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INTRODUCTION |
-Lactamases are bacterial enzymes that confer resistance to
-lactam antibiotics, which include the penicillins and
cephalosporins. These drugs act by inhibiting bacterial penicillin
binding proteins (PBPs)1 that
are essential for the synthesis of the bacterial peptidoglycan layer
(1). Several resistance mechanisms have evolved in bacteria to protect
them from the lethal effects of
-lactam antibiotics.
-Lactamase imparts resistance to the
-lactams by hydrolyzing the
amide bond in the four-membered
-lactam ring (2). Genes encoding
-lactamases may be found on plasmids, transposons, and on the
bacterial chromosome (2-6). There are four classes of
-lactamases
(classes A-D), categorized by their primary sequence homology.
-Lactamase-mediated resistance has increased because of
selective pressure from widespread use of
-lactam antibiotics and is
now a serious threat to antibiotic therapy (7).
TEM-1
-lactamase, encoded by the blaTEM-1
gene, is among the most prevalent plasmid-encoded
-lactamases found
in Gram-negative bacteria (8). The name TEM is derived from the name of
the patient carrying the pathogen from which the enzyme was isolated (9). It efficiently hydrolyzes penicillins and most cephalosporins (2).
However, it is a poor catalyst for hydrolysis of the newer third-generation cephalosporins, such as ceftazidime, that were designed to circumvent TEM-1-mediated inactivation.
In addition to developing new drugs that are unable to be cleaved by
-lactamase, another method to counter the hydrolytic activity of
this enzyme has been to administer
-lactamase inhibitors, such as
sulbactam and clavulanic acid. Use of an inhibitor along with an
existing
-lactam antibiotic is an effective means to treat various
-lactamase producing bacterial pathogens (10). Clavulanic acid was
originally found as a metabolite of the Gram-positive soil bacterium
Streptomyces clavuligerus and, today, is one of the most
commonly used
-lactamase inhibitors.
-Lactamase-mediated resistance has been exacerbated by the fact that
specific mutations in TEM-1
-lactamase enable the enzyme to
hydrolyze the newer third-generation antibiotics (11, 12). These
evolved
-lactamases, called extended spectrum
-lactamases (ESBLs), provide clinically relevant levels of resistance to even the
most recently developed
-lactams. Furthermore, other mutants have
been found with substitutions that allow
-lactamase to avoid inactivation by the
-lactamase inhibitors (13). A similar result has
been observed with SHV-1
-lactamase, another class A enzyme that is
68% identical to TEM-1
-lactamase (7). Recently, a
-lactamase
mutant (TEM-50) has been recovered from clinical isolates with both
types of mutations, enabling
-lactamase to hydrolyze extended
spectrum antibiotics and avoid inactivation by inhibitors (14).
In 1990, the
-lactamase inhibitor protein (BLIP) was isolated
from S. clavuligerus culture supernatants (15). BLIP is a 165-amino acid protein in its secreted form and is a potent inhibitor of TEM-1
-lactamase (Ki = 0.6 nM)
(16). BLIP is able to inhibit several
-lactamases, as well as weakly
inhibit a penicillin-binding protein (PBP) from Enterococcus
faecalis (16). The DNA sequence and crystal structure of BLIP
have been determined, as well as the co-crystal with TEM-1
-lactamase (16, 17).
The BLIP crystal structure shows that the protein has a novel fold with
two similar domains. The BLIP mechanism of inhibition appears to be
two-fold. At 2636 Å2, the surface area of the
enzyme/inhibitor interface is one of the largest known for
protein/protein complexes as BLIP essentially clamps over the active
site of
-lactamase (17). In addition, an aspartic acid residue at
position 49 of BLIP aligns itself in the active site pocket and forms
strong hydrogen bonds with four catalytic residues of
-lactamase.
Furthermore, a phenylalanine at position 142 of BLIP appears to mimic
the benzyl group of the
-lactam antibiotic penicillin G (PenG) and
further stabilizes the inhibitory complex (17). The result is potent
inhibition of TEM-1
-lactamase.
