 10-
min isocratic step in a (26 : 74) acetonitrile/water mixture,
followed by a 26 : 74 to 28 : 72 acetonitrile/water flat linear
gradient (0.4% acetonitrileÆmin
)1
). The second HPLC con-
sisted of an isocratic elution with a (30 : 70) acetonitrile/
water mixture. Absorbance was monitored at 226 nm.
Antibacterial assays
Antibacterial activity of MccJ25, MccJ25-L
1)21
, sMccJ25-
L
1)21
and sMccJ25-L
12)11
was assayed against two bacteria
highly sensitive to MccJ25. The test microorganisms,
E. coli MC4100 tolC
–
and S. enteritidis, were kindly
provided by M. Lavin

expressed as the interval of concentration [a]–[b], where
[a] is the highest concentration tested at which microbial
growth can be observed and [b] is the lowest concentration
that causes 100% growth inhibition [23].
CD spectroscopy
CD spectra were recorded at room temperature from 250 to
190 nm on a Jobin-Yvon Mark V dichrograph (Longjum-
eau, France), using a 0.05-mm path cell. The spectra were
measured for methanolic solutions at peptide concentra-
tions of 0.05–1 m
M
.
NMR spectroscopy
Samples(0.5mL)of6m
M
MccJ25-L
1)21
, sMccJ25-L
1)21
and sMccJ25-L
12)11
in methanol (CD
3
OH) were placed in
5-mm Wilmad tubes for the NMR experiments. Data were
acquired on Bruker AVANCE 400 and DMX 600 spec-
trometers, equipped with
1
H-broad-band reverse gradient
and triple resonance

1
H-
13
C heteronuclear experiments, optimized for J-values of
135 Hz (HSQC) and 7 Hz (HMBC). Methods of spectra
recording and data processing are described elsewhere [20].
NOE buildup curves for MccJ25-L
1)21
(mixing times of 50,
100, 150, 200 and 400 ms) showed that the correlation
remained linear for the 100 ms mixing time, which was
selected for distance calculation.
Temperature coefficients of amide protons were obtained
in the range of 10–35 °C, by acquiring six series of one-
dimensional (1D)-
1
H and TOCSY spectra at 400.13 MHz,
using 5 °C temperature increments. Exchange of amide
protons was monitored as described previously [20]. Briefly,
a normal isotopic sample (either MccJ25-L
1)21
or sMccJ25-
L
1)21
) was dissolved in CD
3
OD at 0 °C. It was analysed for
2hat0°C and over 3 days at 20 °C by the acquisition of a
series of 1D-
1

at 10 °C from the 1D- and the high digital resolution
DQF-COSY spectra (CD
3
OH), were restrained to
)120 ± 25° for
3
J
NHCaH
P 9.5 Hz (Phe9, Val17),
)120 ± 45° for a
3
J
NHCaH
in the range 8.1–8.9 Hz (Ser8,
Tyr20, Phe21) and )120 ± 50° for a
3
J
NHCaH
6 8.0 Hz
(Thr5, Tyr10, Glu19). Two v
1
dihedral angles, derived from
the
3
J
CaHCbH
coupling constants measured in the
DQF-COSY (CD
3
OD) as well as from the intraresidue

and
the NOE intensities were averaged with the ÔsumÕ option. Of
the 100 structures generated, 80 had a total energy less than
25 kcalÆmol
)1
and led in all cases to systematic distance and
dihedral angle violations lower than 0.2 A
˚
and 5°, respect-
ively. Refinement of the structures was achieved using the
conjugate gradient Powell algorithm with 7000 cycles of
energy minimization and the
CHARMM 22
force field [29]. The
30 best structures on the basis of their total energy including
the electrostatic term with no systematic distance violation
larger than 0.2 A
˚
and no dihedral angle violation greater
than 5° were selected as the final structure of MccJ25-L
1)21
.
The structures were visualized and analysed on the Silicon
Graphics O2 and Gateway workstations, using the
X
-
PLOR
[25],
MOLMOL
[30] and

fi CO
13
,NH
13
fi CO
9
), or
distance < 3.0 A
˚
, deviation angle < 50° (NH
11
fi CO
9
,
NH
14
fi CO
12
) in all the selected structures.
The coordinates for the family of 20 refined lowest energy
structures were deposited in the Brookhaven Protein Data
Bank, under the accession code 1GR4.
6214 A. Blond et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Stability of MccJ25 and MccJ25-L
1)21
to denaturing
conditions
Thermal stability of MccJ25-L
1)21
and MccJ25 was exam-

