(Received for publication, June 16, 1995)
From the
Escherichia coli microcin C7 (MccC7) is an antibiotic
that inhibits protein synthesis in vivo. It is a heptapeptide
containing unknown modifications at the N and C termini
(García-Bustos, J. F., Pezzi, N., and
Méndez, E. (1985) Antimicrob. Agents Chemoth. 27, 791-797). The chemical structure of MccC7 has been
characterized by use of H homonuclear and heteronuclear (
C,
N,
P) nuclear magnetic
resonance spectroscopy as well as mass spectrometry (1177 ± 1
Da). The heptapeptide Met-Arg-Thr-Gly-Asn-Ala-Asp is substituted at the
N terminus by a N-formyl group. The C-terminal substituent
consists of the phosphodiester of 5`-adenylic acid and n-aminopropanol (AMPap), which is linked via the phosphorus
atom to an amide group, thus forming a phosphoramide. The main chain
carbonyl of the C-terminal aspartic acid residue is connected via this
amide bond to the modified nucleotide unit. MccC7 and the peptide unit
inhibit protein translation in vitro while a synthetic analog
of the AMPap substituent is not active. Neither the peptide nor the
AMPap molecule has an effect on the growth of MccC7-sensible cells. Our
results strongly suggest that the peptide is responsible for MccC7
antibiotic activity while the C-terminal substituent is needed for
MccC7 transport. Implications of the structure determined in this work
for MccC7 synthesis and mode of action are discussed.
Microcin C7 (MccC7) ()is a small peptide antibiotic
produced by Escherichia coli during the stationary phase of
growth(1) . The spectrum of activity of this microcin includes
several members of the Enterobacteriaceae family(2) .
MccC7 exerts its bacteriostatic action by blocking protein synthesis.
Like for other microcins, the bacterial strains that produce MccC7 are
immune (resistant) to this microcin(3) . The heptapeptide of
MccC7 is synthesized in ribosomes (4) and undergoes
post-translational modifications to yield the mature molecule.
The
genetic determinants for MccC7 synthesis, export, and immunity have
been cloned from the 43 kilobases E. coli pMccC7 plasmid into
multicopy plasmids that overproduce MccC7 and express MccC7
immunity(5) . These determinants lie on a 6.2-kilobase region
of pMccC7, which has been entirely sequenced. ()Different
complementary approaches, such as physical and phenotypical
characterization of insertion mutations and complementation studies,
have shown that this region contains six genes (mccABCDEF) (5) .
Genes A, B, C, D, and E are involved in the production of mature
extracellular microcin. Genes C, E, and F code for self-immunity bestowing products. Genes mccA-D are directly involved in the synthesis and export of MccC7 and
constitute an operon transcribed from a promoter (mccp),
located upstream of mccA(4, 6) . Expression
of these genes is regulated by the cAMP-cAMP regulatory protein complex
and by the stationary phase
factor RpoS (also called
AppR)(6, 7) . (
)
mccA codes for
the unmodified peptide of MccC7, MRTGNAN (MccA)(4) . The
predicted gene polypeptide product of mccB (350 residues) is
strikingly homologous to a 81-residue fragment of the
ubiquitin-activating enzyme from different eucaryotic species (UBA1) (8) , ThiF (9) and ChlN (10) from E. coli which participate, respectively, in the biosynthesis of thiamine
pyrophosphate and of molibdopterin, and HesA, an enzyme required by Anabaena for nitrogen fixation(11) . The predicted mccC product (404 residues) contains 11 potential
transmembrane domains and displays significant similarity with
stretches of transport proteins, suggesting that MccC is
responsible for MccC7 export and explaining why it also confers
resistance to exogenous MccC7. The carboxyl end of the expected MccE
521-residue long polypeptide is highly similar to RimL, an enzyme that
acetylates the ribosomal protein L12 from E.
coli(12) . Principally on the basis of this homology with
an acetylating protein, and because target alteration is a common
mechanism of antibiotic resistance(13) , it has been proposed
that MccE might confer MccC7 immunity to producing cells by acetylating
the target of microcin C7.
No similarity was found for the
predicted MccD (267 residues) and MccF (334 residues) polypeptides.
The knowledge of the chemical structure of MccC7 is necessary to understand its mode of action, biosynthesis, export out of the producing cell and intake into the target cell, and the self-immunity mechanisms. For these reasons, we undertook the elucidation of the microcin C7 chemical structure, one of the best genetically and functionally characterized microcins.
Amino acid analysis and sequencing of peptides derived from MccC7 tryptic digestions showed that the sequence of the MccC7 peptide is MRTGNAD(2) . The N and C termini are both modified, and it has been suggested that the N-terminal might be acetylated, while the C-terminal modification remains unknown(2) .