The ongoing problem of targeting bacteria with antimicrobial agents
able to circumvent ESBL-mediated antibiotic inactivation creates a need
for new potent inhibitors of
-lactamases. The ability to engineer
BLIP to tightly bind the ESBLs and PBPs would aid in the design of
effective new antimicrobials. New protein-protein interactions found to
be effective in inhibiting
-lactamases and PBPs could be duplicated
by a peptide inhibitor or synthetic compound designed to mimic the
interactions. It would be easier to perform such studies with BLIP if
the inhibitor could be expressed in a more manageable genetic and
molecular system, such as Escherichia coli. This report
describes the efficient expression of functional BLIP in E. coli and the use of this system to determine the importance of
residues Asp-49 and Phe-142 for BLIP inhibition of TEM-1
-lactamase, two extended spectrum TEM-1 mutants, and the SHV-1
-lactamase.
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EXPERIMENTAL PROCEDURES |
Strains and Plasmids--
E. coli XL1-Blue
(recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1, lac,
(F' proAB, lacIq lacZDM15,
Tn10(tetr))) was used to propagate plasmid DNA
(Stratagene, Inc.). E. coli RB791 (= strain W3110
lacIQL8) was used to express BLIP and the D49A
and F142A BLIP mutants (18, 19). Plasmid pTP123 is a cmpr
amps derivative of pTrc 99A (Amersham Pharmacia Biotech)
(see Fig. 1). It was created by ligating the HincII cassette
from pKRP10 into BsaI- digested pTrc 99A (20). The
BsaI site was filled in using the Klenow fragment of DNA
Polymerase I prior to ligation. This cloning step inserts a
chloramphenicol acetyltransferase (cat) gene into the
rrnBT1T2 transcriptional terminators and part of the
-lactamase gene encoded by pTrc 99A. As a result, functional
-lactamase is not expressed, and potential difficulty in BLIP purification because of binding of endogenous
-lactamase is avoided. The cat gene in pTP123 is in the same orientation as the
trc promoter.
BLIP Cloning and PCR Mutagenesis--
A 6-histidine (6XHis) tag
was first inserted between the
-lactamase signal sequence and the
BLIP coding sequence of pG3-BLIP (21) by overlapping PCR mutagenesis
(22). The internal mutagenic PCR primer sequences are: BLIPHIS-1 (a top
strand primer),
5'-CACCACCACCACCACCACGCGGGGGTGATGACCGGGGCGAAG-3'; and BLIPHIS-2
(a bottom strand primer),
5'-CGCGTGGTGGTGGTGGTGGTGTTCTGGGTGAGCAGCAAAAACAGGAAGGCA-3'. The external
PCR primers used to amplify the construct are: PD-bla1 (top strand,
N-terminal), 5'-CGGGGAGCTCGTTTCTTAGACGTCAGGTGGC-3'; and MALBLI-2
(bottom strand C-terminal), 5'-GGGAAATCTAGATTATACAAGGTCCCACTGCCG-3'. A
SacI site in PD-bla1 and a XbaI site in MALBLI-2
allowed the PCR product to be cloned into SacI- and
XbaI-digested pTP123 following treatment of the vector with
calf intestinal alkaline phosphatase. The final
SacI/XbaI fragment contains, from 5' to 3', the
TEM-1
-lactamase constitutive promoter, the TEM-1
-lactamase
signal sequence, and a 6XHis tag at the N terminus of BLIP followed by the mature BLIP coding sequence. The sequence of this clone was confirmed by the dideoxy chain-termination method and was named pGR32
(Fig. 1). The positioning of this
construct in pTP123 allows the N-terminal His-tagged BLIP to be
expressed either under the TEM-1
-lactamase constitutive promoter or
by induction of the trc promoter with IPTG. The 6XHis tag
facilitates the purification of BLIP using an appropriate nickel- or
cobalt-based affinity column, while the
-lactamase signal sequence
enables BLIP to be transported to the periplasmic space, thus
eliminating the need to isolate whole cell extracts for BLIP
purification.

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Fig. 1.
Plasmid maps for pTP123 and pGR32. The
BLIP cassette, possessing an N-terminal His-tagged BLIP gene, under
control of the -lactamase promoter, was inserted into
SacI/XbaI-digested pTP123. This construct also
features the -lactamase signal sequence fused to the N terminus of
the His-BLIP construct.