M
guanidinium hydro-
chloride or 1–8
M
urea in water) and temperature
conditions (25–95 °C) were used to assay MccJ25-L
1)21
stability. For high temperature conditions, sealed tubes
were used. Over the reaction time course, aliquots of the
peptide/denaturant mixtures were withdrawn at different
incubation times and analysed by RP-HPLC on a C18
lBondapak column (4.6 · 250 mm; Waters, France)
under isocratic conditions with 0.05% CF
3
COOH-con-
taining (32 : 68) acetonitrile/water mixture at a flow rate
of 1 mLÆmin
)1
. At the end of the incubation period, the
reaction mixture was cooled to room temperature and
applied onto a C8 Sep-Pak cartridge (Waters, France),
which stopped the reaction by removing the chaotropic
agent. Elution was performed in a stepwise manner by
0.05% CF
3
COOH-containing 0 : 100, 25 : 75, and 45 : 55
acetonitrile/water mixtures. MccJ25-L
1)21
, found in the
(45 : 55) fraction, was dried under vacuum and analysed

1)21
and
sMccJ25-L
12)11
; Fig. 1). The sequences chosen for the
synthetic peptides are identical to those of the thermolysin-
cleaved MccJ25 (sMccJ25-L
1)21
) and of the 21-residue
C-terminal end of the MccJ25 precursor (sMccJ25-L
12)11
).
The three linear peptides were purified by RP-HPLC and
their purity was ascertained by MALDI-TOF MS. The
measured masses for all three peptides (MH
+
at m/z
2126.08 for MccJ25-L
1)21
, 2125.70 for sMccJ25-L
1)21
and
[M+Na]
+
at m/z 2147.47 for MccJ25-L
12)11
)werein
agreement with the expected molecular masses at 2125 Da.
Antibacterial activity
The antibacterial activity of MccJ25 linear variants was

1)21
, following the hypo-
thesis that structural features essential to MccJ25 activity
had most likely been retained in this linear form.
Peptide solubility and aggregation state
Due to insolubility of MccJ25 and its linear variants in
aqueous medium in the absence of denaturing agents, CD
and NMR spectroscopic analyses were performed utilizing
methanol, a solvent in which MccJ25 is extremely soluble
[20]. The CD spectrum of MccJ25-L
1)21
at 0.1 m
M
(data not
shown) was very similar to that obtained previously for
MccJ25 [20]. It presented a strong negative band at 193 nm,
as well as a positive band centred at 210 nm, which did not
enable the assignment of any defined secondary structure.
The aggregation state of MccJ25-L
1)21
was evaluated by
recording several CD spectra at concentrations ranging
from 0.05 to 1 m
M
. The similarity in the patterns obtained
at the various concentrations indicated the absence of
aggregation below 1 m
M
. In addition, 1D-
1

Ó FEBS 2002 Structure of thermolysin-linearized microcin J25 (Eur. J. Biochem. 269) 6215
proton chemical shifts, ensuring that MccJ25-L
1)21
will not
aggregate in methanol when performing NMR.
sMccJ25-L
1)21
was also quite soluble in methanol,
showing weak negative and positive bands centred at
190 nm, and 197 nm, respectively (data not shown). CD
spectra could not be acquired for sMccJ25-L
12)11
, due to its
poor solubility in methanol. Dimethylsulfoxide-d6was
finally chosen for the NMR study of this variant, and was
also used to assay the thermal stability of MccJ25- L
1)21
.
Sequential assignments and secondary structures
All proton resonances of MccJ25-L
1)21
were obtained at
10 °C, to ensure a good signal separation. The standard
sequence-specific assignment strategy was used [32]. TOC
SY and DQF-COSY spectra enabled the identification of
amino acid spin systems, and NOESY data provided the
sequential connections between these spin systems. In
addition, backbone and side-chain
13
C resonances were

