From the Department of Microbiology and Immunology
and the § Department of Biochemistry, Baylor College of
Medicine, Houston, Texas 77030
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ABSTRACT |
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Since the introduction of An additional strategy that has been employed to combat antimicrobial
resistance is the use of The small molecule -Lactamase inhibitory protein (BLIP) is a potent
inhibitor of several
-lactamases including TEM-1
-lactamase
(Ki = 0.1 nM). The co-crystal structure
of TEM-1
-lactamase and BLIP has been solved, revealing the contact
residues involved in the interface between the enzyme and inhibitor. To
determine which residues in TEM-1
-lactamase are critical for
binding BLIP, the method of monovalent phage display was employed.
Random mutants of TEM-1
-lactamase in the 99-114 loop-helix and
235-240 B3
-strand regions were displayed as fusion proteins on the
surface of the M13 bacteriophage. Functional mutants were selected
based on the ability to bind BLIP. After three rounds of enrichment,
the sequences of a collection of functional
-lactamase mutants
revealed a consensus sequence for the binding of BLIP. Seven loop-helix
residues including Asp-101, Leu-102, Val-103, Ser-106, Pro-107,
Thr-109, and His-112 and three B3
-strand residues including
Ser-235, Gly-236, and Gly-238 were found to be critical for tight
binding of BLIP. In addition, the selected
-lactamase mutants
A113L/T114R and E240K were found to increase binding of BLIP by over 6- and 11-fold, respectively. Combining these substitutions resulted in
550-fold tighter binding between the enzyme and BLIP with a
Ki of 0.40 pM. These results reveal
that the binding between TEM-1
-lactamase and BLIP can be improved
and that there are a large number of sequences consistent with tight
binding between BLIP and
-lactamase.
INTRODUCTION
Top
Abstract
Introduction
References
-lactam antibiotics, bacterial
resistance to these agents has become an increasing problem (1, 2). The
production of the enzyme
-lactamase by both Gram-positive and
Gram-negative bacteria is the most common mechanism of resistance to
-lactam antibiotics (3).
-Lactamases hydrolyze the amide bond of
the
-lactam antibiotic to create an ineffective antimicrobial agent
(3) and have been grouped into four classes (A, B, C, and D) based on
primary sequence homology (4, 5). Genes encoding these enzymes have
been found on plasmids, transposons, and bacterial chromosomes (4, 6).
The most prevalent plasmid-mediated
-lactamase in Gram-negative
bacteria is the TEM-1
-lactamase from class A (7). Like other class
A
-lactamases, TEM-1
-lactamase is capable of hydrolyzing both
penicillins and cephalosporins (2, 8, 9). To overcome the drug
resistance mediated by TEM-1
-lactamase, extended-spectrum
antibiotics, including aztreonam, cefotaxime, and ceftazidime were
developed. However, soon after their introduction, selective pressure
resulted in the emergence of variant
-lactamases able to hydrolyze
these antibiotics (10). Examination of these enzymes revealed that
amino acid substitutions such as R164S, G238S, and E240K in TEM-1
-lactamase resulted in altered substrate specificity of the enzyme
(11-13). The emergence of these enzymes is an additional threat to
antimicrobial therapy since plasmids encoding these
-lactamase
variants are easily transferable to unrelated bacteria (1).
-lactamase inhibitors such as clavulanic
acid, sulbactam, and tazobactam (14). Although not capable of
antimicrobial activity themselves, these suicide inhibitors are used in
conjunction with various
-lactam antibiotics to bind
-lactamase
and prevent the hydrolysis of the antibiotic, thereby restoring the
therapeutic value to the antimicrobial agent. Unfortunately,
-lactamase variants have been identified that are resistant to these
enzyme inhibitors while still retaining the ability to hydrolyze
-lactam antibiotics (15, 16).
-lactamase inhibitor, clavulanic acid, was
initially purified from the soil bacterium Streptomyces
clavuligerus (17). This organism also produces a protein inhibitor
of
-lactamase called the
-lactamase inhibitor protein
(BLIP)1 (18). BLIP is a 165-amino
acid protein composed of two tandemly repeated domains (19). It has
been shown to be a potent inhibitor of class A
-lactamases from both
Gram-positive and Gram-negative bacteria, including TEM-1
-lactamase
(Ki = 0.1 nM) (20). The co-crystal
structure of TEM-1
-lactamase complexed with BLIP has been solved,
revealing the mode of inhibition by BLIP and the contact residues
involved in the interface between the enzyme and inhibitor (21). The
x-ray structure shows that BLIP binds to a negatively charged
loop-helix region composed of residues 99-112 just outside the active
site pocket of TEM-1
-lactamase (Fig. 1).