Here, we report on the chemical
structure of microcin C7 (Fig. 1) based on complete NMR
assignment of H,
C,
N, and
P resonances at natural abundance, as well as on the
determination of the molecular mass of the molecule (1177 ± 1
Da). We also show that MccC7 inhibits protein synthesis in a cell-free
coupled transcription-translation system. Furthermore, we demonstrate
that the peptide of MccC7 is responsible for protein synthesis blocking in vitro, while the C- and N-terminal substituents do not have
any effect on this function. Finally, some implications of the chemical
structure of MccC7 relative to its biosynthesis and mode of action are
discussed.
Figure 1: Chemical structure of microcin C7.
The phosphodiester of 5`-adenylic acid
and n-aminopropanol (AMPap) was obtained by reacting overnight
the tetrabutyl ammonium salt of 5`-adenylic acid with
1-bromo-3-aminopropane in dry CHCN at 80 °C. After
evaporation, the reaction mixture was dissolved in H
O and
desalted on a G-25 column. The first eluted fractions that were
ninhydrin-positive and contained the AMPap molecule, were pooled and
lyophilized. AMPap was then purified by C18 RP-HPLC using a linear
gradient of CH
CN in 0.1% trifluoroacetic acid.
Final purity (>95%) of synthetic molecules was verified by analytical RP-HPLC. The molecular mass of the formylated peptides was determined by electrospray mass spectrometry and was in every case in accordance with the expected mass. NMR spectra analysis of the peptides and the AMPap molecule was also in agreement with the expected formulas.
NMR
experiments were performed on a Varian Unity 500 MHz spectrometer
equipped with a Sun Sparc 2 station. An indirect detection probe or a
multinuclei broad band direct probe were used. Spectra were processed
on a Sparc 2 station with Varian's Vnmr software (Varian, Inc.).
Sweep widths were for H, 5200 Hz in H
O or
D
O, and 7200 and 5200 Hz in Me
SO; for
C, 32,000 Hz; for
N, 4000 Hz; and for
P, 7500 (direct detection) and 600 Hz (indirect
detection). Chemical shifts were referenced to:
3-(trimethylsilyl)-propane sulfonic acid, sodium salt, for
H and
C,
NH
Cl for
N, and 85% H
PO
for
P,
used as external references. All heteronuclear experiments were carried
out at isotope natural abundance. When needed, the water peak was
eliminated by presaturation during the appropriate delays.
Phase-sensitive COSY(15, 16) , and TOCSY (17) using the MLEV17 sequence for spin-locking (18) and mixing times of 80 or 100 ms were performed to
identify short and long range scalar couplings. NOESY experiments (19) were run with a 500-ms mixing time. A pulse at
variable times was used during the mixing time to reduce zero quantum
contributions. (
)ROESY experiments were carried out with a
mixing time of 500 ms and a spin lock field of 2.5 kHz to reduce
Hartmann Hahn transfers(20) . Two-dimensional spectra were
obtained with quadrature detection in both dimensions using the
hypercomplex method in the f
dimension(19) . Usually, 2048 points were acquired in the f
dimension, and 400 or 512 complex points in the f
dimension. For each complex data point in the
first dimension 8, 16, or 24 free induction decays were accumulated
with a relaxation delay of at least 2.4 s. All spectra were apodized
with a shifted sine-bell function in both dimension or with a sine-bell
function for the COSY experiments.
One bond, H-
C,
H-
N, and one to
three bonds,
H-
P, correlation maps were
obtained from
H detected HSQC
experiments(21, 22) . For
H-
C
HSQC experiments, a BIRD pulse sequence followed by a 0.9-s delay was
used to increase the cancellation of signals of protons attached to
C(23) .
H-
N spectra in
dimethyl sulfoxide were acquired without water suppression to observe
the maximum of
N nuclei bearing a labile hydrogen.
H-
P HSQC spectra were acquired in one and two
dimensions for various conditions of solvent and pH. Multiple bond
H-
C interactions were obtained from HMBC
experiments(22, 24) . The delay preceding the
C pulse for the creation of multiple quanta coherences
through several bonds was set to 60 ms. Heteronuclear coupling constant
values used in the HSQC and HMBC experiments to establish the delays
needed to select the protons coupled to the heteronuclei were the
following: 150 Hz (
H-
C), 90 Hz (
H-
N), and 15 Hz (
H-
P). All reverse detection spectra were
obtained with the WALTZ-16 pulse scheme for heteronuclear decoupling (25) . Heteronuclear two-dimensional spectra were obtained with
quadrature detection in both dimensions using the hypercomplex method (19) . An exponential function in the t
dimension and a shifted sine bell in the t
dimension were used to Fourier transform the heteronuclear
spectra.
One-dimensional C direct detection spectra
were obtained in D
O at different pD values (pD 3.4, 4.3,
and 7.3) with microcin sample concentrations higher than 7 mM.
A long delay, 16 s, as well as a
/3 pulse, were used to allow an
efficient relaxation of the quaternary carbons, thus permitting a
reliable integration of the signals. Spectra were recorded with 5,800
(pD 3.4), 8,944 (pD 4.3), and 20,032 (pD 7.3) scans over 64 K data
points using broad band proton decoupling. One-dimensional
P spectra were acquired in different conditions of pH and
solvent, always with a 3.5-s relaxation delay.
