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INTRODUCTION |
Staphylococci are the most common cause of bacterial infections in
the United States (1). Among these organisms,
methicillin-resistant Staphylococcus aureus has received
notoriety since it is currently the scourge of hospitals. Staphylococci
have acquired multiple drug resistance genes over the past few decades
such that methicillin-resistant S. aureus can usually be
treated only by glycopeptides, such as vancomycin (2), or by
oxazolidinones, such as linezolid (3). It is disconcerting that
variants of methicillin-resistant S. aureus that have become
at least partially resistant to glycopeptide (4) and oxazolidinone
antibiotics are being identified (5).
-Lactam antibiotics target penicillin-binding proteins
(PBPs)1 for inhibition.
Staphylococci have become resistant to
-lactam antibiotics by two
parallel mechanisms. First, they produce
-lactamases, enzymes that
hydrolytically destroy these antibiotics (6, 7). Second, they have
acquired a novel PBP (referred to as PBP 2a) that carries out the
physiological functions of the four existing PBPs in
staphylococci yet is not inhibited in vivo by any of the clinically used
-lactam antibiotics. The acquisition of the gene for
PBP 2a in staphylococci took place only once from an unknown source
(8).
The blaZ and mecA genes encode the staphylococcal
-lactamase and PBP 2a, respectively. Transcription of these genes is
regulated by signal-transducing integral membrane proteins BlaR and
MecR and their respective transcriptional repressor proteins BlaI and MecI. BlaR has a
-lactam-binding domain on the surface of the plasma
membrane and a zinc protease domain in the cytoplasm (Fig. 1) (9). The
-lactam antibiotic
acylates a specific active site serine on the cell surface domain,
which transduces a signal to the cytoplasmic domain. The
zinc-dependent cytoplasmic protease domain, which
hydrolyzes the repressor proteins, precipitates transcription of the
genes for the two resistance proteins. This proteolytic signaling
pathway is unique in bacteria (10). We have cloned, expressed, and
purified to homogeneity the cell surface sensor domain of the BlaR
protein (referred to as BlaRS hereafter). As an
unusual feature, this protein is carboxylated at the side chain of an
active site lysine (Lys-392; a carbamate on the side chain), which is
critical for active site serine acylation by the
-lactam antibiotic.
The kinetics of these processes are reported herein for the first time.
Furthermore, binding of the antibiotic to BlaRS entails a
significant conformational change, a process that is likely to play a
role in the signal transduction mechanism from the cell surface to the
cytoplasm.

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Fig. 1.
Signal-transduction system
for the onset of -lactam antibiotics
resistance in staphylococci. The green band is the
cytoplasmic membrane, which displays BlaR and PBP 2a on its external
surface. The -Lactamase (BlaZ) is excreted to the milieu in
staphylococci.
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EXPERIMENTAL PROCEDURES |
Materials--
Antibiotics and other reagents were purchased
from Sigma, unless otherwise stated. The growth medium was purchased
either from Difco Laboratories (Detroit, Michigan) or Fisher
Scientific. The chromatography media were either from Bio-Rad
Laboratories or Amersham Biosciences. Escherichia coli
DH5
was from Invitrogen, and E. coli BL21(DE3) and
plasmid pET24a+ were from Novagen. The radioactive sodium bicarbonate
(NaH14CO3; 58 mCi/mmol) was purchased from
Amersham Biosciences, and NaH13CO3 was from
Cambridge Isotope Laboratories. BOCILLIN FL, a derivative of penicillin
V, was purchased from Molecular Probes, Inc.
Cloning of the Gene for the Cell Surface Domain of S. aureus BlaR
Protein--
We used the plasmid pI258 from S. aureus as
the source of the blaR gene (11). We utilized high fidelity
Pfu Turbo polymerase (Stratagene®) to amplify the sequence
that corresponds to the periplasmic portion of the BlaR protein (amino
acids 331-385). Two oligonucleotide primers, Dir,
5'-ACATATGGGGCAATCCATAACTGATTATTATAATTA-3', and Rev,
5'-TATAAGCTTATTGGCCATTTAAAACACCCAT-3', were utilized for this purpose. These primers contain the recognition
sequences for NdeI and HindIII restriction
endonucleases, respectively (italicized in the nucleotide sequence).