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PCR Mutagenesis--
Construction of the BLIP D49A and F142A
mutants was accomplished by overlapping PCR mutagenesis (22). PD-bla1
and MALBLI-2 were used as external primers in these mutagenesis
reactions. The internal top and bottom primers for constructing the
D49A mutant are: BP-D49A (top),
5'-CGGGGGCACGCGGCAGGGGCTTACTACGCCTACGCCACC-3'; and D49A (bottom),
5'-GGTGGCGTAGGCGTAGTAGGCCCCTGCCGCGTGCCCCCG-3'. The internal top and
bottom primers for constructing the F142A mutant are: BP-F142A (top),
5'-GGTTACTCGTCGACGGGGGCTTACCGAGGCTCGGCGCA-3'; and F142A-BOT
(bottom), 5'-GTGCGCCGAGCCTCGGTAAGCCCCCGTCGACGAGTAACC-3'. PD-bla1 and
MALBLI-2 were used to amplify the full-size mutagenized product. Both
mutagenic PCR products were cloned into
SacI/XbaI-digested and calf intestinal alkaline
phosphatase-treated pTP123. The plasmid containing the D49A mutant was
named pJP128, and the plasmid containing the F142A mutant was named
pJP129. The DNA sequence of each mutant was confirmed by the dideoxy
chain-termination method.
BLIP and
-Lactamase Expression and Purification--
Plasmids
pGR32, pJP128, and pJP129 were transformed into E. coli
RB791 by electroporation. An overnight culture of each was grown
shaking in 40 ml of Luria-Bertani (LB) medium at 37 °C in the
presence of 12.5 µg/ml chloramphenicol. The 40 ml of overnight culture were used to inoculate 2 liters of LB medium containing 12.5 µg/ml chloramphenicol. The bacteria was then grown shaking at
25 °C until A600 = 1.2. For induction of
BLIP, 3 mM IPTG was added to each culture, and the cultures
were then allowed to grow an additional 5 h.
Following the 5-h induction, the cells were pelleted and resuspended in
15 ml of sonication buffer (20 mM Tris-HCl (pH 8.0) and 500 mM NaCl). The cells were then sonicated in two batches, and
insoluble material was pelleted by centrifugation. The soluble protein
in the supernatant was purified over a 4-ml TALON column (CLONTECH) according to the manufacturer
instructions. A 4 mM imidizole wash step was utilized to
remove protein from the column which bound less tightly than the
His-tagged BLIP. BLIP was eluted using an elution buffer consisting of
50 mM imidizole added to the sonication buffer (pH 8.0).
Fractions were examined by SDS-PAGE to estimate purity and yield
(Fig. 2). Approximately 500 µg of >90% pure BLIP could be isolated for every two liters of culture using this strategy. Quantitative amino acid analysis was performed to
calibrate a Bradford assay for determining BLIP and
-lactamase concentrations (23).

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Fig. 2.
BLIP was purified to >90% homogeneity.
BLIP and the D49A and F142A mutants were purified in a single step
using a TALON (CLONTECH) His-tag affinity column.
First lane, molecular mass marker (M. Wt., sizes
indicated to the left); second lane, wild-type
(W.T.) BLIP eluate from column; third lane, D49A
eluate; fourth lane, F142A eluate.
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Wild-type
-lactamase and the G238S and E104K extended spectrum
mutants were expressed and purified as described previously (24). The
location of the two extended spectrum mutations, with respect to Asp-49
and Phe-142 of BLIP, is shown in Fig.
3.

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Fig. 3.
Crystal complex between BLIP and TEM-1
-lactamase. BLIP amino acids Phe-142 and
Asp-49 are shown in orange and are situated in the
-lactamase active site pocket. TEM-1 residues Glu-104 and Gly-238
are shown in green. S70, the residue responsible for
acylation of -lactams, is colored purple as an active
site landmark. The figure is based on the structure in Ref. 17.
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BLIP Inhibition Assay--
Varying concentrations of BLIP were
incubated with 1 nM
-lactamase for 2 h at 25 °C.
2 nM of the
-lactamase were used in the G238S studies.
The enzyme-inhibitor incubation was done in 0.05 M
phosphate buffer (pH 7.0) containing 1 mg/ml bovine serum albumin.