Sequence alignment of
-lactamases that are inhibited by BLIP reveals
an overall lack of amino acid sequence conservation in this region
(21). The ability of BLIP to bind to such a variety of class A
-lactamases is believed to be due in part to an extensive layer of
water molecules that are trapped between the inhibitor and the 99-112
loop-helix region of the enzyme. The relative importance of the 99-112
residues of TEM-1
-lactamase for binding BLIP is unknown. It has
been proposed, however, that the only critical residues in this region
for binding with BLIP are the aromatic residue at position 105 and the
conserved proline at position 107 (21).
View larger version (57K):
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Fig. 1.
TEM-1
-lactamase. The main chain of TEM-1
-lactamase is shown with the residues examined in this study
highlighted. Gln-99 and Thr-114 flank the loop-helix region,
and Glu-240 is the C-terminal residue of the B3
-strand. The
catalytic Ser-70 residue is displayed to show the location of the
active site. Coordinates are from PDB file 1BTL.
In addition to blocking the enzyme-active site by binding the 99-112
loop-helix region of TEM-1 -lactamase, BLIP also inserts two turns,
one from each domain, directly into the active site of the enzyme (21).
The main contact residues in the active site of TEM-1
-lactamase
involve the B3
-strand residues 234-240 (Fig. 1). The
-hairpin
turn of domain 1 of BLIP is stabilized through several van der Waals
contacts made with residues 235-238 of
-lactamase in addition to
four strong hydrogen bonds made by Asp-49 of BLIP with residues in the
enzyme that are critical for substrate binding and catalysis. The turn
of domain 2, in particular Phe-142, also makes several contacts with
TEM-1
-lactamase. Since
-lactamases inhibited by BLIP are highly
conserved in the 234-240 region, these residues have been hypothesized
to be the crucial determinants for the binding and inhibition by BLIP
(21).
In order to circumvent bacterial resistance and thereby continue the
effectiveness of antimicrobial therapy, new antibiotics and inhibitors
are needed. Understanding the molecular details of TEM-1 -lactamase
and BLIP binding may aid in the development of inhibitors and
antibiotics designed to mimic the BLIP-
-lactamase interaction (20).
In this study we have developed and used a phage display system to
determine the residues in the 99-112 loop-helix and the 234-240 B3
-strand of TEM-1
-lactamase that are critical for the tight
binding interaction with BLIP (Fig. 1).
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EXPERIMENTAL PROCEDURES |
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Bacterial Strains and Plasmids--
Escherichia coli
XL1-Blue (22) [recA1, endA1, gyrA96,
thi-1, hsdR17, supE44,
relA1, lac, [F' proAB
lacIq lacZDM15, Tn10
(tetr)]] (Stratagene, Inc.) was used for transformation
of ligation reactions and to produce the initial, unpanned
bacteriophage stocks. E. coli TG1 (23) [F'
traD36+, lacIq, (lacZ)
M15
proA+B+/supE
(hsdM
mcrB
)
(r
m
McrB
) thi
(lac
proAB)] was used for
production, amplification, and titering of bacteriophage stocks.
E. coli RB791 (24, 25) (strain W3110 lacIqL8) was used to express and purify
-lactamase mutants. The
-lactamase random libraries encompassing
regions 99-114 and 232-240 were previously constructed in the pBG66
plasmid as described (26, 27). The pBG66 vectors harboring the
-lactamase mutants A237T and the double mutant L113A/T114R were
selected from random libraries of
-lactamase as described previously
(27, 28). The phagemid vector pG3-SPT was constructed previously by the
insertion of a spectinomycin cassette into the cat gene of
pG3-CMP as described (29).
Construction of Phage Display Libraries--
For monovalent
phage display of -lactamase random libraries including regions
99-114 and 232-240, the phagemid vector pG3-CMP was used (29). The
-lactamase random library 103-105, consisting of three consecutive
randomized codons 5'-NNN-NNN-NNN-3' (N = A, G, C, or T) in the
vector pBG66, was constructed as described previously (30). The
remaining
-lactamase gene libraries were constructed as described
(27), each consisting of three consecutive randomized codons
5'-NNS-NNS-NNS-3' (S = G or C) in vector pBG66. The
-lactamase
gene for each library was polymerase chain reaction-amplified using the
following primers: PD-bla1,
5'-CGGGGAGCTCGTTTCTTAGACGTCAGGTGGC-3'; PD-bla2,
5'-CCCCGTCTAGACCCAATGCTTAATCAGTGAG-3'. Enzyme restriction sites are underlined; the primer PD-bla1 contains a SacI
site, and the primer PD-bla2 contains an XbaI site. The
polymerase chain reaction product from each
-lactamase random
library was digested with SacI and XbaI,
gel-purified, and ligated into the SacI/XbaI sites of pG3-CMP to generate pG3-C4 (Fig. 3). Plasmid DNA was electroporated into E. coli XL1-Blue according to
manufacturer's instructions (Stratagene, Inc.) and plated on LB agar
containing 12.5 µg/ml chloramphenicol. The resulting colonies were
pooled using 1 ml of LB medium. To determine that the pool size of each constructed library was large enough to have a 99% probability of
containing the least probable sequence combination (e.g.