OH and dimethyl-
sulfoxide-d6, respectively. In both sMccJ25-L
1)21
and
sMccJ25-L
12)11
NOESY spectra, the aH
i-1
-dH
i
cross-peaks
that characterize a trans conformation of the X-Pro amide
bonds were observed for Pro6 and Pro18, while no
contribution from cis conformation could be detected. In
addition, the proline c-carbon
13
C chemical shifts were in
agreement with two trans proline residues in sMccJ25-L
1)21
.
The Ha and Ca secondary chemical shifts (chemical shift
deviations, CSD), which represent the difference between
the observed chemical shifts and the random coil values of
Wishart [33,34], were determined for MccJ25-L
1)21
.Mostof
the residues had chemical shifts that differed from the
random coil values by more than 0.1 p.p.m., indicative of a
structured peptide. The CSD did not show any clear
evidence of a-helix (upfield shifts) or b-sheet (downfield

of MccJ25-L
1)21
appeared disordered, considering the
complete lack of medium- and long-range NOE connectiv-
ities in these two parts.
The sequential assignments obtained for sMccJ25-L
1)21
were completely different from those for its enzymatically
generated equivalent, MccJ25-L
1)21
. This strongly suggests
that despite an identical sequence, the two peptides adopt
distinct conformations. Indeed, a small chemical shift
Fig. 2. Comparison of NMR conformational parameters for MccJ25
(black), MccJ25L
1)21
(grey) and sMccJ25-L
1)21
(white). The intensities
of the secondary chemical shifts of the Ha protons (CSD
Ha
)andCa
carbons (CSD
Ca
), of the
3
J
NHCaH
coupling constants and temperature
coefficients of the NH protons (Dd/DT

1)21
NOESY spectrum
(data not shown). Only a few sequential NOEs of low
intensity, a series of daNi,i+1 (or dadi,i+1 in the case of
prolines) and a few dNNi,i+1 (Gly4–Thr5, and
Gly15–His16) and dbNi,i+1 (Ile7–Ser8, Phe9–Tyr10,
Ala14–Gly15, His16–Val17) were observed. The absence
of the 8–10/17–19 b-sheet in the sMccJ25-L
1)21
structure
was demonstrated by: (a) temperature coefficients of amide
protons in the range of 6–10 p.p.b.ÆK
)1
, as usually found in
unstructured peptides; and (b) rapid NH–ND exchange
rates of all the amide protons, including those of Phe9,
Tyr10 and Glu19 that could be observed at less than 2 h in
sMccJ25-L
1)21
vs. more than 3 days in both the thermoly-
sin-generated MccJ25-L
1)21
and the native cyclic MccJ25
(Fig. 2).
The conformational parameters of sMccJ25-L
12)11
obtained in dimethylsulfoxide-d6 (data not shown) also
argued in favour of an unstructured peptide, with Ha and
NH chemical shifts in the random coil range and tempera-
ture coefficients between )4and)7.5 p.p.b.ÆK

significant deviation from ideal covalent geometry. The 80
structures with the lowest energy were used in the last run of
energy minimization. An evaluation of the quality and
precision of the 30 lowest energy structures chosen to
represent the MccJ25-L
1)21
solution structure is given in
Table 2.
From Thr5 to Tyr20, the individual backbone confor-
mation of all nonglycine residues was located in the
energetically allowed regions of the /, w space. Glycine
residues assembled either in the specific glycine-allowed
Fig. 4. NOE distribution per residue (A) and values of / and w angles in
the 30 final structures (B) for MccJ25-L
1)21
. Intraresidual, sequential,
and medium- and long-range NOEs are in black, grey and white,
respectively.
Table 2. Structural statistics for the 30 final structures of MccJ25-L
1–21
.
The van der Waals’ energy is calculated with a switched Lennard–
Jones potential and the electric energy with a switched Coulomb
potential and a dielectric constant e ¼ 32.7. The experimental NOE
energy is calculated with a square-well potential and a force constant of
50 kcalÆmol
)1
ÆA
˚
)2