Figure 11: Protein synthesis inhibition by MccC7 (1) and the peptides f-MRTGNAN (2), f-MRTGNAD (3), and MRTGNAD (4) at three different concentrations, using a coupled transcription-translation system. An unrelated synthetic octapeptide, which is a modulator of a neuronal-membrane opioid receptor, was used as negative control and, as expected, did not have any influence on protein synthesis.
MccC7
concentration was determined by measuring the absorbance at 259 nm,
using an extinction coefficient of 16,500 M cm
, determined through the quantitative amino
acid analysis of a MccC7 solution of known absorbance. The
concentration of peptides was determined with the BCA protein assay
reagent (Pierce), using a solution of MccC7 of known concentration as
standard.
H homonuclear shift correlation spectra COSY and
TOCSY allowed the identification of three distinct units in the
molecule: the peptide chain modified in its N-terminal (X-peptide), a
sugar ring, and an aliphatic chain. In addition, there are two singlets
in the low field region of the spectrum resonating at 8.41 and 8.35
ppm, and integrating for one proton each (Fig. 2). We will
describe these different units in the following sections.
Figure 2:
One-dimensional proton spectrum of MccC7
in HO at pH 3.4. (a) Low field and (b)
high field regions. Amide (8.1-8.6 ppm) and
proton
(3.9-4.6 ppm) signals are identified by the amino acid one-letter
code; f-M
, formyl proton; R
, arginine guanidinium; standard nomenclature is
used for peptide side chain, ribose, and adenine proton resonances. H2`
(4.75 ppm) and N
(4.68 ppm) signals are obscured by the residual
water signal and are not shown in this
figure.
One-bond H-
C correlation maps (HSQC) and
two- or three-bond
H-
C heteronuclear couplings
(HMBC) obtained in D
O allowed the assignment of
C signals including those of the quaternary carbons.
Chemical shifts are reported in Table 2.
In previous
studies(1) , it was not possible to perform the N-terminal
sequence of MccC7 by the Edman degradation method, suggesting the
presence of an NH blocking group at the N terminus of the
peptide. The singlet observed at 8.12 ppm in the one-dimensional proton
spectrum is in fact scalar coupled (J = 1.5 Hz) to the
methionine amide proton resonating at 8.45 ppm as observed in COSY
spectra. In addition, these two protons are in dipolar contact (rOe).
The 8.12 ppm proton is borne by a carbon resonating at 166.8 ppm as
shown in a
H-
C HSQC experiment run in
D
O (pD 4.3). This position is compatible with a carbonyl
group such as that of formamide(27) . From the HMBC experiment,
it was possible to (i) determine a
H-
C
coupling constant of 198 Hz, in agreement with an aldehyde such as
formamide whose coupling constant is 188 Hz (28) and (ii) to
link the proton resonating at 8.12 ppm with the methionine
-carbon
at 54.1 ppm. Taken together, these data are in favor of a N-formyl-Met concatenation. This is further confirmed by
analysis of the NMR spectra of the synthetic peptide f-MRTGNAD. Indeed,
the one-dimensional proton spectrum of this synthetic peptide also
contains a singlet at 8.12 ppm which shows the same coupling pattern
(COSY, TOCSY, and ROESY) as the analogous signal in the MccC7 spectra.
In addition to the N- and C-terminal substitutions, the peptide of
MccC7 might be modified at other positions. It is thus necessary to
identify all the peptide signals, especially those of exchangeable
protons. It must be mentioned though, that the H and
C chemical shifts are in agreement with values expected
for nonsubstituted amino acids in unstructured
peptides(26, 29) . As rapidly exchanging proton
signals might not be observed in aqueous solvents, proton as well as
nitrogen 15 (
H-
N, HSQC) assignments were
completed in dimethyl sulfoxide
(Me
SO-d
). For this purpose, MccC7 in
aqueous solution at pH 3.2 was freeze-dried and dissolved in
Me
SO-d
. Assignments are reported in Table 3. All the expected exchangeable protons could be
identified. Indeed, the threonine hydroxyl proton (4.95 ppm), the
arginine NH
(7.56 ppm), and guanidinium group (7.15 ppm)
integrating, respectively, for one and four protons, and the two
asparagine side chain amide protons (7.52 and 7.03 ppm) were assigned
via chemical shift analysis, through-bond (COSY, TOCSY) and
through-space (NOESY, ROESY) interactions (Fig. 3a).
The broad signal at 4.95 ppm corresponding to the threonine hydroxyl
proton, sharpened upon addition of a small amount of NaOH and became a
doublet with a coupling constant of 4.2 Hz, as a result of through-bond
coupling with the threonine H
proton.
Figure 3:
One-dimensional proton spectrum of MccC7
in MeSO. (a) Low field and (b) high field
regions. Ad
, amino proton signal of adenine; NHp, phosphoramide proton signal; Rib
and Rib
, respectively, 2` and 3` ribose
hydroxyl proton resonances. The carboxyl proton signal of Asp at 12.5
ppm is not shown. The signal at 2.50 ppm belongs to Me
SO.