Trp-Trp-Trp), the Poisson distribution was used (26). A 1/100 volume of
the pooled colonies was used to prepare bacteriophage library stocks.
Preparation of Phagemid Particles and Titering-- For packaging phagemid, E. coli infected with the phagemid to be packaged were inoculated into 25 ml of 2YT containing 12.5 µg/ml chloramphenicol along with 1 × 109 VCS M13 helper phage (Stratagene, Inc.). After 15 min incubation at room temperature, the culture was grown shaking overnight at 37 °C. The bacteria were pelleted after overnight growth, and the phages were precipitated from the supernatant with 1/5 volume of 20% PEG-8000, 2.5 M NaCl (31). The phages were pelleted by centrifugation and resuspended in 1/50 of the original volume of STE (0.1 M NaCl, 10 mM Tris-Cl, pH 8.0, 1 mM EDTA, pH 8.0) (31). The phage titer was determined by making serial dilutions of the phage stock and adding 0.2 ml to 0.2 ml of an E. coli TG1 mid-log phase culture. After 30 min of incubation at 37 °C, aliquots of 0.2 ml were plated on LB agar supplemented with 12.5 µg/ml chloramphenicol. After overnight growth at 37 °C, colonies were counted and the titer determined. Titers between 5 × 1012 and 5 × 1013 phages per ml were generally obtained.
Enrichment of -Lactamase Mutants Capable of Binding
BLIP--
Purified BLIP was the target for binding of
-lactamase
mutants displayed on phage particles. BLIP was expressed and purified as a fusion to the C terminus of maltose-binding protein. Binding experiments demonstrated the mal-BLIP fusion has the same
Ki for
-lactamase inhibition as wild-type BLIP.
Therefore, the mal-BLIP fusion protein was immobilized as the target
for binding enrichment experiments. The only exception to this was the
use of an N-terminal 6×-His BLIP protein (20) for the panning
experiments for the 106-108 and 112-114 libraries that were repeated.
BLIP was immobilized for panning by attaching it covalently to
oxirane-acrylic beads (Sigma) (31). BLIP was suspended at 0.2 mg/ml in
0.1 M sodium carbonate buffer, pH 8.6, with 50 mg of
oxirane-acrylic beads and gently mixed at room temperature for 2 h
followed by overnight incubation at 4 °C. After overnight
incubation, bovine serum albumin (BSA) was added at a final
concentration of 10 mg/ml to block the remaining oxirane sites. Beads
were stored at 4 °C. Oxirane-acrylic beads conjugated to polyclonal
anti-
-lactamase antibody and to BSA were prepared as described for
the BLIP-oxirane-acyrlic beads using a final volume of 0.50 and 2.0 mg/ml, respectively, of each protein.
To prepare protein-bound oxirane-acrylic beads for phage panning, 5 mg
of each bead solution was washed 4× with 0.8 ml of buffer A
(Tris-buffered saline containing 1 mg/ml BSA and 0.5 g/liter Tween 20)
followed by 1 h incubation at room temperature in 1 ml of
Superblock (Pierce). The beads were then pelleted by centrifugation and
washed an additional 5 times with buffer A. For panning, 1 × 1011 of a -lactamase phage library and 1 × 1011 pG3-SPT phage were added to the 5 mg of the washed
BLIP, polyclonal anti-
-lactamase antibody, or BSA-conjugated oxirane
beads in a final volume of 0.8 ml in buffer A. The mixture was gently
rocked at room temperature for 2 h. The beads were then washed 10 times in 0.8 ml of buffer A. Bound phage were removed from the beads by
incubating with 0.2 ml of elution buffer (0.1 M glycine, pH 2.2, 1 mg/ml BSA, 0.5 g/liter Tween 20, 0.1 M KCl) for 15 min. The beads were pelleted, and the supernatant was removed and
neutralized with 25 µl of 1 M Tris-HCl, pH 8.0. The titer
of the phage eluted from the beads was determined as described above by
plating on LB agar containing chloramphenicol (12.5 µg/ml) to
determine the number of phage encoding
-lactamase and by plating on
LB agar containing spectinomycin (50 µg/ml) to determine the number
of pG3-SPT phage that do not display a protein. The eluted phage were
amplified by adding 0.18 ml of the neutralized elution mixture to 1 ml
of mid-log phase E. coli TG1 cells and used to prepare phage
particles for the next round of binding enrichment.
Construction of the -Lactamase Triple Mutant
L113A/T114R/E240K--
The TEM-1
-lactamase mutants L113A/T114R and
E240K had previously been constructed in pBG66 (11, 27). These
-lactamase mutants were used to construct the triple mutant
L113A/T114R/E240K. Internal
-lactamase restriction sites,
EcoRI and PstI, allowed for the cloning of the
mutated L113A/T114R
-lactamase gene fragment into the
EcoRI/PstI-digested pBG66 vector harboring the
-lactamase mutation E240K. The sequence of this clone was confirmed
by restriction enzyme analysis and DNA sequencing. The newly
constructed vector was named pGR35.