NOE restraint
3.5 ± 0.6
E
Dihedral restraint
0.00 ± 0.01
Mean rmsd from idealized covalent geometry 0.012 ± 0.001
Bond (A
˚
) 0.012 ± 0.001
Angle (deg) 2.10 ± 0.06
Dihedral (deg) 57.6 ± 0.4
Improper (deg) 2.5 ± 0.2
Average rmsd values (A
˚
)
N, Ca,C¢, for residues 8–19 0.20 ± 0.07
N, Ca,C¢, for residues 8–10 and 17–19 0.18 ± 0.07
N, Ca,C¢, for residues 11–16 0.07 ± 0.03
Ó FEBS 2002 Structure of thermolysin-linearized microcin J25 (Eur. J. Biochem. 269) 6217
regions (Gly12, Gly13), or in the b-turn region (Gly11)
together with Tyr10, His16 and Glu19, or in the extended
region (Gly15) where residues 5, 7–9, 14, 17, 18 and 20 were
also located. The //w couples were very dispersed for the
residues at positions 1–4, in contrast with those observed for
the remaining MccJ25-L
1)21
amino acids (Fig. 4B). These
//w couples reflected a certain degree of heterogeneity in the
MccJ25-L
1)21

10
fi CO
17
,NH
19
fi CO
8
)thatare
also found in the MccJ25 structure. These involve the Tyr10
and Glu19 amide protons, which remained strikingly
unexchanged at room temperature for more than 3 days,
a feature also reported for the native cyclic MccJ25 (Fig. 2).
These two hydrogen bonds are therefore believed to be
particularly strong. Residues 11–16 are folded into a loop
(Fig. 5). This region, which contains a series of turns, is
strongly stabilized by five hydrogen bonds that involve
amide protons exhibiting medium to very slow exchange
rates (Fig. 2). The NH
11
fi CO
9
and NH
14
fi CO
12
hydrogen bonds define a reverse c-turn (Phe9-Tyr10-
Gly11), with /
10
¼ )78.8 ± 1°/w
10

9
). The 11–16 loop is finally stabilized by the
NH
9
fi CO
13
hydrogen bond, which enables a tight
connection between the 11–14 region and the 8–10 strand.
Interestingly, despite the preservation of MccJ25 global
structure in this region of MccJ25-L
1)21
, the hydrogen bond
network does not fit between the two peptides, except for
the two bonds that stabilize the b-sheet (NH
10
fi CO
17
,
NH
19
fi CO
8
). This was expected from the NH–ND
exchange rates and temperature coefficients of the amide
protons in the Ile7–Gly15 region that differ from those
found for the cyclic MccJ25, while they fit very closely for
the residues belonging to the b-sheet (Fig. 2). The slow
exchange rates exhibited by some other amide protons,
chiefly by Ser8 and Val17, can probably be likely attributed
to their low level of accessibility as a result of nearby

) is engaged in the concave face
of the boot and is flanked by the Ile7 side chain (mean
rmsd ¼ 1.22 A
˚
) and the Pro18 ring (mean rmsd ¼ 0.35 A
˚
)
(Fig. 7). The side-chains of Tyr10, His16 and Val17 (mean
rmsd ¼ 0.89, 1.21 and 0.26 A
˚
) form a cluster at the bottom
of the loop (Fig. 7). As was observed previously with
MccJ25, the hydrophobic side chains are not packed in a
core, but are distributed over the periphery of the structure,
as is often found in larger proteins. In addition, the aromatic
residues are not stacked. Several hydrophobic side chains
adopt a specific location on the two strands of the b-sheet.
Fig. 5. Superimposition of backbone atoms (N, Ca,C¢) of the 30 final
NMR-derived lowest-energy structures of MccJ25-L
1)21
(best overlap
for residues 8–19), whose geometric and energetic statistics are given in
Table 2. The view exhibits the 8–10/17–19 antiparallel b-sheet and the
11–16 loop.
6218 A. Blond et al. (Eur. J. Biochem. 269) Ó FEBS 2002
The side chains of Ile7, Phe9 and Tyr10 located on one
strand are facing those of Phe21 (in half of the selected
structures), Pro18 and Val17 on the other strand, respect-
ively. The resulting hydrophobic interactions supported by
a few long-range NOEs seem to maintain MccJ25-L

