From the School of Biological Sciences, University of
Sussex, Falmer, Brighton BN1 9QG, United Kingdom,
§ Department of Structural Biology, St. Jude Children's
Research Hospital, Memphis, Tennessee 38105, ¶ Department of Biochemistry, University of Tennessee, Memphis,
Tennessee 38163, and
Department of Pharmacology,
University of North Carolina at Chapel Hill, Chapel Hill,
North Carolina 27599-7365
Received for publication, May 24, 2000, and in revised form, August 15, 2000
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ABSTRACT |
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Penicillin-binding protein 5 (PBP 5) of
Escherichia coli functions as a D-alanine
carboxypeptidase, cleaving the C-terminal D-alanine residue
from cell wall peptides. Like all PBPs, PBP 5 forms a covalent
acyl-enzyme complex with Penicillin and other In Escherichia coli, at least 10 PBPs have been identified.
These PBPs can be split into two classes: the high molecular mass PBPs
(PBPs 1A, 1B, 1C, 2, and 3) and the low molecular mass PBPs (PBPs 4, 5, 6, 6b, and 7) (3). High molecular mass PBPs are essential for cell
viability and are involved in the physiological processes of cell
elongation, cell division, and the maintenance of cell shape (4). The
role of low molecular mass PBPs in bacterial physiology is less clear.
These PBPs catalyze D,D-endopeptidase or
D,D-carboxypeptidase activity both in
vitro and in vivo (5, 6), but although they account for
>90% of the amount of penicillin G bound to membranes, they are not
essential for cell viability (7). However, a recent study utilizing
strains with multiple deletions of low molecular mass PBPs
revealed a role for PBP 5 in the proper synthesis of peptidoglycan
(8).
Production of a The reaction mechanism of the interaction of -lactam antibiotics; however, PBP 5 is
distinguished by its high rate of deacylation of the acyl-enzyme
complex (t1/2 ~ 9 min). A Gly-105
Asp
mutation in PBP 5 markedly impairs this
-lactamase activity
(deacylation), with only minor effects on acylation, and promotes
accumulation of a covalent complex with peptide substrates. To gain
further insight into the catalytic mechanism of PBP 5, we determined
the three-dimensional structure of the G105D mutant form of soluble PBP
5 (termed sPBP 5') at 2.3 Å resolution. The structure is composed of
two domains, a penicillin binding domain with a striking similarity to
Class A
-lactamases (TEM-1-like) and a domain of unknown function. In addition, the penicillin-binding domain contains an active site loop
spatially equivalent to the
loop of
-lactamases. In
-lactamases, the
loop contains two amino acids involved in
catalyzing deacylation. This similarity may explain the high
-lactamase activity of wild-type PBP 5. Because of the low rate of
deacylation of the G105D mutant, visualization of peptide substrates bound to the active site may be possible.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-lactam antibiotics exert their lethal
effect by inhibiting the proteins that synthesize bacterial cell wall
peptidoglycan (1). These proteins, known as penicillin-binding proteins
or PBPs,1 utilize
lipid-linked disaccharide peptide substrates to catalyze both the
polymerization of glycan chains (transglycosylation) and cross-linking
of peptide chains (transpeptidation) during cell wall synthesis. In the
latter reaction, a serine residue on the PBP reacts with the
acyl-D-Ala-D-Ala C terminus of the peptide
chain to form a transient acyl-enzyme complex, releasing the C-terminal
D-alanine residue. This complex reacts with an amino group
from another peptide chain to form a cross-link, which is crucial to
the integrity and rigidity of the cell wall. An additional activity
catalyzed by some PBPs, carboxypeptidation, occurs when the acyl-enzyme
complex reacts with water. Penicillin and other
-lactam antibiotics
mimic the structure of the acyl-D-Ala-D-Ala C
terminus of the peptide chain (2) and react with PBPs to form an
acyl-enzyme complex. Unlike the transient nature of the PBP-peptide
complex, the acyl-enzyme complex formed between PBPs and
-lactam
antibiotics is much more stable and results in prolonged inhibition of
the enzyme.
-lactamase is the most common mechanism by which
bacteria become resistant to
-lactam antibiotics (9). The initial
step of the reaction catalyzed by these enzymes is analogous to that
mediated by PBPs, but instead of accumulating a stable acyl-enzyme
complex,
-lactamases destroy the antibiotic by rapidly hydrolyzing
the acyl-enzyme bond. Analysis of the sequences and crystal structures
of PBPs and Class A and class C
-lactamases reveals strong
structural similarities in the two classes of penicillin-interacting proteins (10-12). The hallmark of all active site serine-based penicillin-interacting proteins is the presence of three well conserved
motifs in the active site (13). These motifs are the SXXK
tetrad containing the active site serine residue, the
(S/Y)XN triad, and the KT(S)G triad.