DNA Sequencing--
DNA sequencing of -lactamase mutants was
performed directly on polymerase chain reaction products of
blaTEM amplified from single colonies (32).
Oligonucleotides used for DNA sequencing were designed to prime
specific sites within blaTEM.
Purification of -Lactamase Mutants--
The
-lactamase
mutants P107I/V108I, L113A/T114R, A237T, and E240K were selected for
further characterization based on the phage panning results. For
kinetic analysis and inhibition studies, the
-lactamase proteins
were purified to greater than 90% homogeneity as described previously
(28). All enzymes were expressed under the control of the constitutive
-lactamase promoter in the non-amber suppressor E. coli
strain RB791. Kinetic studies of the purified double mutant P107I/V108I
and the single mutant A237T revealed that
-lactamase variants
expressed from the pG3-C4 phagemid vector were inactive for unknown
reasons. To express functional
-lactamase mutants, alternative
expression vectors were utilized. Previous studies by our laboratory
(11) have shown that the
-lactamase variant E240K is expressed as an
active enzyme from the expression vector pBG66. Based on these
findings, the previously constructed
-lactamase mutants E240K and
L113A/T114R in pBG66 were used to purify the corresponding
-lactamase enzymes (11, 27). For expression of the active
-lactamase single mutant A237T and double mutant P107I/V108I, the
expression vector pTP123 was used (20). This vector was utilized since
the
-lactamase variants from pG3-C4 could easily be cloned into the
multi-cloning site of pTP123 and placed under the control of the
inducible Ptrc promoter for increased
-lactamase
expression if desired. The
-lactamase variants A237T and P107I/V108I
were digested from the pG3-C4 vector with SacI and
XbaI to release the
-lactamase promoter and gene (Fig.
3). The fragments were gel-purified and ligated into the
SacI/XbaI sites of the expression vector pTP123
to generate pGR34 and pGR36, respectively. The resulting plasmids were
transformed into E. coli RB791 and selected on LB medium
supplemented with 12.5 µg/ml chloramphenicol. The
-lactamase
mutants were expressed under the
-lactamase constitutive promoter
and purified as described with the pBG66 clones. The triple mutant
L113A/T114R/E240K was constructed in pBG66 as described above and
purified from E. coli RB791.
Enzyme Kinetics--
The kinetic parameters of the -lactamase
mutant enzymes were determined with cephaloridine as a substrate using
a 0.1-cm path length cuvette as described previously (28). Values
reported are based on velocity measurements at 100, 200, 300, 400, 500, 600, 700, and 800 µM substrate concentrations.
BLIP Inhibition Assay--
Inhibition assays were performed
using the substrate cephaloridine as described previously with the
following modifications (20). For the -lactamase mutants
L113A/T114R, E240K, and L113A/T114R/E240K, 0.1 nM
-lactamase was incubated with varying concentrations of 6×
histidine-tagged BLIP. For the wild-type
-lactamase and
-lactamase mutants P107I/V108I and A237T, 1.0 nM
-lactamase was incubated with varying concentrations of the 6×
histidine-tagged BLIP.
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RESULTS |
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Phage Display of -Lactamase--
The crystal structure of the
TEM-1
-lactamase-BLIP complex shows that BLIP associates mainly with
the 99-112 loop-helix and the 234-240 B3
-strand lining the active
site pocket of TEM-1
-lactamase; however, the relative importance of
these residues for binding BLIP remains unknown (21). In previous
experiments by this laboratory (27) to determine the amino acid
residues that are critical for the structure and function of TEM-1
-lactamase, blocks of three contiguous codons were randomized to
create a library containing all possible amino acid substitutions.
Constructing a set of 88 individual libraries randomized the entire
coding sequence of the gene (263 codons). Nine of these libraries,
encompassing residues 99-114 and 232-240, cover the surface of TEM-1
-lactamase that interacts with BLIP (Fig.
2). To determine which residues in the
loop-helix and B3
-strand regions of TEM-1
-lactamase are
critical for tight binding with BLIP, functional random mutants were
selected from these libraries based on the ability to bind BLIP using
monovalent phage display.
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In order to display the -lactamase variants from the random
libraries on the surface of the M13 bacteriophage, the pG3-C4 phagemid
was constructed (Fig. 3). This vector encodes
a fusion of
-lactamase to the N terminus of the M13 gene III protein
(gIIIp), which is transcribed by the constitutive
-lactamase
promoter. An amber codon present between the
-lactamase gene and
gene III allows for expression of the fusion protein in amber
suppressor strains of E. coli and expression of only
-lactamase in non-amber suppressor strains (31). Because the vector
encodes chloramphenicol resistance, bacteria harboring this vector can
be selected on medium containing chloramphenicol.