thermal denaturation of MccJ25 was also probed by NMR.
MccJ25-L
1)21
was first treated with 1–6
M
guanidinium
hydrochloride or 1–8
M
urea for 10 h at 25 °Candwasthen
separated from the denaturing agent on a C8 cartridge.
RP-HPLC analysis did not show any variation in
MccJ25-L
1)21
retention time during the entire incubation.
In addition, the
1
H-1D, TOCSY and NOESY spectra
recorded after removal of the denaturing agents were
identical to those of the untreated reference. Thus, MccJ25-
L
1)21
is resistant to chaotropic agents.
Fig. 7. Stereoview of the mean MccJ25-L
1)21
structure showing the position and orientation of the side-chains. The main chain is in grey, aromatic
residues are in magenta, negatively charged or polar Glu19 and Ser8 are in orange, hydrophobic Val17 and Ile7 are in blue, His16 is in green and the
Pro6 and Pro18 heterocycles are in black.
Fig. 6. Superimposition of the solution structures of the cyclic MccJ25
(cyan) and the linear MccJ25-L
1)21

only. MccJ25-L
1)21
was recovered in its original form
after treatment with 6
M
guanidinium hydrochloride at
65 °C for 16 h. Treatment with 8
M
urea at 65 °Cfor
16 h resulted in the coupling of one urea molecule at the
peptide N terminus, but did not induce conformational
changes. Indeed, the NOE contacts were maintained
between residues of the 8–10 and 17–19 regions, including
the da,a
9)18
NOE typical of the b-sheet present in both
MccJ25 and MccJ25-L
1)21
(data not shown). Further-
more, those strongly denaturing conditions showed only
poor effect on the antibacterial activity (data not shown).
Complete denaturation of MccJ25-
L1)21
could not be
obtained up to 40 h at 95 °Cin8
M
urea. Thus, the
MccJ25-L
1)21
structure is highly resistant to both chemical

Phe21–Val1 peptide bond. This area was shown in the
MccJ25 three-dimensional structure to be less protected by
both the side chains and the compactness of the structure.
This cleavage results in the complete disruption of the
Phe21–Pro6 loop, and is accompanied by a net decrease in
antibacterial activity. However, the remaining portion of the
linear MccJ25-L
1)21
structure is remarkably unaltered as
compared with that of MccJ25. In particular, the region
8–19 shows a very well defined structure, with an irregular
double-stranded antiparallel b-sheet folded into a twisted
b-hairpin. Both MccJ25 and MccJ25-L
1)21
contain a stable
arrangement of cross-linking hydrogen bonds associated
with very low NH–ND exchange rates (over 3 days)
exhibited by several amide protons. Those which stabilize
the b-sheet (NH
10
fi CO
17
,NH
19
fi CO
8
) are identical in
both peptides. The structure of both forms is also stabilized
by hydrophobic interactions involving mainly the aromatic
side chains of Phe9, Tyr10 and Phe21 facing the Pro18 ring