The amplified fragment was separated by electrophoresis on a 1%
agarose gel and further purified using a ZymocleanTM gel DNA recovery
kit (Zymo Research, Orange, CA). This fragment was cloned into
HincII sites of plasmid pUC19, and the resulting construct
pUC19:blaRS was used to transform E. coli DH5
. Transformants were selected on agar plates
supplemented with
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-gal),
isopropyl-1-thio-
-D-galactopyranoside, and 100 µg/ml ampicillin. Plasmid DNA from several transformants was isolated, and
the presence of the inserted blaRS gene was
verified by digestion with NdeI and
HindIII. Subsequently, the nucleotide sequence of
the blaR gene was verified by sequencing of both DNA strands.
To overexpress the cell surface domain of the BlaR protein
(BlaRS) in cytoplasm, we released the corresponding DNA
fragment from the plasmid pUC19:blaRS by
HindIII and NdeI digestion and reinserted the
fragment into the HindIII-NdeI sites of the
expression vector pET24a(+). The DNA from the ligation mixture was used
to transform E. coli DH5
, and the plasmids from several
transformants were analyzed for the presence of the insert.
Subsequently, E. coli BL21(DE3) was retransformed with the
pET24a+ vector containing the cloned blaRS gene.
Mutational Alteration of Ser-389 and Lys-392 to Ala in the
BlaRS Protein--
The S389A and K392A mutant variants
were generated using the QuikChange site-directed mutagenesis
protocol (Stratagene®). Specifically, the
pUC19:blaRS vector was amplified using
Pfu Turbo DNA polymerase and two mutagenic primers,
Ser389AlaDir, 5'-GGTATTCTCCTAATGCAACTTATAAAATT-3',
and Ser389AlaRev, 5'-AATTTTATAAGTTGCATTAGGAGAATACC-3', to
introduce Ser-389 to Ala substitution. Two other mutagenic primers,
Lys392AlaDir, 5'-CTCCTAATTCAACTTATGCAATTTATTTAGCTATGTTTGG-3', and
Lys392AlaRev, 5'-CCAAACATAGCTAAATAAATTGCATAAGTTGAATTAGGAG-3', were used to
mutate Lys-392 to Ala. The PCR products were treated with restriction endonuclease DpnI that digests only the methylated
template DNA, and the resulting mixture was used to transform
E. coli DH5
. The nucleotide sequences of the entire
mutated blaRS genes were verified. These genes
were released by digestion with restriction enzymes NdeI and
HindIII, recloned into the corresponding sites of the
pET24a(+) vector, and transformed into E. coli BL21(DE3).
Purification of the BlaRS Protein--
A single
colony of E. coli BL21(DE3) harboring pET24a(+) vector with
the cloned blaRS gene (or the mutated versions)
was used to inoculate 5 ml of the LB medium containing 30 µg/ml
kanamycin A ,and bacteria were allowed to grow overnight at 37 °C.
The cell culture was diluted 100-fold into fresh Terrific Broth,
supplemented with 30 µg/ml kanamycin A. The culture was grown
at 37 °C to an A600 of ~0.8 and then
was cooled to 25 °C. Expression of the BlaRS protein was
induced by the addition of
isopropyl-1-thio-
-D-galactopyranoside to a final
concentration of 0.4 mM. The induced cultures were shaken
at 25 °C for 16 h.
The cells were removed by centrifugation at 8,300 × g
for 15 min. The pellet was suspended in 25 mM TES buffer,
pH 7.5, and the cytoplasmic content was liberated by sonication. The
suspension was centrifuged at 21,000 × g for 1 h,
and the supernatant was loaded onto a Macro-Prep S-support column
(2.5 × 20 cm) equilibrated with 25 mM TES buffer, pH
7.5. The BlaRS protein was eluted at 60% of a linear
gradient of 0-1.0 M NaCl in 25 mM TES buffer
(1.6 liters), pH 7.5. The fractions containing the protein were
concentrated and loaded onto a Sephacryl S-200 column (2.5 × 75 cm) equilibrated with 10 mM sodium phosphate buffer,
pH 7.0. Subsequent to elution of the protein from the column by the
same buffer, the protein-containing fractions were analyzed by
SDS-PAGE, and the protein was concentrated to 5 mg/ml and kept at
4 °C for further use. At this point, the protein was pure to
apparent homogeneity. The above operations were all carried out at
4 °C.
13C NMR Experiments--
The wild-type
BlaRS protein (10 mg) was dialyzed against several changes
of degassed 25 mM sodium acetate buffer (pH 4.5) and then
against degassed 10 mM sodium phosphate buffer, 0.1 mM EDTA, pH 7.5. Finally, the protein was dialyzed against
the last buffer containing 10% D2O and 20 mM
NaH13CO3 (as the source of CO2).