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Specific Enrichment of Randomized -Lactamase Phage
Libraries--
In order to sample all amino acid substitutions in the
loop-helix region and B3
-strand of TEM-1
-lactamase, each of the nine
-lactamase randomized libraries were inserted into the pG3-C4 vector to create the
-lactamase phage display libraries. To
determine the
-lactamase residues important for binding with BLIP,
phage from each library were selected based on their ability to bind immobilized BLIP. Three controls were used to show that the phage binding to the immobilized BLIP was dependent on the
-lactamase-BLIP interaction. First, phage displaying a library of
-lactamase mutants
were panned on immobilized BSA in addition to the immobilized BLIP.
After three rounds of binding and selection of the random library
112-114, over 1600-fold more
-lactamase phage bound to immobilized
BLIP than to the BSA control (Fig. 4). These
data indicate that the
-lactamase displaying phage can be rapidly enriched when panned against immobilized BLIP and that the enrichment is not due to nonspecific binding to BSA.
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As a second control, phage displaying libraries of -lactamase
mutants were incubated with immobilized polyclonal anti-
-lactamase antibody. Since the polyclonal anti-
-lactamase antibody is capable of recognizing multiple epitopes on
-lactamase, it was expected that
the majority of the displayed
-lactamase molecules would still bind
the polyclonal antibody even if they contained substitutions in the
BLIP-binding surface. After one round of panning of the 112-114
randomized library, 70-fold more phage bound the polyclonal anti-
-lactamase antibody than bound to BLIP (Fig. 4). However, after
three rounds of panning only 5-fold more phage bound the antibody
versus BLIP. Similar results were obtained with the
remaining phage displaying random libraries (data not shown). These
data indicate that the
-lactamase mutants from the libraries were efficiently displayed on the phage and provide further evidence that
after several rounds of panning
-lactamase mutants that bind BLIP
can be selected.
Finally, an internal control was used in which phage displaying
-lactamase libraries were incubated with an equal number of phage
that did not display a protein in the presence of the immobilized
targets BLIP, BSA, or polyclonal anti-
-lactamase antibody. The
pG3-SPT vector used to produce non-displaying phage is similar to the
randomized
-lactamase phage display vector pG3-C4 except the
chloramphenicol cassette is replaced with the spectinomycin cassette,
and no fusion is made with gene III (29). This results in the
production of phage that do not display a fusion protein and whose
presence can be determined by spreading infected E. coli on
agar plates containing spectinomycin. The advantage of this system is
that the number of nonspecific phage interactions versus
interactions dependent on
-lactamase can be determined by titering
the phage recovered from a binding experiment on spectinomycin medium
as well as chloramphenicol medium. After one round of panning library
112-114, 190-fold more
-lactamase-displaying phage than
non-displaying phage were recovered after binding BLIP (Fig. 4). After
three rounds of panning, 28,000-fold more
-lactamase-displaying phage bound BLIP compared with non-displaying phage. These data show
that the majority of the binding interactions are specific for BLIP and
-lactamase and not due to nonspecific phage-protein interactions.
Since similar numbers of pG3-SPT non-displaying phage were recovered
from BSA and
-lactamase antibody binding experiments, it is believed
that these numbers represent the background level of nonspecific
protein interactions or trapping of phage by the beads.
Selecting for High Affinity -Lactamase-BLIP Complexes--
The
residues in the 99-114 and 232-240 regions of TEM-1
-lactamase
that are most critical for binding BLIP were determined by screening
each of the nine libraries covering these regions for the ability to
bind tightly to BLIP. After three rounds of binding enrichment
representative clones were sequenced (Fig. 2). At several positions a
certain residue type was more prevalent among functional binding
mutants than was expected from a random distribution of amino acids in
the starting library (Table I). For example,
after three rounds of binding enrichment on immobilized BLIP, 97% of
the clones sequenced from the 112-114 random library contained a T114R
substitution. This was 17 standard deviation units (17
) above the
expected frequency based on a random chance occurrence for Arg at this
position in the library. Following a similar procedure for the
remaining libraries, a consensus sequence for the loop-helix and B3
-strand regions of TEM-1
-lactamase was established for the
mutants retaining the capacity to bind BLIP (Fig. 2).
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Out of the 16 residues randomized in the 99-114 loop-helix region,
seven positions exhibited a strong bias for the wild-type residue
including Asp-101, Leu-102, Val-103, Ser-106, Pro-107, Thr-109, and
His-112 (Fig. 2 and Table I). The results indicate that these residues
are critical for tight binding with BLIP or for maintaining the
structure and function of -lactamase. At 4 of the 16 residues, the
wild-type residue as well as one or two substitutions appear frequently
among the selected clones. These include positions Gln-99, Glu-104,
Tyr-105, and Val-108. In all cases except for Gln-99, non-wild-type
substitutions are preferred (Table I). For example, Asn was favored
over the aromatic side chain Tyr-105 by 3
and Ile was favored over
Val-108 by almost 6
. For Glu-104, both Glu and Trp were distributed
almost equally among the mutants (10.1
and 10.9
, respectively)
indicating that both are consistent with binding of BLIP. The results
suggest that these positions are important for tight binding with BLIP; however, the sequence requirements are not as stringent since a limited
number of substitutions are tolerated without dramatically effecting
binding with BLIP. Three positions, Lys-111, Leu-113, and Thr-114,
exhibited a bias for a non-wild-type residue. The preferred
substitutions were K111T (9.6
), L113A (21.2
), and T114R (16.9
)
(Table I). The bias toward non-wild-type residues at these positions
suggests that the substituted residues provide for a tighter binding
complex between TEM-1
-lactamase and BLIP compared with the
wild-type residue. The two remaining residues, Asn-100 and Glu-110, had
no strong bias for any particular residues. This suggests that these
positions are not important for binding BLIP.