The water signal corresponds to the base line deformation between 6 and
3.5 ppm.
All these signals are also present in the spectra of the synthetic peptide f-MRTGNAD (data not shown). Hence, the peptide is not substituted and the remaining spin systems identified in the proton homonuclear experiments must belong to the C-terminal modification.
(i) The six non-exchangeable
sugar ring protons were assigned mainly from COSY and TOCSY spectra
analysis and their chemical shifts are reported in Table 1(HO, pH 3.4), Table 2(D
O,
pD 4.3), and Table 3(Me
SO-d
).
The sugar corresponds to a ribose ring. The chemical shift values
obtained for the H5`5" protons, downfield shifted by
0.7 ppm
relative to ribose, suggest that the corresponding carbon is
substituted(26) . For the same reason, the C2` and C3` carbons
do not seem to be substituted. This is validated by the presence of two
broad signals of exchangeable protons at 5.58 and 5.38 ppm (Fig. 3b), chemical shifts that are in agreement with
that of hydroxyl protons. These two broad signals sharpened upon
addition of NaOH. Using a short mixing time of 10 ms in TOCSY spectra
to limit the transfer to two or three bonds, it was possible to observe
cross-peaks from the signal at 5.58 ppm to the H2` sugar proton at 4.57
ppm, and from the signal at 5.38 ppm to the H3` sugar proton at 4.19
ppm.
Having established the proton network in the H
spectra, the sugar ring carbon signals were identified from a
H-detected
H-
C HSQC experiment
carried out in D
O (Table 2). The chemical shifts
values obtained are in accordance with a C5` substituted ribose bearing
a N-glycosyl bond in C1`. The H1` ribose proton (6.13 ppm)
gives an interaction with a quaternary carbon at 151.6 ppm and with a
signal at 142.7 ppm (HMBC, Fig. 4a) to which the low
field proton resonating at 8.37 ppm is attached (HSQC). In addition, in
ROESY experiments, a dipolar interaction between the ribose H1` sugar
proton and the singlet at 8.37 ppm is observed, suggesting that the
latter could correspond to a purine proton. Furthermore, this low field
proton resonating as a singlet at 8.37 ppm showed an interaction in the
HMBC spectrum with two quaternary carbons resonating at 121.3 and 151.6
ppm (Fig. 4b). Finally, the remaining low field proton
resonating as a singlet at 8.29 ppm and attached to the carbon
resonating at 154.4 ppm (HSQC) is linked via scalar interactions to two
quaternary carbons resonating at 157.4 and 151.6 ppm (Fig. 4b). Thus, the proton singlets at 8.37 and 8.29
ppm are both coupled to a carbon resonating at 151.6 ppm and belong to
the same coupling network. The coupling pattern observed for the base
and the sugar carbons and protons, the proton and carbon chemical
shifts(27, 30) , and the number of protons and carbons
for this unit are characteristic of an adenine or a hypoxanthine linked
to a ribose via a N-glycosyl bond. The fact that the MccC7 UV
absorbance spectrum presents a maximum at 259 nm (H
O, pH 7)
is in favor of an adenine rather than hypoxanthine. Indeed, adenosine
presents a UV absorbance maximum at 260 nm at neutral pH, while
hypoxanthosine, under the same conditions, absorbs maximally at 249.5
nm(31) . These results are strengthened by the identification
of the two adenine amino protons (Fig. 3a) as well as
of the nitrogen bearing those protons in an
H-
N HSQC experiment in dimethyl sulfoxide (Fig. 5). Under more basic conditions, the signal corresponding
to the two amino protons at 7.82 ppm (Fig. 3a)
sharpened up to become a singlet integrating for two protons while
moving upfield at the same time that the H2 and H8 singlets titrated.
Under these conditions, a through-bond interaction (TOCSY) with the H2
base proton is observed. Titration of the base amino, H2 and H8 protons
is surely caused by the imino nitrogen deprotonation.
Figure 4:
Low
field regions of the H-
C HMBC spectrum of
MccC7 (pD 4.3, D
O). The spectrum was obtained with 288
scans per increment, 2048 points in the
H dimension
(f
), and 96 transients in the
C dimension
(f
). Spectral width was 5,200 Hz for
H and
32,000 Hz for
C. a and b are plotted in
absolute value mode at different contour levels. a,
interactions of the H1` proton with purine base and ribose carbons. b, interactions of the aromatic protons with the purine base
carbons. The correlations of the H8 proton with the C8 carbon, and of
the formyl proton and carbon of f-Met, are less intense and are not
observed at this contour level. Two signals, separated by the
heteronuclear
H-
C coupling constant, can be
seen for a proton and the carbon to which it is
attached.