chaotropic agents, proteolysis). However, the circular
backbone is needed to reach the highly potent antibacterial
activity of MccJ25. It could also play an essential role in the
resistance to the numerous exoproteases encountered in the
gut microflora ecosystem where MccJ25 is naturally
encountered.
Considering the absence of activity of the synthetic
variant sMccJ25-L
1)21
, which lacks the MccJ25 zipper-like
structured core, it is tempting to speculate that the very
stable hydrogen bonding of MccJ25 is involved in both the
peptide structure and activity. This structured-core makes
MccJ25 very different from other cyclic antimicrobial
peptides. The mammalian antimicrobial peptide RTD-1,
and the trypsin inhibitor from sunflower seeds SFTI-1
[38,42] instead contain disulfide bridges to stabilize the
double-stranded antiparallel b-sheet. To date, no general
rule as to which factors lead to increased protein stability
has emerged, except that cumulative effects of hydrogen
bonding, hydrophobic, coulombic and van der Waals’
interactions are all involved [43,44]. Most likely, the stability
of the MccJ25-L
1)21
structure is ensured by both (a) the
hydrophobic interactions that involve the aliphatic and
aromatic residues on opposing strands of the b-sheet and (b)
the hydrogen bond network that stabilizes the b-sheet and
6220 A. Blond et al. (Eur. J. Biochem. 269) Ó FEBS 2002
were shown to be maintained in the thermolysin-linearized

the 21-amino acid C-terminal part of the propeptide. The
propiece together with the processing enzymes might be
involved in structural maturation. However, MccJ25 fold-
ing could also involve unidentified molecular partners.
Similar to the E. coli microcin B17 synthase, which copu-
rifies with an uncharacterized chaperone protein [46,47], it is
possible that MccJ25 folding is also assisted by a helper
molecule.
ACKNOWLEDGEMENTS
We thank J P. Briand (UPR 9021 CNRS, Strasbourg, France) for
peptide synthesis, A M. Pons (Universite
´
de La Rochelle, France) and
M. Lavin
˜
a (Facultad de Ciencias, Montevideo, Uruguay) for providing
the bacterial strains used in this study, and L. Dubost for MS
measurements. We are grateful to B. Gilquin (CEA, Saclay, France) for
helpful and stimulating discussions and to A. Cole (University of
California, Los Angeles, USA) for critical reading of the manuscript.
This work was supported in part by the ÔProgramme de Recherche
Fondamentale en Microbiologie et Maladies Infectieuses et ParasitairesÕ
of the French Ministry for Research and Technology. The 400-MHz
NMR spectrometer and the mass spectrometer used in this study were
funded jointly by the Re
´
gion Ile-de-France, the French Ministry for
Research and Technology and by CNRS (France); the 600-MHz NMR
spectrometer was funded by the Re
´

(1986) The DNA replication inhibitor microcin B17 is a forty-
three-amino-acid protein containing sixty percent glycine. Proteins
1, 230–238.
10. Vizan, J.L., Hernandez-Chico, C., del Castillo, I. & Moreno, F.
(1991) The peptide antibiotic microcin B17 induces double-strand
cleavage of DNA mediated by E. coli DNA gyrase. EMBO J. 10,
467–476.
11. Guijarro, J.I., Gonzalez-Pastor, J.E., Baleux, F., San Millan, J.L.,
Castilla, M.A., Rico, M., Moreno, F. & Delepierre, M. (1995)
Chemical structure and translation inhibition studies of the anti-
biotic microcin C7. J. Biol. Chem. 270, 23520–23532.
12. Lagos, R., Wilkens, M., Vergara, C., Cecchi, X. & Monasterio, O.
(1993) Microcin E492 forms ion channels in phospholipid bilayer
membrane. FEBS Lett. 321, 145–148.
13. Yang, C.C. & Konisky, J. (1984) Colicin V-treated Escherichia coli
does not generate membrane potential. J. Bacteriol. 158, 757–759.
14. Salomo
´
n, R.A. & Farı
´
as, R.N. (1992) Microcin 25, a novel anti-
microbial peptide produced by Escherichia coli. J. Bacteriol. 174,
7428–7435.
15. Portrait, V., Gendron-Gaillard, S., Cottenceau, G. & Pons, A.M.
(1999) Inhibition of pathogenic Salmonella enteritidis growth
mediated by Escherichia coli microcin J25 producing strains. Can.
J. Microbiol. 45, 988–994.
16. Rintoul,M.R.,deArcuri,B.F.&Morero,R.D.(2000)Effectsof
the antibiotic peptide microcin J25 on liposomes: role of acyl chain
length and negatively charged phospholipid. Biochim. Biophys.