After the third round of panning the -lactamase phage libraries
106-109 and 112-114, only one sequence was identified from all clones
sequenced for each library. Library 106-109 converged on the amino
acid sequence Ser-Ile-Ile and library 112-114 converged on the
sequence His-Ala-Arg. To verify that these results were not due to
"sibbing" in which a single clone was amplified during the binding
enrichment based on factors other than binding to the target molecule,
the libraries were re-panned (31). After each round of panning,
representative clones were sequenced to verify the diversity of the
library. After the first round of panning library 106-109, 11 clones
were sequenced, and 10 different amino acid sequences were identified,
with only the mutant Ser-Pro-Ile being represented twice in the library
(Fig. 2). Even after one round of panning a clear consensus for each
position was seen in that 72% of the clones contained Ser at position
106, 54% contained Pro at position 107, and 100% contained a
hydrophobic residue at position 108. Round two sequencing results
identified four different clones, with both Ser-Pro-Val and Ser-Pro-Ile
being equally represented and making up 66% of the population. After round three, the sequences converged on the wild-type Ser-Pro-Val sequence and the non-wild-type Ser-Pro-Ile sequence with various non-wild-type triplicate codons being represented in each case. These
data indicated that during the initial panning of the library sibbing
occurred and the Ser-Ile-Ile sequence overtook the phage population
during the selection process. The re-panning and sequencing of library
112-114 revealed similar results as library 106-108 for the first two
rounds of panning in that the library was initially diverse but quickly
converged on certain residues at each position randomized (Fig. 2). By
round three, the sequence converged on the same amino acid sequence,
His-Ala-Arg, as that selected in the first set of binding experiments
indicating that this sequence was strongly preferred for the tight
binding of BLIP and was not a result of sibbing.
Three -lactamase random libraries were used to study the B3
-strand residues 232-240 (Fig. 2). Contamination of the library 232-234 by wild-type
-lactamase sequence prevented analysis of these positions. Out of the five residues studied, four exhibited a
very strong bias for the wild-type residue among the sequences of
functional mutants. These include residues Ser-235 (12.0
), Gly-236
(14.9
), Ala-237 (12.7
), and Gly-238 (15.5
) (note: TEM-1
-lactamase has no residue 239 (33)) (Table I). In addition to Ala at
position 237, Thr was also observed but to a much lesser degree
(3.5
) (Table I). This suggests that the B3
-strand residues 235, 236, and 238 of TEM-1
-lactamase are critical for the tight binding
of BLIP. The only position that did not exhibit a strong bias for the
wild-type residue among functional mutants was Glu-240, which lies just
outside the active site pocket (Fig. 1). Instead of the wild-type
negative charged Glu residue, positive charged residues were selected
at this site with a strong bias toward E240R (9
) and a weaker bias
for E240K (3.6
) (Fig. 2 and Table I). This finding suggests that
either a positive charge or an extended side chain at position 240 may
result in more efficient binding of BLIP.
Quantitation of -Lactamase-BLIP Binding Affinity--
Because
the phage display binding experiments represent a competition among
random sequences for BLIP binding, it is assumed that the selected
variants bind BLIP as tight or tighter than wild-type
-lactamase.
This assumption was tested by purifying several of the selected
-lactamase variants and determining the affinity for BLIP. The two
loop-helix mutants studied were P107I/V108I and L113A/T114R. These
mutants were strongly selected during the first set of phage binding
experiments, suggesting a tight binding interaction with BLIP (data not
shown). In addition, two B3
-strand variants were studied, A237T and E240K.
The mutant -lactamases to be studied were expressed independently of
M13 gIIIp. Prior to determining the affinity of BLIP for the selected
mutants, kinetic parameters were determined for each purified enzyme
using the cephalosporin, cephaloridine, as a substrate (Table
II). The kinetic parameters for the A237T and E240K enzymes agreed well with published values (11, 28). The
P107I/V108I and L113A/T114R enzymes exhibited slightly lower catalytic
efficiencies (kcat/Km)
compared with wild-type
-lactamase for cephaloridine hydrolysis due
to small decreases in both kcat and
Km.
|
The equilibrium dissociation constants for the interaction between BLIP
and the -lactamase enzymes were determined using cephaloridine as a
substrate in an assay developed previously (20). Wild-type or mutant
-lactamases were incubated in the presence of BLIP for 2 h. By
monitoring the hydrolysis of cephaloridine added to the reaction after
the incubation time, the concentration of free
-lactamase could be
determined. Fitting the data obtained when incubating varying
concentrations of wild-type
-lactamase in the presence of 1 nM BLIP resulted in a Ki of 0.22 nM (Table II), which compares favorably with previously
reported data of 0.1 nM for BLIP purified from E. coli (20).