The protein was concentrated to give a 1.0 mM solution. The
13C NMR spectra of the wild-type BlaRS protein
modified by 13C-labeled carbon dioxide were collected at
25 °C. The same experiment was repeated with the K392A mutant
variant of the BlaRS protein. The procedure did not tamper
with the quality of the protein since we measured pseudo-first-order
rate constants for acylation of the protein by nitrocefin with and
without this treatment, which were the same.
Modification of BlaRS by
NaH14CO3--
A 50-µl portion of the 100 µM protein solution (either wild-type BlaR or the K392A
mutant variant) was diluted in 4 ml of degassed 25 mM
sodium acetate buffer (pH 4.5) and concentrated on an Ultrafree-4
centrifugal filter unit (Millipore) down to ~100 µl. Buffer
exchange was performed twice with the sodium acetate buffer and then
twice with degassed 100 mM sodium phosphate buffer (pH 7.5;
hereafter referred to as the "pH 7.5 buffer"). Concentration of
protein was measured at the end of this procedure
spectrophotometrically. A 30-µg portion of each protein was
reconstituted in the pH 7.5 buffer supplemented with 10 mM
unlabeled sodium bicarbonate and 4 mM
14C-labeled sodium bicarbonate (total volume 40 µl) and
was incubated at room temperature for 15 min. A 2-µl portion from the
reaction mixture was diluted with 28 µl of the pH 7.5 buffer, which
was supplemented to give a final concentration of 10 mM
unlabelled sodium bicarbonate, and the solution was passed through a
desalting column (Micro Bio-Spin 6; Bio-Rad) that was equilibrated with the same buffer (with bicarbonate supplement). A portion of this solution was used for the measurement of the radioactivity, along with
a control experiment, for which protein was withheld from the solution.
Determination of the Dissociation Constant of Carbon Dioxide and
the BlaRS Protein--
Binding of carbon dioxide to
Lys-392 of the BlaRS protein resulted in quenching of
protein fluorescence at 320 nm, when excited at 295 nm. Fluorometric
measurements were performed in a Spex Industries (Metuchen, NJ)
Fluoromax luminescence spectrophotometer. The slit width was kept at 1 nm for excitation and 1.5 nm for emission. Experiments were carried out
at 25 °C in 100 mM sodium phosphate buffer (pH 7.5).
Aliquots of concentrated NaHCO3, prepared in the same
buffer, were added to the protein solution (1 µM) to
provide the desired carbon dioxide concentration. The measurement of
the fluorescence signal was carried out after 2 min from the NaHCO3 addition, which was monitored over 2 min. The
experimental data were fit using the GRAFIT software (Erithacus
Software, Middlesex, UK) for a single binding site model by the
following quadratic equation,
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(Eq. 1)
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where Fo is the initial intrinsic
fluorescence of the protein,
F is the change in
fluorescence,
Fmax is maximum change in
fluorescence after saturation by carbon dioxide, [L]t is the
concentration of total carbon dioxide, and [E]t is the enzyme
concentration in the assay. The experiment was carried out two times,
and analysis of the data was according to literature methods (12, 13).
The same experiment was attempted with the mutant K392A
BlaRS protein.
Circular Dichroism Spectroscopy--
The CD spectra of the
wild-type BlaRS and the K392A and S389A mutant proteins
(2.0 µM each in 10 mM sodium phosphate, pH
7.0, supplemented with 50 mM sodium bicarbonate), were
recorded on a Jasco J-600 (Easton, MD) instrument (5-mm path length) in
the absence and presence of various
-lactam antibiotics. The
contribution of the substrate was subtracted in each case. The
concentrations of the
-lactams were generally 2-fold higher than the
respective Ks values. Prior to recording the spectra
of the proteins with a
-lactam antibiotic, the proteins were
incubated with the
-lactam antibiotics for 15 min at 25 °C.
Determination of the Kinetic Parameters for Interactions of
-Lactam Antibiotics with the BlaRS Protein--
The
BlaRS protein experiences acylation at the active site
serine, and the acyl-protein species slowly undergoes deacylation according to Reaction 1,
where the ratio
k
1/k1 equals
Ks, B represents the BlaRS protein,
B·I is the non-covalent preacylation complex, B-I is the covalent
acyl-protein species, and P denotes the product of hydrolysis of the
-lactam antibiotic.