Following the 2-h incubation, which is sufficient time to achieve
binding equilibrium in a small volume, cephaloridine was added at a
concentration of at least 10-fold lower than the Km
of the
-lactamase being tested (e.g. wild-type TEM-1
-lactamase has a Km of approximately 700 µM for cephaloridine, therefore 70 µM
cephaloridine was added to the TEM-1/BLIP incubation). The final volume
for the reaction was 0.5 ml. Hydrolysis of cephaloridine was monitored
at A260 on a Beckman DU70 spectrophotometer. The extinction coefficient used for cephaloridine was 
= 10,200 M
1 cm
1 (24). Plots of the
concentration of free
-lactamase versus inhibitor
concentration were fit by nonlinear regression analysis to Equation 1,
where [Efree] is the concentration of active
-lactamase calculated from the measured velocity and the activity
and concentration of uninhibited
-lactamase,
[Eo] is the total
-lactamase concentration, and [Io] is the total inhibitor concentration
(25). From the equation, apparent equilibrium dissociation constants
(Ki*) were determined.
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(Eq. 1)
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RESULTS |
Wild-type BLIP Binding--
To determine whether the histidine tag
affects BLIP inhibitory activity and to test the activity of the BLIP
mutants against TEM-1, the ESBLs, and SHV-1
-lactamase, an inhibitor
assay was developed using the cephalosporin cephaloridine as a
substrate. Wild-type or mutant BLIP was incubated with a target for
2 h, which is sufficient time to achieve equilibrium. After
the 2-h incubation, cephaloridine (at a concentration 10-fold less than the cephaloradine Km for the
-lactamase being
tested) was added. Monitoring the hydrolysis of cephaloridine at a
concentration below Km allowed the concentration of
uninhibited
-lactamase to be determined without shifting the binding
equilibrium. The concentration of free
-lactamase was calculated
from the cephaloridine activity in the presence of a given quantity of
BLIP, the cephaloridine activity in the absence of BLIP, and the known
molar concentration of
-lactamase being used. Fitting the data
obtained when incubating varying concentrations of wild-type,
His-tagged BLIP with 1 nM TEM-1
-lactamase resulted in a
Ki of 0.11 nM
(Fig. 4, Table
I). This value compares reasonably well
with the previously reported value of 0.6 nM found with
BLIP purified from S. clavuligerus (16) and suggests that
the N-terminal 6XHis tag has little effect on the binding of the
inhibitor to the TEM-1 enzyme.

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Fig. 4.
Determination of wild-type and mutant BLIP
Ki values for TEM-1
-lactamase. BLIP inhibitory activity is
expressed as the remaining concentration of free -lactamase at
varying inhibitor concentrations. TEM-1 concentration is 1 nM, and cephaloridine concentration is 70 µM
for all experiments. The lines represent the nonlinear
regression fit of the data to Equation 1 to calculate the
Ki. Filled circles, wild-type BLIP;
open circles, D49A; and filled squares, F142A.
Each point is the average of at least three independent
experiments.
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Table I
Inhibition constants (Ki) for wild-type BLIP and the D49A and
F142A mutants with TEM-1, G238S, E104K, and SHV-1 -lactamases
All values are expressed in nM. Error limits are the
standard deviation of the parameter values obtained by non-linear least
squares fitting to Equation 1. ND, value not determined.
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To determine whether wild-type BLIP has similar affinity for extended
spectrum
-lactamases, the Ki of BLIP for two representative ESBLs was determined. The G238S
-lactamase mutation is found in many extended spectrum enzymes (26). This single mutation
increases the catalytic efficiency of
-lactamase for the third
generation cephalosporins ceftazidime and cefotaxime approximately 70- and 40-fold, respectively (27). The E104K mutation, likewise, has been
found in many extended spectrum
-lactamase variants (26). This
mutation increases the catalytic efficiency of
-lactamase
approximately 50-fold for ceftazidime and 10-fold for cefotaxime (28).
Both Gly-238 and Glu-104 are located at the binding interface of BLIP
and
-lactamase (17). Wild-type BLIP was found to have a
Ki of 0.07 nM for G238S, and a
Ki of 140 nM for E104K
(Figs. 5 and
6, Table I). These values suggest that
the G238S mutation has little effect on the binding of wild-type BLIP
to
-lactamase, whereas the E104K mutation interferes with binding in
such a way that the Ki increases 1000-fold.

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Fig. 5.