The enzyme containing the active site B3 -strand substitution A237T
was found to have a Ki for BLIP binding of 0.49 nM, whereas a Ki of 0.019 nM
was determined for E240K (Table II). The Ki value
obtained for the A237T enzyme suggests that the substitution interferes
slightly with the binding of BLIP. This is consistent with the phage
display data that indicated a strong bias for the wild-type Ala residue
over the Thr residue among mutants selected for BLIP binding. The E240K
enzyme binds BLIP 11-fold tighter than wild-type
-lactamase,
indicating the positive charge or extended side chain at position 240 improves the interaction between
-lactamase and BLIP.
As stated above, in the first set of phage binding enrichments, an
enzyme containing the P107I/V108I substitution in the loop-helix region
predominated among selected mutants. However, re-panning of the library
106-108 showed that the predominance of the P107I/V108I mutant was a
result of sibbing and that the wild-type sequence or the single V108I
substitution are the preferred residues at these locations. Consistent
with these observations, the P107I/V108I enzyme bound BLIP 1.5-fold
weaker than wild-type -lactamase (Table II). In contrast, the enzyme
containing the A113L/T114R substitutions bound BLIP approximately
6-fold tighter than wild-type
-lactamase (Fig.
5 and Table II). This is consistent with the
finding that the A113L/T114R enzyme predominated even when the 112-114
library was re-panned on BLIP. These results indicate that the
-lactamase variants identified from the random libraries bind BLIP
as tight or tighter than wild-type
-lactamase. Therefore, the
sequence data obtained from the phage display experiments is an
accurate indication of which residue positions are critical for binding BLIP.
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Combining -Lactamase Mutants to Improve Affinity for
BLIP--
According to additivity principles, residues in
non-interacting regions of a protein should combine to give additive
changes in the free energy of binding (34). Because the
-lactamase mutants A113L/T114R and E240K both exhibited increased binding affinity
for BLIP, we sought to test whether the substitutions act additively by
constructing the triple
-lactamase mutant A113L/T114R/E240K. The
equilibrium dissociation constant of the triple
-lactamase mutant
was determined as a Ki of 0.00042 nM
(Fig. 5 and Table II). This binding was 550-fold tighter than binding
by wild-type TEM-1
-lactamase. Based on simple additivity, it was
expected that the triple mutant would bind BLIP approximately 75-fold
tighter than wild-type
-lactamase. Therefore, the A113L/T114R and
E240K substitutions appear to act synergistically to produce a very tight binding interaction between
-lactamase and BLIP.
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DISCUSSION |
---|
Over the past several years, maintaining the effectiveness of
-lactam antibiotics and
-lactamase inhibitors has become an increasing problem. BLIP has been shown to be a potent inhibitor of
several class A
-lactamases and also exhibits inhibitory activity toward the penicillin-binding protein 5 of Enterococcus
faecalis (19). Understanding the molecular interactions between
BLIP and TEM-1
-lactamase may aid the development of novel
inhibitors of both
-lactamases and penicillin-binding proteins that
are based on the structure of BLIP.
The crystal structure of TEM-1 -lactamase complexed with BLIP
indicates that over two-thirds of all contacts made by BLIP occur in
the 99-112 loop-helix of TEM-1
-lactamase, whereas more than 50%
of the remaining contacts are made with the B3
-strand residues
234-240 in the active site of the enzyme (21). The role of these
residues in the binding of BLIP was determined using a set of eight
-lactamase random libraries that encompassed the loop-helix and B3
-strand. The
-lactamase variants were displayed as fusion
proteins to the gIIIp of M13. A newly constructed vector, pG3-C4,
allows for low level constitutive expression of the
-lactamase-gIIIp fusion from the weak
-lactamase promoter. This low level expression reduces the potential toxicity of the gIIIp fusion and provides for
monovalent display of the protein fusion from M13 phage (35).