The first-order rate constants for protein acylation
(k2) were determined for different
-lactam
compounds using a Cary 50 UV spectrophotometer (Varian Inc.) equipped
with an SFA-20 stopped-flow apparatus (Hi-Tech Scientific,
Salisbury, UK) at room temperature. The parameters for the
reaction between the BlaRS protein and nitrocefin were
determined directly by monitoring the formation of the acyl-enzyme
species at 500 nm (
500 = +15,900 cm
1
M
1) (14). The experiments were carried out in
100 mM sodium phosphate buffer supplemented with 50 mM NaHCO3, and sodium sulfate was added to
maintain an ionic strength of 0.3 M (pH 7.0).
The observed first-order rate constants (kobs)
were measured at a protein concentration of 1.0 µM and at
different concentrations of nitrocefin (5-20 µM). The
reactions were monitored for 15 s each, at which time the protein
was invariably acylated. The stopped-flow instrument had an intrinsic
delay time of 20 ms from the moment of mixing to the time of recording.
The observed first-order rate constants (kobs)
for acylation of BlaRS by nitrocefin were determined by
fitting the change in absorbance as a function of time to the following
equation: At = Amax(1
exp(
kobst)), where
At is the absorbance at time t and
Amax is the maximum absorbance reached after all
the protein is acylated (calculated from fitting of the progress curves with the above equation). The kobs values were
plotted against the nitrocefin concentrations to obtain the apparent
first-order rate constant (k2), as per the
equation: kobs = k2 × [S]/(Ks + [S]). The parameters for the K392A
mutant protein were calculated similarly.
Nitrocefin was used as a reporter molecule to determine the apparent
first-order rate constants for acylation by other non-chromogenic (or
poorly chromogenic)
-lactams in competitive reactions, as described
by Woodward-Graves and Pratt (15). The progress curves, obtained by
monitoring at 500 nm the formation of acyl-enzyme species between
nitrocefin and BlaRS protein (1 µM) at
different concentrations of the
-lactam, were fit to the
following equation: At = Amax{1
exp(
(kn + ki)t)}, where Kn is
the observed first-order rate constant at 12 µM
nitrocefin and Ki is the observed first-order rate
constant of the competing
-lactam at a particular concentration. The
Ki values were plotted against the
-lactam
concentrations, and the data obtained for each
-lactam were
processed using nonlinear regression analysis (GRAFIT) to calculate the
k2 and Ks values.
The deacylation rate constants were determined using BOCILLIN FL as a
reporter molecule (16). A typical reaction mixture (60 µl) contained
either 3 or 10 µM of BlaRS (the higher
concentration was used for the measurement of the deacylation rate
constant for more stable complexes) and a
-lactam antibiotic at a
concentration of at least 2-fold higher than its Ks
value. The mixture was incubated at 25 °C for 15 min in 100 mM sodium phosphate buffer, pH 7.0, supplemented with 50 mM NaHCO3. The excess of
-lactam antibiotic
was removed by passing the mixture through a Micro Bio-Spin®6 column
(Bio-Rad). An aliquot (3 µl) of the mixture was diluted 5-fold with
the buffer and incubated at different time intervals at 37 °C. The
amount of the free protein, liberated from the acyl-protein species,
was assayed by the addition of BOCILLIN FL to afford a final
concentration of 5 µM and incubated for an additional 5 min at 37 °C. A portion of the SDS sample buffer (15 µl) was added
to the reaction mixture, and it was boiled for 3 min. The samples (30 µl in total) were loaded onto 15% Laemmli SDS-PAGE, and the gel
was developed and scanned using Storm840® Fluorimager. The
fluorescent bands were quantified using the NIH IMAGE 1.62 software. A
control sample was also prepared in which the BlaRS protein
was incubated with BOCILLIN FL under the same conditions as with other
-lactam antibiotics, with the exception that the SDS buffer was
added immediately after a 5-fold dilution of the reaction mixture. This
control sample was used to determine the amount of the acylated protein
at time 0. The data were fit to the equation
k3t = ln[B-I]t/[B-I]0, where
[B-I]0 and [B-I]t are the concentrations of the
acylated BlaRS at time 0 and at time t,
respectively, calculated as [B-I]t = [B-I]0
[B-IB]t, where [B-IB]t is
the concentration of the acylated protein with BOCILLIN FL at time
t.
The activity of S389A mutant protein was assessed
spectrophotometrically in reaction with nitrocefin by monitoring the
formation of the acylated species at 500 nm and by fluorescence
detection after incubation with BOCILLIN FL. In subsequent experiments, the protein at 1.0 µM was incubated with BOCILLIN FL (5 µM) for 30 min at 37 °C. The reaction was quenched
with SDS sample buffer, and the sample was analyzed by
SDS-polyacrylamide gel electrophoresis.