Barthe
´
le
´
my, M., Goulard, C., Salomo
´
n, R., Moreno, F., Farı
´
as,
R. & Rebuffat, S. (2001) Solution structure of microcin J25, the
single macrocyclic antimicrobial peptide from Escherichia coli.
Eur. J. Biochem. 268, 2124–2133.
21. Neimark, J. & Briand, J.P. (1993) Development of a fully auto-
mated multichannel peptide synthesizer with integrated TFA
cleavage capability. Pept. Res. 6, 219–228.
22. Destoumieux, D., Bulet, P., Loew, D., van Dorsselaer, A.,
Rodriguez, J. & Bache
`
re, E. (1997) Penaeidins, a new family of
antimicrobial peptides isolated from the shrimp Penaeus vannamei
(Decapoda). J. Biol. Chem. 272, 28398–28406.
Ó FEBS 2002 Structure of thermolysin-linearized microcin J25 (Eur. J. Biochem. 269) 6221
23. Casteels, P., Ampe, C., Jacobs, F. & Tempst, P. (1993) Functional
and chemical characterization of Hymenoptaecin, an antibacterial
polypeptide that is infection-inducible in the honeybee (Apis mel-
lifera). J. Biol. Chem. 268, 7044–7054.
24. Wu
¨
thrich, K., Billeter, M. & Braun, W. (1983) Pseudo-structures
for the 20 amino acids for use in studies of protein conformation

31. Laskowski, R.A., Rullmann, J.A., MacArthur, M.W., Kaptein,
R. & Thornton, J.M. (1996) AQUA and PROCHECK-NMR:
programs for checking the quality of protein structures solved by
NMR. J. Biomol. NMR 8, 477–486.
32. Wu
¨
thrich,K.(1986)NMR of Proteins and Nucleic Acids.John
Wiley and Sons, New-York.
33. Wishart,D.S.,Sykes,B.D.&Richards,F.M.(1991)Relationship
between nuclear magnetic resonance chemical shift and protein
secondary structure. J. Mol. Biol. 222, 311–333.
34. Wishart, D.S., Sykes, B.D. & Richards, F.M. (1991) Simple
techniques for the quantification of protein secondary structure by
1
H NMR spectroscopy. FEBS Lett. 293, 72–80.
35. Galvez, A., Gime
´
nez-Gallego, G., Maqueda, M. & Valdivia, E.
(1989) Purification and amino acid composition of peptide anti-
biotic AS-48 produced by Streptococcus (Enterococcus) faecalis
subsp. liquefaciens S-48. Antimicrob. Agents Chemother. 33,
437–441.
36. Craik, D.J. (2001) Plant cyclotides: circular, knotted peptide tox-
ins. Toxicon 39, 1809–1813.
37. Tam, J.P., Lu, Y A., Yang, J L. & Chiu, K W. (1999) An
unusual structural motif of antimicrobial peptides containing end-
to-end macrocycle and cystine-knot disulfides. Proc. Natl Acad.
Sci. USA 96, 8913–8918.
38. Trabi, M., Schirra, H.J. & Craik, D.J. (2001) Three-dimensional
structure of RTD-1, a cyclic antimicrobial defensin from Rhesus

ing peptide antibiotics: microcin B17 synthase. Science 274, 1188–
1193.
47. Milne,J.C.,Roy,R.S.,Eliot,A.C.,Kelleher,N.L.,Wokhlu,A.,
Nickels, B. & Walsh, C.T. (1999) Cofactor requirements and
reconstitution of microcin B17 synthetase: a multienzyme complex
that catalyzes the formation of oxazoles and thiazoles in the
antibiotic microcin B17. Biochemistry 38, 4768–4781.
SUPPLEMENTARY MATERIAL
The following material is available from http://www.
blackwellpublishing.com/products/journals/suppmat/EJB/
EJB3340/ EJB3340sm.htm
Table S1. The
1
Hand
13
C chemical shifts in CD
3
OH of the
thermolysin-linearized MccJ25-L
1–
21.
Table S2. The
1
Hand
13
C chemical shifts in CD
3
OH of the
synthetic peptide having the same sequence (sMccJ25-
L