-lactam antibiotics
with PBPs and
-lactamases is represented schematically by the
following three-step model.
where E is the PBP or
(Eq. 1)
-lactamase, S is a
-lactam antibiotic, E · S is the Michaelis
complex, E-S* is the covalent acyl-enzyme complex, and
P is the inactive degradation product (14, 15). In PBPs,
k3 is usually quite small compared with
k2, leading to accumulation of the inactive
acylated enzyme. For
-lactamases, both the k2
and k3 rate constants are very large, and the
-lactam antibiotic is hydrolyzed.
In contrast to most other PBPs, E. coli PBP 5 is
distinguished by its high -lactamase activity, with k3
0.07 s
1 (t1/2 < 10 min) for the penicilloyl-PBP 5 complex. A mutant PBP 5, PBP 5-G105D
(termed PBP 5'), shows near normal acylation rates with penicillin G
but displays a 30-fold decrease in the rate of deacylation (16, 17).
Additionally, the mutant protein forms a stable covalent complex with
the depsipeptide substrate,
N,N'-diacetyl-L-lysyl-D-alanyl-D-lactate,
which is not observed with the wild-type protein (16). The phenotype of
PBP 5' is similar to that of wild-type PBP 5 in which Cys-115 has been
modified with sulfhydryl reagents, causing a larger effect on
deacylation than acylation (18). However, since the mutants PBP 5-C115A
and PBP 5-C115S both display wild-type levels of D-alanine carboxypeptidase (CPase) and penicillin binding activities, this residue does not appear to be directly involved in the catalytic mechanism of the enzyme (17). Subsequent attempts to identify residues
involved in deacylation of PBP 5 have been unsuccessful (19).
In an effort to understand the molecular mechanism of deacylation of
PBP 5 and to define further the interactions of -lactam antibiotics
and peptide substrates with PBPs, we have solved the structure of the
G105D mutant of PBP 5 at 2.3-Å resolution, the first structure for PBP
5. It reveals the closest similarity yet identified between a PBP-type
enzyme and a Class A
-lactamase, which may explain the relatively
high
-lactamase activity of PBP 5.
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MATERIALS AND METHODS |
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Protein Purification and Crystallization--
Construction of
the expression plasmid encoding sPBP 5' has been described (17, 20).
The gene encoding the G105D mutant form of PBP 5 missing its last 17 codons (plus six extra codons added during plasmid construction) was
cloned into the PstI-HindIII restriction sites of
pBR322. Overnight cultures of MC1061 harboring the expression plasmid
were subjected to osmotic shock (21), and sPBP 5' was purified from the
shock fluid by ampicillin affinity chromatography exactly as described
(17). The purified protein was dialyzed exhaustively against 20 mM Tris· HCl, 150 mM NaCl, 10 mM
2-mercaptoethanol, 0.2% NaN3, pH 7.5 and concentrated to 6-8 mg ml1. SDS-polyacrylamide
electrophoresis indicated that the protein was >98% pure with very
little of the characteristic 30- and 10-kDa breakdown products
(22).
Crystallization conditions were similar to those described previously (23). Crystals were grown by vapor diffusion in 20% polyethylene glycol 4000, 50 mM Tris·HCl, pH 7.0, 0.2% NaN3. Crystals of bullet-shaped morphology formed at 18 °C within 1-2 days. The crystals belong to space group P32 with cell dimensions a = 50.83 Å and b = c = 140.29 Å. There is one molecule in the asymmetric unit.
Data Collection--
In all cases crystals were mounted in
quartz capillaries, and data were collected at room temperature by the
standard oscillation method. The initial native data set was collected
using a Rigaku RAXIS-II image plate system (Molecular Structures Corp.,
The Woodlands, TX) mounted on a Rigaku RU-300 x-ray generator operating
at 40 kV and 80 mA and fitted with a graphite monochromator. The
crystal-to-plate distance was 130 mm, the oscillation angle was 3°,
and the exposure time was 30 min frame1. A
total of 84° of data was collected.
Derivative diffraction data were collected using a DIP 2030H image
plate detector mounted on a Nonius FR391 rotating anode x-ray generator
fitted with MacScience focusing mirrors (Nonius B. V., Delft, The
Netherlands) and operating at 40 kV and 100 mA. Data were collected at
a crystal-to-plate distance of 150 mm, with an oscillation angle of
1° and an exposure time of 18 min frame1.