Structure-based Computational Modeling--
The
Swiss-Model server (www.expasy.ch/swissmod) was used to
construct a model for the BlaRS protein. The procedure
consisted of first carrying out a BLAST (17) search of the ExNRL-3D
data base of sequences of known structures for suitable templates. A
pairwise sequence alignment of each template to the target sequence was
then carried out using the SIM method (18), and sequence identity was
determined after each alignment. Only those structures with 25% or
greater sequence identity to the target sequence were selected. The
ProMod package (19) was then implemented to use the information from
the SIM sequence alignments and the Cartesian coordinates from the
Protein Data Bank files of the templates to construct a
three-dimensional model of the desired protein.
Three sequences of known structures (Protein Data Bank accession codes:
1H8Y, 1EWZ, 1FOF) with 28% percent or more sequence identity to
BlaRS were found. The sequence alignment and resulting
model generated by the Swiss-Model server were analyzed with the Sybyl
6.7 (Tripos Inc., St. Louis, MO) software package. The
BlaRS three-dimensional model generated by the Swiss-Model
server was locally refined by molecular dynamics simulations using the
AMBER 7 suite of programs (20). The active site Lys-392 was
carboxylated in silico, for which derivation of the force
field parameters was described previously (21). The protein was fully
solvated in a box of TIP3P (22) waters, resulting in a total of 37,585 atoms. A short trajectory was carried out with the protein frozen to
equilibrate water molecules. This was followed by a series of energy
minimization procedures to gradually relax the protein. Subsequently, a
total of 200 ps of molecular dynamics simulation was carried out on the
system. Snapshots were collected every 2 ps, and an average structure
was generated by root-mean-square fitting of all the collected
snapshots to the initial structure. The fitted structures were then
averaged, and the resulting model was subjected to a 20,000-step
conjugate gradient energy minimization procedure.
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RESULTS AND DISCUSSION |
We cloned the sensor domain of the signal transducer protein BlaR
from S. aureus (spans amino acids 331-581), which we refer to as the BlaRS protein. The cloned protein was expressed
in the cytoplasm of E. coli. The protein was purified to
homogeneity in two chromatographic steps and was highly soluble (up to
26 mg/ml). We routinely obtain 50 mg of pure protein from one liter of
growth medium. The C terminus of BlaR from Bacillus
licheniformis has also been cloned (23, 24).
Carboxylation of Lysine Side Chain in the BlaR Protein--
The
BlaRS protein is related to the OXA family of
-lactamases, enzymes of resistance to
-lactam antibiotics (24,
25). The active site peptide sequence of
Ser-X-X-Lys, which is a known minimal motif for
these proteins that undergo acylation at the serine residue, is present
in both (25). The x-ray structures for the OXA-10
-lactamase (26,
27) reveal that the active site lysine is carboxylated on its side
chain (i.e. the carbamate product of reaction with carbon
dioxide). The side chain of lysine in the OXA-10
-lactamase is
sequestered in an unusual environment made up of five hydrophobic amino
acid side chains (Phe-69, Val-117, Phe-120, Trp-154, and Leu-155) that
is believed to lower the pKa of the lysine side
chain such that it exists in the free base form that undergoes reaction
with carbon dioxide (26). The side chain of the carboxylated lysine and
that of serine are in contact, and the former activates the latter for
enzyme acylation by
-lactam antibiotics (26). The requisite amino
acids in the Ser-X-X-Lys motif and the five
hydrophobic sites, among others, are conserved among the many OXA
-lactamases and the BlaR protein (25). A pertinent question now is
whether the sensor domain of the BlaR protein is also carboxylated at
the corresponding lysine residue.
A diagnostic test for carboxylation of the lysine side chain is by
13C NMR, which detects a distinctive signal. The
13C NMR experiment indicated that lysine carboxylation is
seen in the BlaRS protein, as shown by the presence of a
diagnostic resonance at 164 ppm (Fig. 2).
The same experiment was carried out with the K392A mutant protein, and
unexpectedly, we observed that the NMR signal at 164 ppm was not
entirely eliminated (Fig. 2B). The integrations of the
carbamate signals in Fig. 2 indicated that the wild type enzyme (Fig.
2A) had approximately two carboxylated lysines to one in the
mutant protein. Therefore, under the NMR experiment conditions, two
lysines in the wild-type protein exist in the free base forms,
which undergo carboxylation in the presence of the
13C-labeled carbon dioxide, one of which is at position
392.