Determination of wild-type and mutant BLIP
Ki values for G238S
-lactamase. BLIP inhibitory activity is
expressed as the remaining concentration of free -lactamase at
varying inhibitor concentrations. G238S concentration is 2 nM, and the cephaloridine concentration is 7 µM for all experiments. The Ki was
determined as in Fig. 4. Filled circles, wild-type BLIP;
open circles, D49A; and filled squares,
F142A.
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Fig. 6.
Determination of wild-type and mutant BLIP
Ki values for E104K
-lactamase. BLIP inhibitory activity is
expressed as the remaining concentration of free -lactamase at
varying inhibitor concentrations. E104K concentration is 1 nM, and the cephaloridine concentration is 70 µM for all experiments. The apparent
Ki was determined as in Fig. 4. Filled
circles, wild-type BLIP; open circles, D49A;
and filled squares, F142A.
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Mutant BLIP Binding--
The x-ray structure of the BLIP·TEM-1
-lactamase complex shows that BLIP residues Asp-49 and Phe-142 mimic
portions of the
-lactam PenG when bound to
-lactamase (17). This
structural mimicry suggests that these residues maintain important
interactions in the inhibitory complex. To determine the contribution
of these amino acids to inhibition of TEM-1
-lactamase and extended
spectrum-hydrolyzing
-lactamases, the D49A and F142A mutants were
constructed. The Ki of each was determined with
TEM-1, E104K, and G238S
-lactamases (Figs. 4-6; Table I).
Both the D49A and F142A mutants exhibited an approximately
100-300-fold increase in Ki compared with wild-type
BLIP inhibition of TEM-1
-lactamase. The D49A mutant inhibits TEM-1 with a Ki of 8.3 nM, whereas the F142A
mutant inhibits with a Ki of 33 nM. To
ascertain whether these mutations affect inhibition in an additive
manner, the D49A/F142A double mutant was constructed. However, we were
unable to express the double mutant under the same strategy used with
wild-type BLIP and the other single amino acid mutants.
The interactions of the wild-type, D49A, and F142A BLIP inhibitors with
the ESBLs show that BLIP binds G238S with similar strength as that of
wild-type TEM-1, but binds E104K weakly. The Ki
values found for D49A and F142A mutants with G238S
-lactamase were
similar to the Ki values found for the BLIP mutants
binding TEM-1. The D49A BLIP mutant inhibited G238S with a
Ki of 9.4 nM. Therefore, as with TEM-1,
the D49A mutation reduced inhibition 100-fold. Likewise, the F142A BLIP bound G238S
-lactamase approximately 800-fold weaker, with a Ki of 55 nM. The fact that these two
substitutions in BLIP have a similar effect on the
Ki values for the TEM-1 and G238S
-lactamases
suggests that BLIP inhibits G238S in much the same way it inhibits
TEM-1.
Further experiments showed that Asp-49 and Phe-142 do not contribute to
the binding of E104K
-lactamase as they do to the binding of TEM-1
and G238S. The Ki of BLIP D49A with E104K
-lactamase is 1.5 µM, which is only a 10-fold increase compared with that of the wild-type BLIP interaction with E104K. This suggests that BLIP residue Asp-49 is not as critical to inhibition of E104K
-lactamase as it is to the other enzymes tested. This observation is even more pronounced with the BLIP F142A substitution in
that there was little change from the wild-type BLIP
Ki with the BLIP F142A mutant inhibiting E104K
(Ki (F142A) = 240 nM). Therefore, in
contrast to their effect on TEM-1 and G238S binding, the D49A and F142A
substitutions do not have as detrimental an effect on the
Ki for E104K
-lactamase.
BLIP Binding to SHV-1
-Lactamase--
The amino acid sequence
of SHV-1 is 68% identical to TEM-1
-lactamase (7). How this
similarity corresponds to structure homology is unknown as the crystal
structure of SHV-1
-lactamase has not yet been solved. Both the
TEM-1 and SHV-1 enzymes hydrolyze a similar profile of penicillins and
cephalosporins. It is not clear whether the homology between the two
enzymes implies that BLIP should inhibit both equally well. It may be
that even slight differences in the three-dimensional structure of
SHV-1 compared with TEM-1 would effect BLIP binding considerably. These
issues were addressed by performing an additional inhibitory assay with wild-type BLIP and SHV-1
-lactamase. SHV-1 was purified to greater than 90% homogeneity (data not shown) and was bound to increasing concentrations of wild-type BLIP. The Ki of BLIP for SHV-1 was found to be 1.0 µM, 9,000-fold higher than what
was found for TEM-1 (Fig. 7, Table I).