Rotation ranges of 60-75° were sufficient to collect essentially complete data sets.
For model refinement purposes a high resolution data set was collected,
where the crystal-to-plate distance was 150 mm, the oscillation angle
was 1°, and the exposure time was 12 min
frame1. To ensure a high redundancy of data,
the crystal was rotated through a total of 90°. In all cases the
oscillation data were processed using HKL (24). In the case of
derivative data, the Friedel pairs were not merged in order to retain
the anomalous signal.
Phasing-- A search for derivatives was made by soaking crystals in solutions of heavy atom compounds in stabilizing buffer (20% polyethylene glycol 4000 in 100 mM Tris·HCl, pH 7.0). Soaks in various mercury and platinum compounds yielded several promising derivatives, as judged by difference Patterson maps. In each case heavy atom positions were determined and refined, and the data were used in trial phasing. These calculations showed that two derivatives, mercury cyanide (HgCN2) and di-µ-iodobis-(ethylenediamine) di-platinum nitrate (PIP), were of the best quality and sufficient to solve the structure. All calculations to this point were performed using PHASES (25). At this stage the derivative and native data were merged using SCALEIT (26), and the final phasing calculation was performed using SHARP (27) and included anomalous data. The resulting multiple isomorphous replacement (MIR) phases were improved by solvent flattening using SOLOMON (28) with an estimated solvent content of 46%.
Model Building and Refinement--
An MIR electron density map
(Fig. 1B) was calculated using CCP4 programs (26) and
displayed using the program O (29). The map was of excellent quality
and was easily interpreted to build the entire structure, including
side chains, with the exception of residues 1-3 and 74-90. The model
was then refined by alternating rounds of XPLOR and manual revision
using O. In later rounds water molecules were included. The final round
of refinement was performed using REFMAC (26). The stereochemistry of
the final model was evaluated using PROCHECK (30). The numbering of the
final model corresponds to the sequence of the mature processed protein.
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RESULTS |
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Structure Determination
The structure of sPBP 5' was
determined by MIR with anomalous
scattering. The data collection and phasing statistics are
shown in Tables I and II, and
portions of both the MIR and final 2/Fo Fc electron density maps are shown in Fig.
1. The final structure has an
R factor of 19.6% (Rfree = 27.0%)
at 2.3-Å resolution (Table III).
Currently 151 water molecules are included in the model.
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As defined by PROCHECK (30), 90.0% of the residues lie within the most favored region of the Ramachandran plot. Three residues fall within disallowed regions. Of these, Ala-155 and Lys-219 both lie within loop regions with weak density, whereas Ile-212 is a hydrophobic core residue with excellent density. Residues with the highest B factors are located exclusively at the termini or in surface-exposed loops. Certain parts of the molecule have been omitted from the model where they correspond to regions of poor electron density, likely due to disorder. These are residues 1-3 at the N terminus, seven residues at the C terminus (six of which are non-native and result from the genetic construct), and an external loop comprising residues 74-90. The apparent flexibility of these latter residues is consistent with the increased susceptibility of the G105D mutant enzyme to proteolysis compared with PBP 5 wild type. Proteolysis of sPBP 5' generates characteristic 30- and 10-kDa fragments by cleavage between residues 87 and 88, which reside within this external loop (31).
Structure Description
sPBP 5' is composed of two domains that are oriented approximately
at right angles to each other (Fig. 2).
Each domain is formed from contiguous primary sequence, residues 3 to
262 for domain 1 and residues 263 to 356 for domain 2. The N-terminal region of sPBP 5' is a loop that extends away from domain 1 and makes a
slight contact with domain 2 via Lys-6.
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Domain 1--
The principal feature of domain 1 is a five-stranded
anti-parallel sheet that forms the hydrophobic core. This is packed on one side by an extended loop at the N terminus,
10, and a loop
connecting
8 and
9 that contains a small
helix (
9). The
other side is comprised primarily of an array of seven
helices. In
addition, there are three
hairpin-like structures,
3-
4,
5-
6, and
7-
8, one of which (
3-
4) contains the
disordered residues 74-90. The active site of PBP 5 is located within
this domain at the boundary of the five-stranded
-sheet and the
helical array (see below). It is immediately apparent that domain 1 has the same fold as Class A and Class C
-lactamases (discussed below).