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Fig. 2.
The 13C NMR spectra of the
wild-type BlaRS (A) and the K392A mutant
proteins (at 1 mM) (B) in 10 mM sodium phosphate, 0.1 mM EDTA,
supplemented with 20 mM
NaH13CO3.
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We resorted to binding of radioactive carbon dioxide to the
BlaRS and the K392A mutant proteins. Analogously to the
case of the OXA-10
-lactamases, the expectation was that the active
site carboxylated lysine would be stabilized by specific interactions. On the other hand, the other carboxylated lysine seen in the NMR experiment might have experienced carboxylation in an adventitious process and could be back-titrated by non-radioactive carbon dioxide. Here, each protein was incubated at pH 4.5 to facilitate the
decarboxylation of lysine followed by reconstitution of the protein by
the radioactively labeled carbon dioxide. The workup was made in the
presence of non-labeled carbon dioxide. We were able to measure an
average of 0.9 equivalents of radioactive label incorporated per each of the wild-type protein molecule. By the same procedure, no label was
introduced into the K392A mutant protein. This argued that carboxylation of the protein was indeed at the Lys-392 position and
that there may be another exposed lysine with reduced
pKa in this protein that may be partially
and unstably carboxylated under the conditions of the NMR
experiment (i.e. high carbon dioxide concentration in the
NMR tube). The cloned protein has a total of 31 lysine residues.
In the case of the OXA-10
-lactamase, proximity of one of the oxygen
atoms of the lysine carbamate to Trp-154 was useful in quantitative
fluorescence quenching studies of the interaction of carbon dioxide
with the enzyme (26). Tryptophan 475 of the BlaR protein corresponds to
Trp-154 of the OXA-10
-lactamase, so we felt that the dissociation
constant for carbon dioxide of the BlaRS protein may be
evaluated by fluorescence analyses. The intrinsic fluorescence of
BlaRS was quenched upon addition of sodium bicarbonate as
the source of carbon dioxide in a saturable fashion. Data fit to
Equation 1 revealed a Kd of 0.6 ± 0.2 µM (Fig. 3). Considering that the physiological concentration of carbon dioxide is 1.3 mM (28), this indicates that BlaRS is fully
carboxylated in vivo. It is significant to note that the
K392A mutant protein did not give the tryptophan fluorescence quench,
which is indicative of the fact that the dissociation constant that was
evaluated for the wild-type protein was for carboxylation of residue
392 and is a further validation that this residue is indeed
carboxylated in the wild-type protein. As will be described below,
carboxylated Lys-392 is the active site base that activates the serine
for protein acylation. This is now only the second example of a
protein, after the OXA-10
-lactamases (26), that uses the highly
uncommon carboxylated lysine as a basic residue to facilitate reactions
in the active site. The few other proteins having carboxylated lysine
use the modified amino acid as metal ligand or for hydrogen bonding in the protein structure.

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Fig. 3.
Relative quenching of the intrinsic
tryptophan fluorescence of BlaRS protein (1 µM) versus total
concentration of carbon dioxide (µM). Data were fit
to a single binding site model as per the equation described under
"Experimental Procedures."
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The collective information in the preceding paragraphs made possible
the generation of a homology-based computational model for the sensor
domain of the BlaR protein (Fig. 4). In
comparison with the structure of the OXA-10
-lactamase, the
arrangements of the side chains of serine and the carboxylated lysine
and the hydrophobic environment around the lysine, including the
proximity of the carboxylated lysine and the tryptophan residue, are
preserved.

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Fig. 4.
A, stereo view of the
computational model for the -lactam binding site of the BlaR
protein. A Connolly water-accessible surface (in green)
was constructed around the hydrophobic pocket of the binding site
residues. The side chains of the residues that make up the hydrophobic
pocket are shown in orange-capped sticks. Carboxylated
Lys-392 (shown in the middle of the Connolly surface),
Ser-389, and Trp-475 are color-coded according to atom types
and shown in capped-sticks representation (white,
red, and blue correspond to carbon, oxygen, and
nitrogen, respectively). Hydrogen bonding interactions between
carboxylated Lys-392 and Ser-389 and Trp-475 are represented with a
white dashed line. The protein is shown in a purple
tube representation. In B, a similar perspective from
the x-ray structure for the OXA-10 -lactamase is given for
comparison.