Figure 5:
15N-edited HSQC spectrum of MccC7 in
MeSO. Acquisition parameters were: 5200 Hz and 4000 Hz
spectral widths for
H (f
) and
N (f
), 832 accumulations for each of
the 30 transients, and 2048 points in the
H
dimension.
(ii) The
aliphatic chain contains three methylene groups coupled one to another,
as shown by integration curves and COSY spectra. In
MeSO-d
, these methylene proton signals
were assigned at 4.07, 1.88, and 2.89 ppm (Fig. 3b).
The methylene group at 2.89 ppm is scalar coupled to three exchangeable
protons resonating at 7.78 ppm (COSY), and which are attached to a
nitrogen resonating at 88.4 ppm (
H-
N HSQC, see Fig. 5). Thus, this group is a n-aminopropane chain
(-CH
-CH
-CH
-NH
).
(iii) For the Z substituent, taking into account the chemical shift
values observed for the ribose H5`5" and the C5` signals, as well as
the presence of heteronuclear couplings in the one-dimensional
proton-decoupled carbon spectrum for the C5` and C4` signals (Fig. 6), it was important to check if the molecule contained
any phosphorus atom. One signal is indeed observed, at -2.36 ppm
in the one-dimensional phosphorus spectrum obtained in
MeSO. From a
H-
P HSQC experiment,
it was possible to link the phosphorus nucleus to the ribose H5`5"
protons (4.12 and 4.26 ppm), to the aliphatic chain
(CH
)
protons (4.07 ppm) as well as to a signal at 9.46
ppm (Fig. 7). The intensity of the signal at 9.46 ppm
corresponds to that of one proton. Its coupling constant of 10.5 Hz (Fig. 3a) disappears in proton one-dimensional spectra
acquired with phosphorus broad band decoupling, indicating that this
signal is coupled to the phosphorus nucleus but not to any other
proton. A
H-
N HSQC shows that this proton is
attached to a nitrogen resonating at 92.8 ppm (Fig. 5). Thus,
MccC7 contains a P-NH group. In addition, the C5` ribose and the C
aliphatic chain chemical shifts (69.3 and 68.5 ppm, respectively) in
D
O (pD 4.3), together with the phosphorus chemical shift
(-1.63 ppm) are in favor of P-O-C bonds rather than of phosphine
bonds (P-C)(27) . The
C-
P coupling
constants C5`-P (4.7 Hz) and C
-P (5.5 Hz) are also in agreement
with two-bond C-O-P coupling constants(27) . This holds true
for the three-bond coupling constants C4`-P (7.9 Hz) and C
-P (7.6
Hz). Hence, the MccC7 phosphorus group is a phosphoramide. Together,
these results lead to the formula shown in Fig. 1for the Z
substituent.
Figure 6:
Four regions of the one-dimensional
carbon-13 spectrum of MccC7 (DO, pD 4.3). The spectrum was
obtained with proton broad band decoupling. Acquisition conditions were
as described under ``Materials and Methods.'' The presence of
doublets in this spectrum indicates that MccC7 contains a heteronucleus
in high natural abundance.
Figure 7:
One-dimensional P-edited HSQC
proton spectrum of MccC7 (Me
SO). 192 scans were
accumulated. The free induction decay was Fourier-transformed with a
3-Hz line broadening.
The comparative analysis of the chemical shifts (and
coupling patterns) of the proton signals of the MccC7 Z substituent and
of the analogous molecule AMPap (phosphodiester of 5`-adenylic acid and n-aminopropanol) is in agreement with this chemical structure.
Indeed, in DO and Me
SO, chemical shifts are
similar for the corresponding proton signals in both molecules, with
the exception of those protons that are closer to the differently
substituted phosphorus atom: the ribose H5`5" protons and the
(CH
)
aliphatic protons.
We will now describe how the Z substituent is linked to the peptide.
The proton
one-dimensional spectrum in MeSO displays a broad signal at
12.5 ppm, a typical position for a carboxylic proton (not shown).
However, this signal is too broad to produce scalar or dipolar
interactions. The chemical shift variation as a function of pD in
D
O, or of NaOD added in Me
SO, indicates that
the aspartic residue contains a ionizable group. In addition, in the
one-dimensional
C spectrum (D
O), nine carbonyl
signals are observed (Fig. 8): six belong to the peptide amide
groups f-M, R, T, G, N, and A, and one to the asparagine side chain.
Thus, the two remaining carbons must belong to the aspartic residue. In
agreement with this hypothesis, the
H-
C HMBC
experiment shows an interaction between the Asp
proton and a
carbon signal at 177.5 ppm as well as an interaction between the Asp
protons and a carbon signal at 177.8 ppm. (
)It should
be mentioned though, that the digital resolution in the carbon
dimension was quite low (2.65 ppm/point) and it is not possible to know
if this correlation peaks correspond to one or two carbons. Finally,
one of these two carbons is scalar coupled to the phosphorus (J
Figure 8:
Carbonyl region of the carbon-13 spectrum
of MccC7 obtained in DO at pD 4.3. Broadband proton
decoupling was used. An exponential decay function with a 2-Hz constant
was applied for Fourier transformation. The main chain carbonyl signal
of Asp (D
) is a doublet with a 3.2 Hz
C-
P coupling
constant.