Therefore, the high degree of identity between these enzymes does not
translate to similar binding properties by BLIP.

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Fig. 7.
Determination of wild-type BLIP
Ki for SHV-1
-lactamase. BLIP inhibitory activity is
expressed as the remaining concentration of free -lactamase at
varying inhibitor concentrations. SHV-1 concentration is 1 nM, and the cephaloridine concentration is 70 µM for all experiments. The apparent
Ki was determined as in Fig. 4.
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DISCUSSION |
The development of novel inhibitors for
-lactamases as well as
penicillin-binding proteins would provide new options for the treatment
of bacterial infections. As
-lactamases are believed to have evolved
from PBPs, it is conceivable that minor changes in the structure of
BLIP could enable it to bind and inhibit PBPs (29). It has already been
observed that wild-type BLIP weakly inhibits PBP5 from
Enterococcus (16). Understanding how the amino acid sequence
of BLIP encodes its tight binding affinity for certain
-lactamases,
and its weaker affinity for PBPs, would facilitate the development of
novel inhibitors with potent activity for the ESBLs and for the PBPs.
The identification of amino acids most responsible for inhibition and
those critical for binding specificity will pinpoint the residues that
need to be targeted for engineering BLIP mutants with higher inhibitory
activity for different
-lactamases and PBPs.
The crystal structure of the BLIP·TEM-1 complex suggests that two
BLIP amino acids, Asp-49 and Phe-142, are critical for the inhibition
of TEM-1 through interactions in the active site pocket (17). In
addition, the BLIP amino acids that bind the 99-112 loop of
-lactamase (Ser-35, Phe-36, Ser-39, His-41, His-45, Ala-47, Gly-48,
Tyr-50, Tyr-53, Ser-71, Glu-73, Lys-74, Ser-113, Gly-141, Tyr-143,
Trp-150, Trp-112, Arg-160, Trp-162) represent the majority of the
surface interactions between the two proteins (2636 Å2 is
buried at the intermolecular surface) (17). These residues are the
primary candidates for mutational analysis to distinguish whether
"hot spots" consisting of a subset of these amino acids are
involved in specificity and binding or if all of these residues contribute to the binding of BLIP to TEM-1
-lactamase.
The first step in being able to identify amino acids important for BLIP
specificity and inhibitory activity is to develop an expression system
to produce functional BLIP. BLIP expressed in its native S. clavuligerus produces large quantities of protein, whereas
expression in another Streptomyces species,
Streptomyces lividans, produces limited quantities of BLIP
(15, 30). Successful expression of soluble BLIP in E. coli
would facilitate the study of BLIP mutants and would also allow
protein engineering techniques to be performed.
Histidine-tagged proteins are able to be purified in a relatively
simple manner, while usually maintaining the native activity of the
tagged protein. Therefore, an expression system centered around an
N-terminal 6XHis-tagged BLIP was constructed. Expression is directed by
the inducible trc promoter, and a cat gene is
inserted into the
-lactamase gene of the plasmid to avoid possible
complex formation during purification of BLIP (Fig. 1). This system
enabled BLIP to be purified to >90% homogeneity in one step (Fig. 2). Wild-type His-tagged BLIP was found to have a Ki of
0.11 nM. This value is slightly lower than the previously
calculated value of 0.6 nM for BLIP isolated from S. clavuligerus (16). This difference could be attributed to the
manner in which the Ki was calculated, and confirms
that the N-terminal His tag has no effect on BLIP binding.
Once expression of functional BLIP was achieved, the roles of BLIP
residues in the inhibition of different targets could be determined.
The crystal structure of BLIP with TEM-1
-lactamase shows that
Asp-49 of BLIP makes strong hydrogen bond contacts with four conserved
residues in the TEM-1 active site pocket: Ser-130, Lys-234, Ser-235,
and Arg-244 (17). These four amino acids are involved in the binding
and catalysis of
-lactam antibiotics and are conserved in all class
A
-lactamases. Mutation of the aspartic acid to an alanine removes
the carboxylate moiety that serves as a hydrogen bond acceptor for the
four active site TEM-1 residues. Elimination of the carboxylate reduced
the inhibitory activity of BLIP approximately 100-fold, indicating
residue Asp-49 does make an important contribution to BLIP inhibition
of TEM-1
-lactamase.