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Kinetics of Interactions of
-Lactam Antibiotics and the
BlaRS Protein--
In light of the information that the
sensor domain of the BlaR protein has a carboxylated lysine, it is
conceivable that the protein at the end of each individual purification
protocol would be carboxylated to varying degrees, since the process is
reversible. This point was documented by observing typically a 2-fold
enhancement of the rate of interactions of the BlaRS
protein with
-lactam antibiotics by supplementation of the buffer with sodium bicarbonate (as a source of carbon dioxide). As argued above, the BlaR protein is fully carboxylated in vivo, and
the fact that some of the carboxylation of the protein is reversed during the purification is an artifact. Therefore, we have supplemented the reaction mixtures for the kinetic studies with bicarbonate to
generate the fully carboxylated and active form of the protein for all
kinetic determinations.
BOCILLIN FL, a fluorescent penicillin, was used to further study and
analyze the mode of action of BlaRS protein. This molecule
modifies BlaRS covalently, as would any
-lactam
antibiotic, whereby the protein would migrate through an
SDS-polyacrylamide gel to allow quantitative detection by Fluorimager.
Titration of the BlaRS protein with BOCILLIN FL revealed
saturation and also indicated a one-to-one modification of the protein
by the antibiotic. The wild-type and K393A mutant BlaRS
proteins were acylated by BOCILIN FL, as revealed by Fluorimager. The
K393A has a residual level of activity (see below) that accounts for this observation. In contrast, incubation of the S389A mutant variant with BOCILIN FL did not give a fluoregenic band. A previous study based on sequence analysis of the BlaR from B. licheniformis with a class D
-lactamase had suggested that
residue Ser-389 (BlaR numbering according to S. aureus)
might be the modification site by
-lactams (23). The experiments
reported herein clearly reveal Ser-389 to be the serine-active site
residue that is acylated.
The kinetics of interactions of several
-lactam molecules
(three penicillins, three cephalosporins, and one carbapenem) with the
BlaRS protein were investigated (Table
I). Stopped-flow kinetics distinguished between a rapid enzyme acylation event and a substantially slower deacylation step. Acylation of the active site proceeded with microscopic rate constants (i.e. k2)
of 1-26 s
1, which indicate rapid t1/2
values for acylation of 27-690 ms for the
-lactams that we studied. The deacylation rate constants (i.e.
k3) for the same
-lactam molecules are listed
in Table I, corresponding to t1/2 values of
~12-240 min. The k3 for oxacillin would
appear to be representative of most of the substrates studied, with a
value of (4.8 ± 0.6) × 10
5 s
1,
which corresponds to a t1/2 value of 240 min. In
light of the fact that typical strains of S. aureus double
their population sizes in 20-30 min under favorable growth conditions,
this indicates that a single acylation event per each molecule of the
-lactam signal sensor-transducer protein accounts for the biological
consequences per each generation of bacterial growth.
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Table I
Kinetic parameters of BlaRS with the -lactam antibiotics
The rate constants were determined in the presence of 50 mM
NaHCO3 at pH 7.0. The concentration of the protein in the
assays was 1 µM. The kinetic parameters for substrates
other than nitrocefin were determined in competition experiments with
nitrocefin. (See "Experimental Procedures" for more details.)
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It is a feature of the signal sensor-transducer protein that it is
activated by all
-lactam antibiotics (29). Consistent with this
information, the dissociation constants (i.e.
Ks) for various
-lactam antibiotics are in the
micromolar range, which are attainable in the milieu where the bacteria
grow. The dissociation constants are practically in the same range for
the three penicillins and three cephalosporins that we tested, whereas the carbapenem imipenem shows a higher value (Table I). The
second-order rate constants (i.e.
k2/Ks) for the encounter of
the
-lactam molecules and the BlaRS protein were
typically 104 to 106
M
1 s
1, indicative of a very
favorable process.
As per the computational model and the foregoing evidence, we decided
to evaluate the effect of Lys-392 on the kinetics of BlaRS
acylation. In the case of the OXA-10
-lactamase, the mutational change of the corresponding carboxylated lysine resulted in an inactive
enzyme that did not experience acylation in the active site by the
-lactam antibiotics (26). Similarly to the case of the OXA-10
-lactamase, a mutational change of Lys-392 to Ala in
BlaRS resulted in a protein that was severely impaired in
acylation of the active site serine as evaluated for oxacillin
(k2 = 0.0026 ± 0.0005 s
1 and
Ks = 43 ± 14 µM). The rate
constant for acylation was attenuated for the mutant protein by
6730-fold with no change in Ks. Since the mutant
protein has the same conformation as the wild-type protein by circular
dichroic analyses (see below), the attenuation on
k2 may be ascribed to poor activation of serine in the mutant protein.