Taking into account the MccC7 molecular weight, the number of
carbons determined from the one-dimensional C spectrum and
the assignments already established, there is only one solution for the
Z substituent branching: an amide peptide bond to the phosphoramide NH
as shown in Fig. 1.
Several experimental evidences support
the proposed branching and lead to the conclusion that the side chain
carboxylic group is free. Indeed, (a) the Asp C is scalar
coupled to the phosphorus nucleus (10.6 Hz) while the C
is not.
This coupling constant value is of the same order of magnitude as the
three-bond
C-
P coupling constant observed
between the phosphorus and the sugar ring C4` or the aliphatic chain
C
. In agreement with this observation, the sugar ring C3` and the
aliphatic chain C
, separated by four bonds from the phosphorus
atom, are not scalar coupled to it. Furthermore, in D
O, the
Asp C
signal broadened at pD 4.3, that is, at a pD value close to
the carboxylic group pK
value, whereas the C
signal remained sharp. In addition, the Asp C
signal broadened (23
Hz at half-height) at pD 7.3 in the phosphoramide pK
value range.
(b) In MeSO, a larger
chemical shift variation is observed upon the carboxylic group
ionization for the Asp H
protons (-0.26 ppm and -0.13
ppm) than for the
proton (-0.10 ppm). It is well known that
for protons, the inductive effect decreases as the number of bond
increases. This observation is also valid in aqueous solution but the
interpretation is not straightforward, as in this environment the
aspartic residue and the adenine base titrated with identical
pK
values.
(c) In DO, the
Asp
proton chemical shifts remained constant when the pD was
changed in the phosphoramide group pK
range (Fig. 9), while the Asp H
chemical shift varied
(-0.09 ppm) between pD 7.2 and 9. Hence, the Asp
protons
are not in the direct environment of the phosphoramide group. Moreover,
the Asp
proton displayed the same chemical shift variation
pattern as a function of pD than the H5`5" protons and the aliphatic
chain
protons which are at a two-bond distance from the
phosphorus (Fig. 9).
Figure 9:
Proton and phosphorus chemical shifts
(, in ppm) of MccC7 in D
O at 27 °C as a function
of pD. Chemical shifts reported at 2.8, 3.0, and 5.0 pD values come
from measurements in H
O at the pH values of 3.2, 3.4, and
5.4 after isotope correction (pD = pH 0.4, (32) ). In
the titration region of the phosphoramide group, the phosphorus signal
splits into two signals (open and filled circles).
Only one set of chemical shift values is shown for the ribose H5`5"
signals although these protons are unequivalent at some pD
values.
(d) There is a dipolar coupling
between the Asp proton and the phosphoramide NH
proton in Me
SO (Fig. 10). This interaction is
analogous to the interaction observed in the peptide of MccC7 between
the H
proton of residue i and the amide proton of residue
i+1.
Figure 10:
Footprint region of the MccC7 ROESY
spectrum (MeSO, 27 °C). Eight scans were acquired for
each of the 400 complex points that constitute the t
dimension. Only negative contours are shown. The
NH
-D
cross-peak shows two components separated by the
proton-phosphorus coupling constant of NH
(10.5 Hz).
, this signal belongs to a minor form of the formyl and
protons of f-Met and might correspond to the cis form of the
formyl group.
It was previously reported that MccC7 does not react with ninhydrin (2) , a compound that is commonly used to reveal aliphatic amines. However, in the MccC7 structure determined here, there is a primary amine group that should react with ninhydrin. We thus performed the ninhydrin test on thin layers and found that MccC7 is ninhydrin-positive.
It is interesting to note that the
phosphorus signal splits into two broad signals when the pD value is
near 6. Close to pD 7.8, the phosphorus signal spreads over several
units of ppm. At pD values higher than 9, it becomes a single sharp
peak once again. These line width changes and splitting are
reproducible and reversible, indicating that these are not due to
hydrolysis phenomena or other possible modifications of the molecule.
The splitting of the phosphorus signal also occurs in MeSO.
However, in this solvent, both phosphorus signals remain sharp, and the
splitting is observed after DCl addition to Me
SO samples
prepared from MccC7 ``acid'' samples (see ``Materials
and Methods''). Although we cannot explain this phenomenon, it is
likely to be due to a pH-dependent conformational equilibrium. This is
substantiated by the lack of scalar coupling between one of the
phosphorus signals when splitting occurs, and the phosphoramide proton
NH
. That MccC7 was not modified by the exposure to
different conditions of pH and solvent is further corroborated by the
fact that, after NMR experiments (in H
O, D
O,
and Me
SO), MccC7 samples conserved the same antibiotic
activity than fresh samples.