The -lactamase random libraries were used to construct a consensus
sequence for the loop-helix and active site B3
-strand regions
showing the preferred residues at each position for tight binding with
BLIP (Fig. 2). This consensus sequence differs from the wild-type
-lactamase sequence by as much as 52%, suggesting that the binding
between TEM-1
-lactamase and BLIP has not been optimized and can be
improved. This is not surprising in that BLIP most likely did not
evolve under selective pressure to bind TEM-1
-lactamase since TEM-1
-lactamase is prevalent in enteric bacteria, and BLIP is produced by
a soil bacterium (18). In addition, TEM-1
-lactamase is found only
in the periplasmic space of Gram-negative bacteria and the
Gram-negative outer membrane would prevent BLIP from reaching the
periplasm. The biological target of BLIP is unknown; however, S. clavuligerus also produces a class A
-lactamase, which has been
proposed to be the natural target of BLIP (36). The amino acid sequence
in the loop-helix and B3
-strand regions of the S. clavuligerus
-lactamase is 52% identical to the TEM-1
-lactamase sequence and 52-66% identical to the consensus sequence
depending on the selected consensus residues. Seven out of the 10 residues identified as critical in TEM-1
-lactamase for binding BLIP
are conserved in the S. clavuligerus
-lactamase including
Asp-101, Ser-106, Pro-107, Thr-109, His-112, Gly-236, and Gly-238. The
remaining critical TEM-1
-lactamase residues Leu-102, Val-103, and
Ser-235 consist of Val, Glu, and Thr, respectively, in the S. clavuligerus
-lactamase. Although a Ki for
S. clavuligerus has not been determined, it has been
reported that the S. clavuligerus
-lactamase is strongly inhibited in the presence of 5 µM BLIP. This is
approximately 100-fold higher than the amount of BLIP required to
strongly inhibit TEM-1
-lactamase (36). These few amino acid
differences may account in part for the reduced inhibitory effects of
BLIP for the S. clavuligerus
-lactamase compared with
that of TEM-1
-lactamase.
Amino acid substitutions at Leu-113, Thr-114, and Glu-240 exhibited a
bias for non-wild-type residues, which appear to improve the binding
interaction between -lactamase and BLIP. This is evident by the
in vitro binding studies in which L113A/T114R and E240K
-lactamase variants were shown to bind BLIP 6- and 11-fold tighter
than the wild-type
-lactamase, respectively (Fig. 5 and Table II).
Examination of the BLIP-
-lactamase interface in the 113-114 region
indicates that the T114R substitution could introduce interactions with
Asp-68, Ser-69, or Tyr-115 of BLIP. Molecular modeling also suggests
these new interactions could displace one or more ordered water
molecules that are present at the BLIP-
-lactamase interface (21).
Ordered water molecules at a protein-protein interface can fill in gaps
between imperfectly packed regions (21, 37). Displacement of these
water molecules by improved packing should result in tighter binding
because of the more favorable entropy of the complex.
In contrast, the E240K/E240R substitution is not in a region of ordered
water molecules. Rather, the Glu-240 residue makes van der Waals
contacts with the BLIP residue Phe-142 (Fig.
6). BLIP Phe-142 is inserted in the active
site pocket of TEM-1 -lactamase and is critical for inhibition (20).
Molecular modeling suggests that Arg or Lys at position 240 could
increase contacts with Phe-142 or could alter its position slightly in
the active site. It is of interest to note that both the L113A/T114R
and E240K residues are at the periphery of the
-lactamase-BLIP
interface. This result suggests that new interactions can be engineered
between residues that line the edges of the
-lactamase-BLIP
interface.
|
Construction of a -lactamase containing the L113A/T114R and the
E240K substitutions resulted in an enzyme that was bound very tightly
by BLIP (Ki = 0.4 pM). This affinity is approximately 7-fold tighter than that expected based on simple additivity of the effects of the individual substitutions (34). A
possible explanation for the synergy between the substitutions observed
in
-lactamase is that the 113-114 and 240 regions of
-lactamase
interact with different domains of BLIP. A hinging motion between the
domains of BLIP has been proposed to allow it to bind several different
-lactamases (21). This flexible hinging motion may also contribute
to non-additive interactions between the domains.
Almost one-half of the extended spectrum TEM-based -lactamases
contain one or more amino acid substitutions in the 99-112 loop-helix
and/or 234-240 B3
-strand regions. These mutations include E104K,
A237G, A237T, G238S, and E240K (38). The data collected here and from
previous studies indicate that BLIP remains a potent inhibitor of
-lactamases harboring the mutations A237T, G238S, and E240K (20).
These findings suggest that BLIP is not only a potent inhibitor of
TEM-1
-lactamase but may also be a potent inhibitor of many of the
61 currently known TEM-based
-lactamases.
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ACKNOWLEDGEMENTS |
---|
We thank Carlos Cantu III for assistance with
enzyme kinetics, Joseph Petrosino for providing purified mal-BLIP
fusion protein, and Wanzhi Huang for technical assistance. We also
thank Hiram Gilbert for providing assistance with BLIP inhibition data
analysis and Natalie Strynadka for the coordinates of the BLIP/TEM-1
-lactamase structure.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant AI32956.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Tel.: 713-798-5609; Fax: 713-798-7375; E-mail: timothyp{at}bcm.tmc.edu.
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ABBREVIATIONS |
---|
The abbreviations used are:
BLIP, -lactamase
inhibitor protein;
BSA, bovine serum albumin;
mal, maltose.
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REFERENCES |
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