Conformational Change in the BlaRS Protein--
Signal
transduction from one side of the membrane to the other necessitates
communication between the surface and the cytoplasmic domains. A means
to this communication is by conformational change of the membrane-bound
protein after binding to the
-lactam antibiotic. As shown in Fig.
5, the BlaRS protein is prone
to significant conformational change on binding to the
-lactam
antibiotics. The conformational change commences upon binding to the
-lactam antibiotic at the preacylation complex and reaches its full
extent on protein acylation. If the acyl-protein species is allowed to
undergo its sluggish deacylation, the protein returns to the native
conformation (data not shown). As indicated earlier, the K392A mutant
variant of the BlaRS protein is severely deficient in the
acylation step. This mutant variant would not experience acylation by
-lactam antibiotics during the course of the CD experiment.
However, the non-covalent binding by a
-lactam antibiotic, for
example by oxacillin (Fig. 5B), resulted in a discernable
change in the CD spectrum of the protein (similar results were seen
with the S389A mutant protein, which does not have the opportunity to
give the acyl-protein species; see Supplemental Material). The minima
at 208 and 222 nm, which are due to the helices, were enhanced, and the
maximum at 195 nm, due to
-sheets, sharpened (Fig. 5B).
These data argue for the enhancement of secondary structures (helicity
and
-sheets) in the protein on non-covalent binding by the
antibiotic. Upon acylation of the protein by oxacillin (Fig.
5A), these effects were enhanced further, but the native
state returned upon deacylation. Similar results and trends were noted
for all
-lactam antibiotics shown in Table I (see Supplemental
Material), so the effects of the conformational change on the
BlaRS protein are shared by all of these antibiotics. In
light of the fact that this conformational change is significant and is
generally seen regardless of the nature of the
-lactam antibiotic,
we believe that it is likely that it plays a role in the signal
transduction process. However, we acknowledge the fact that in the
whole cell context, other factors may play a role as well.

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Fig. 5.
Circular dichroic spectra of the
wild-type BlaR protein (2 µM, solid line) and
the wild-type BlaRS protein (2 µM) incubated
with oxacillin (30 µM, broken line)
(A) of the K392A mutant variant of the BlaRS
protein (2 µM, solid line) and the K392A
mutant (2 µM) incubated with oxacillin (30 µM, broken line) (B) and of the
S389A mutant variant of the BlaR protein (2 µM,
solid line) and the S389A mutant (2 µM)
incubated with oxacillin (30 µM, broken line)
(C). All the spectra were corrected for the small
contribution from the antibiotic in the mixture.
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The essence of signal transduction is the switch between inactive and
active forms of a given protein. In the case of the
-lactam signal
sensor-transducer, a key question is how binding of the
-lactam
antibiotic to the sensor domain facilitates signal transduction. Two
classical models for signal transduction have been proposed (30). In
one, ligand binding induces the formation of a new conformation in the
protein. In the other, an equilibrium mixture of conformational states
exists, and the ligand binding shifts the equilibrium in favor of the
active form. The data presented here for the BlaR protein are
consistent with either the induced-fit or the population shift model.
It is conceivable that the protein switches its conformation on binding
by the
-lactam antibiotic during the formation of the preacylation
complex, which reaches its maximal effect after acylation of the enzyme
in the active site (the induced-fit model). Alternatively, the CD
spectrum of the native BlaR protein may be due to the distribution of
several preexisting conformational states, which binding of the
-lactam antibiotics to one would shift the equilibrium in favor of
the active structure (the population shift model). The discrimination between these two models for the case of the BlaR protein should await
availability of structural information in the future.
We have described in this report the dynamic nature of the sensor
domain of the BlaR protein from staphylococci. This protein undergoes
structural rearrangement on binding to a wide range of
-lactam
antibiotics, the implications of which for the signal transduction
event remain to be studied by structural biologists. We have shown that
-lactam antibiotics modify the protein covalently and essentially
irreversibly within a bacterial population doubling time at Ser-389.
The covalent modification of the sensor domain is facilitated by an
uncommon carboxylated lysine at position 392 within the antibiotic
binding site. The means by which the BlaR system carries out its signal
sensing and transducing processes is unique to Gram-positive bacteria.
We have provided herein insights into how this specific protein in
S. aureus facilitates the manifestation of resistance to
-lactam antibiotics, a process that remains a challenge in clinical
treatment of infections caused by this organism.