We also checked these peptides and AMPap for cell growth inhibition. Typically, the growth of E. coli MccC7-susceptible cells is inhibited at a 20 µM concentration of MccC7. In contrast, AMPap and the peptides did not show any effect on growth when used at a 10-fold higher concentration. These results suggest that the unmodified peptide of MccC7 is not able to reach the MccC7 intracellular target, and that the AMPap group may be required for microcin transport through the cell envelope.
Results described here allow us to conclude on the structure of microcin C7 as presented in Fig. 1. The interpretation of the MccC7 NMR spectra is reinforced through comparative analysis with NMR spectra of the synthetic molecules f-MRTGNAD and AMPap, analogs of the MccC7 formylated peptide and of the C-terminal substituent Z, respectively.
That the C-terminal residue of mature MccC7 is an aspartic acid while the gene mccA codes for an asparagine at this position is not surprising to us: chemical deamidation of Asn to produce Asp has been recognized in proteins and peptides for many years(36, 37, 38) . In principle, the side chain carbonyl carbon of the C-terminal asparagine should be susceptible to nucleophilic attack by the phosphoramide nitrogen. Hence, chemical deamidation, which is not usually observed on peptide C-terminal asparagines lacking a neighboring main chain amide nitrogen, is conceivable for MccC7. However, we do not know whether this deamidation occurs enzymatically or chemically, in vivo or during MccC7 purification, whether it is important for peptide modification or not, or whether it is concomitant with peptide modification (adenylation) or not.
The peptide coded by the gene mccA, f-MRTGNAN, inhibits protein synthesis in vitro like the peptide found in mature microcin: f-MRTGNAD. Hence, the deamidation of the asparagine residue is not important for translation inhibition. However, as already noted, this deamidation might be necessary for (or parallel to) the modification of the peptide.
The peptide of MccC7 (f-MRTGNAD) is
similar to the leader peptide (MSTSKNAD) involved in the regulation by
translation attenuation of the gene cmlA from E.
coli, which codes for non-enzymatic resistance to
chloramphenicol(39) . This peptide inhibits the peptidyl
transferase activity of the ribosomal 50 S subunit, and binds the
peptidyl transferase center of the 23 S rRNA. Given that the peptide of
MccC7 inhibits protein synthesis, that both peptides have five residues
in common, that the side chain of residue 2 in f-MRTGNAD is not
important for protein synthesis inhibition, ()and that the
unformylated peptide (MRTGNAD) has the same in vitro activity
that the formylated peptide, it is tempting to think that the target of
MccC7 might be the same as that of the cmlA leader peptide.
Further experimental work is needed to test this hypothesis.
Principally based on the molecular
mass of MccC51 (1177.5 Da), analysis of MccC51 NMR spectra obtained in
DO at 40 °C, and amino acid sequencing (and mass
determination) of MccC51 proteolytic peptides (MRTGNAX, where X represents N or D), Metlitskaya and co-workers (41) have proposed a chemical structure for MccC51. MccC51
would consist of the peptide f-MRTGNAN, a three-methylene chain, and
the nucleotide nebularine 5`-monophosphate. The side chain of the
N-terminal asparagine would be linked via an amide bond to the
three-methylene chain. The aliphatic chain would be linked to the
nucleotide phosphate. Finally, the hydroxyl oxygen of threonine would
be involved in an hydroxylamine group (-O-NH
). However, we
have several arguments to conclude that this proposed structure is not
correct. Indeed, the one-dimensional proton spectra of MccC7 (40
°C, H
O, pH 3.2, not shown) and of MccC51 (40 °C,
D
O, pD not specified) are very similar. The differences can
be accounted for by impurities, labile proton signals which are not
present in D
O spectra, line shape of proton signals coupled
to exchangeable protons, and a systematic
0.05 ppm shift for all
proton signals (which is probably linked to the different references
used in both studies).
In the region between 8.0 and 8.5 ppm, the
proton spectrum of MccC51 contains three singlets that integrate for
one proton each (8.44, 8.40, and 8.18 ppm). These signals were assigned
to the H2, H6, and H8 protons of the nucleoside nebularine, without any
experimental proof that these belong to the same system of
connectivities. It is astonishing to note that the chemical shift of
these resonances corresponds, respectively, to the adenine H8, H2
protons, and to the formyl proton of f-Met in the MccC7 spectrum. A
resonance at 12.20 ppm was attributed to the formyl proton of f-Met,
arguing that 12.20 ppm is a chemical shift compatible with a formyl
signal. No information of scalar coupling with other protons or carbons
of the methionine residue is mentioned. However, for MccC7, we were
able to demonstrate unambiguously that the signal at 8.12 ppm
corresponds to the formyl proton. This assignment is grounded on the
scalar (and dipolar) connectivities observed for this proton signal and
the methionine protons (and even the carbon), its intensity and
the chemical shift of the carbon attached to it. Furthermore, this
signal is also present in spectra of the synthetic peptide f-MRTGNAD
and shows the same pattern of connectivities than that seen in MccC7
spectra. That the chemical shift of the formyl proton (and of the rest
of the methionine residue protons) is the same in the synthetic peptide
and in MccC7, indicates that the C-terminal substituent has no
influence on it. Given the similarity of MccC7 and MccC51 spectra (and
molecules) it is very unlikely that the chemical shift of the formyl
proton could shift as much as
4 ppm. Hence, the signal at 8.18 ppm
in the MccC51 spectrum could correspond to the formyl proton of f-Met.