The crystal structure also leads to the prediction that Phe-142 is also
important for inhibition of TEM-1
-lactamase (17). Phe-142 is in
contact with
-lactamase residues Glu-104, Tyr-105, Asn-170, Ala-237,
Gly-238, and Glu-240 in the complex. As in the case of Asp-49, most of
these residues are either conserved in class A
-lactamases or are
involved in catalysis. It was also found that in other protein
complexes, such as between human growth hormone and its receptor, the
most critical interactions are hydrophobic (31). Therefore, the
contribution of Phe-142 to binding and inhibition was examined. The
F142A substitution removes the hydrophobic side chain that mimics the
benzyl group in PenG from the TEM-1·PenG complex. This change also
increases the Ki approximately 100-fold, which
suggests that the interactions mediated by Phe-142 are important for
inhibitor binding, and that they are similar in magnitude to the
contributions made by Asp-49. The D49A/F142A double mutant was
constructed to test additivity between these two residues, but the
protein was not expressed.
The ability of wild-type BLIP and the D49A and F142A mutants to inhibit
two extended spectrum
-lactamase mutants was also examined. G238S,
the
-lactamase mutation found in TEM-19, and E104K, the mutation
found in TEM-17, were the two representative extended spectrum mutants
tested with BLIP and the BLIP mutants (26). The prevalence of the G238S
and E104K substitutions in many of the extended spectrum
-lactamases
makes these two single mutants ideal candidates for study.
Interestingly, wild-type BLIP, D49A, and the F142A BLIP mutant each
inhibited G238S at similar levels to that which they inhibited
wild-type TEM-1
-lactamase. According to the crystal structure, the
only contact made with Gly-238 of TEM-1 is by Phe-142. The fact that no
change in the inhibition profile was observed between the
-lactamase
enzymes suggests that this contact between Gly-238 and Phe-142 is not critical for BLIP binding and inhibition. If this interaction played a
role in BLIP inhibition, then replacement of the glycine side-chain at
position 238 of TEM-1 would have resulted in an increased
Ki with wild-type BLIP. However, the possibility that the substituted serine at position 238 makes new interactions with
BLIP, and corrects exactly for the loss of interaction with Phe-142,
cannot be excluded.
In contrast to BLIP binding to G238S, significant changes in the
inhibitory profile were observed when E104K was used as the target
-lactamase. Wild-type BLIP inhibited E104K approximately 1000-fold
worse than for TEM-1, suggesting that the interactions made between
BLIP and Glu-104 are critical for wild-type levels of activity. This
result indicates that some, or all, of the BLIP residues interacting
with Glu-104 (BLIP-Glu-73, Lys-74, Phe-142, and Tyr-143) are making
important interactions. However, results from the BLIP
F142A/
-lactamase E104K binding experiment suggest that the lysine
substitution at position 104 of
-lactamase disrupts critical
interactions made by Phe-142 of BLIP. This disruption of the BLIP
Phe-142 interaction with
-lactamase results in the pronounced loss
of inhibition observed with the E104K enzyme.
It is apparent from this study that while BLIP residues Asp-49 and
Phe-142 are critical for wild-type BLIP inhibitory levels, the D49A and
F142A variants still bind TEM-1
-lactamase with nanomolar affinity.
Therefore, other interactions must also contribute to the strong levels
of inhibition observed. The identification of the epitopes responsible
for the remaining binding energy will facilitate the engineering of
tighter, smaller inhibitors for these
-lactamases.
Determination of the Ki of BLIP with SHV-1
-lactamase shows that even though TEM-1 and SHV-1 are both class A
-lactamases and are 68% identical, the interactions which make BLIP
a tight inhibitor of TEM-1 are not conserved with SHV-1. Although no
crystal structure is available for SHV-1, the level of identity between TEM-1 and SHV-1 suggests that both enzymes share a similar protein fold. However, the fact that BLIP is a poorer inhibitor of SHV-1 shows
that small differences between the
-lactamases are significant with
respect to BLIP binding.