If this were the case, the aromatic base of MccC51 would contain only
two protons. Because the chemical shifts of the aromatic protons of
MccC51 are the same as those observed for the H8 and H2 protons of the
MccC7 base, we think that MccC51 might also contain an adenine and not
the base corresponding to the nebularine nucleoside. Unfortunately,
Metlitskaya et al.(41) do not report the chemical
shifts of the aromatic-base carbons of MccC51, which could confirm or
invalidate this proposal.
The proton chemical shifts of the ribose
and the three-methylene chain are identical in MccC7 and MccC51
spectra, taking into account the 0.05 ppm correction. Moreover,
the phosphorus signal is coupled to the same proton signals in both
molecules spectra (H5`5" at 4.49-4.43 ppm and
(CH
)
at 4.23 ppm for MccC51). A comparison of the
phosphorus chemical shift is not possible because Metlitskaya and
co-workers (41) did not specify the phosphorus reference used
in their study. The ribose and three-methylene chain carbon chemical
shifts are also very similar for both molecules. (
)The only
significant difference is found for the methylene carbon that bears the
protons resonating at 3.13 ppm (MccC51 chemical shift), which resonates
at 54.5 ppm for MccC51 and at 39.1 ppm for MccC7. Such a big difference
in this carbon chemical shift while all the surrounding nuclei resonate
at the same position seems surprising to us. In the proposed structure
of MccC51, the phosphorus atom is implicated in a disubstituted
phosphate. However, the H5`5" (4.49-4.43 ppm) and the
phosphate-bound methylene (4.23 ppm) proton chemical shifts are in
disagreement with this structure. Indeed, in the phosphodiester of
5`-adenylic acid and n-aminopropanol (AMPap), which contains
this atom arrangement, the H5`5" and methylene protons are observed,
respectively, at 4.15 and 3.94 ppm (27 °C, D
O, pD 3.3).
With respect to the branching of the peptide and the C-terminal substituent of MccC51, no evidence is given either to support the decision to link them together via the side chain of the C-terminal residue rather than via the peptide main chain or to back the bridging of the peptide and the nucleotide via the three-methylene chain. As the chemical shifts of the C-terminal residue and the methylene protons are the same in the spectrum of MccC7 and MccC51, it is very likely that the peptide and the substituent are linked together in the same way in both molecules.
Finally, it was proposed that the threonine residue
of MccC51 contains a hydroxylamine group. Three arguments were put
forward to support this conclusion: (i) the observation of ``some
deviation from normal threonine mobility'' for the product of the
first cycle of the Edman degradation of the proteolytic peptide
``Thr-Gly-Asn-Ala-Asx.'' (ii) ``The mass spectrometry of
threonine-containing proteolytic fragments'' which ``revealed
the molecular mass increasing by 15'' (which fragments? what
proteases were used? Did the proteolytic fragments contain the
C-terminal substituent?). (iii) NMR data, which was not shown nor
specified. In the present work, we showed unambiguously that the
threonine residue of MccC7 is not modified (see ``Results'').
It is thus surprising to note that the chemical shift of the
proton signal of Thr, as well as that of its
and
protons
are identical for MccC7 and MccC51: the replacement of a hydrogen by an
amine group having an electronegative nitrogen should influence the
chemical shift of the surrounding protons. However, this might not be
the case and MccC7 and MccC51 might be different at this position.
Nevertheless, it should be pointed out that small variations in elution
time are often observed for amino acid phenylthiohydantoin derivatives
relative to reference compounds during the sequencing of peptides. In
addition, there is a priori no reason to expect that the
mobility of a threonine-phenylthiohydantoin derivative might be similar
to that of a phenylthiohydantoin derivative of a
hydroxylamine-substituted threonine.
In summary, although at present
we cannot know if MccC7 and MccC51 are the same molecule or not, they
are very similar. The comparative analysis of the results presented in
this work (obtained in different conditions of solvent and pH, with
many NMR experiences that produced complementary data, and with
synthetic analogs of the peptide and the C-terminal substituent of
MccC7), with those reported for MccC51 (41) (only in
DO, at a single pD value and with relatively few NMR data),
permits us to conclude that the chemical structure proposed for MccC51
is not correct. It is noteworthy that it has been suggested that the
antibiotic activity of MccC51 might be due, at least in part, to its
nebularine unit(41) , nebularine being a well known antibiotic (42) . However, as stated by Metlitskaya et
al.(41) , MccC51 and nebularine have different antibiotic
activity spectra. This apparent contradiction might be easily
explained, if as argued above, MccC51 does not contain a nebularine
unit.