Domain 2--
This domain, which is almost exclusively comprised
of structure, is a sandwich of two anti-parallel
sheets, one
three-stranded and the other two-stranded. The two-stranded sheet is
kinked at residues Gly-272 and Asp-293, causing the two sides of the
sheet to be at right angles. The effect of this is to form a loose
barrel at the C-terminal end of the domain. Compared with domain 1, domain 2 has a relatively hydrophobic surface as judged by an
electrostatic plot (data not shown), although none of these hydrophobic
residues are particularly well conserved in other CPases. The
significance of this apparent hydrophobicity and the role of this
domain in the function of the protein are unknown. A search of the
Protein Data Bank using the DALI server (32) revealed no genuine
structural similarities to domain 2.
Domain Interface-- Since the hydrophobic cores of domains 1 and 2 extend to include the domain interface and because this region has comparatively low B factors, significant movements between domains 1 and 2 seem unlikely, suggesting that the relative juxtaposition of the two domains is conserved.
Sequence Alignment to Other CPases and Secondary Structure Assignment
A Blast search of the GenBankTM data base with
E. coli PBP 5 as a query sequence identified two other
E. coli PBPs (PBP 6 and PBP 6b) and PBPs from
Salmonella typhimurium and Haemophilus
influenzae as its closest matches. Alignment of these PBPs is
shown in Fig. 3 along with secondary
structure assignments from the three-dimensional structure of sPBP 5'.
As expected, the regions of highest identity reside in and around the
conserved sequence motifs (shown in parentheses) associated with the
active site in domain 1: residues 42-53
(Ser44-X-X-Lys47),
residues 110-114 (Ser110-X-Asn112),
and residues 210-217
(Lys213-Thr214-Gly215) (19). Two
additional highly conserved regions, comprising residues 149-154 and
195-203, also are located in close proximity to the active site (see
Fig. 4). In general, the residues in
domain 2 are less conserved than domain 1, and most of the conserved residues are within the hydrophobic core.
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Active Site
The location of the active site is readily identified by plotting
the conserved sequence motifs onto the sPBP 5' structure. In common
with other PBPs and -lactamases, the active site is located in the
cleft between the five-stranded anti-parallel
sheet and the large
helical cluster. As viewed in Fig. 2, the entrance to the active
site is at the opposite end of the molecule from the C terminus and the
membrane anchor (which is missing in our construct), in an ideal
position to interact with cell wall peptides. The architecture of
active site (Fig. 4) is comprised of the following elements: helix
2
(yellow), containing Ser-44 and Lys-47 of the
SXXK tetrad; the turn between helices
4 and
5
(green), containing Ser-110 and Asn-112 of the SXN triad;
and
9 (orange), the edge strand of the
sheet
containing Lys-213, Thr-214, and Gly-215 of the KT(S)G triad. In
addition, the loops between
5 and
6 and between
8 and
9
also contribute residues to the active site. These include His-151,
present on the extended loop (blue) at the bottom of the
cavity, and Arg-198, located on the loop (purple) at the top
of the cavity. Both of these latter residues are conserved in related
CPases (Fig. 4). The identification of Ser-44, Lys-47, Ser-110,
Asn-112, and Lys-213 as active site residues is consistent with data
showing that mutation of any of these renders PBP 5 highly deficient
or, in some cases, completely inactive in both antibiotic binding and
carboxypeptidation (19, 33).
The hydrogen-bonding network within the active site is extensive (Fig.
4). The -NH2 group of Lys-47 plays a central role in
this network, forming hydrogen bonds with the hydroxyl group of Ser-44,
the amide carbonyl group of Asn-112, and the backbone carbonyl groups
from both Ser-110 and His-151. In common with other
penicillin-interacting enzymes, the
-NH2 group of
Lys-213 forms a hydrogen bond with Ser-110. In contrast to Class A
-lactamases, in which two structurally conserved water molecules are
typically observed within the hydrogen bonding network (34), there are no visible water molecules in the immediate vicinity of the active site. However, further refinement and higher resolution data of sPBP 5'
may be necessary to firmly establish the presence or absence of
potential active site water molecules.
Structural Homology of sPBP 5' and TEM-1 -Lactamase
One of the most outstanding features of the sPBP 5' structure is
the similarity of domain 1 with the fold of Class A -lactamases, as
represented by TEM-1 (35) and PC1 (36). Notably, this structural similarity is considerably more pronounced than previously reported PBP
structures, namely the Streptomyces R61
D,D-peptidase (37) and PBP 2x from
Streptococcus pneumoniae (38). The main chain atoms in the
209 residues of domain 1 of sPBP 5' composing the common elements of
secondary structure of penicillin-interacting proteins can be
superimposed onto the PC1
-lactamase fold (36) with an r.m.s.
deviation of 2.7 Å and the TEM-1
-lactamase (39) fold with an
r.m.s. deviation of 2.5 Å.
The similarity of domain 1 of sPBP 5' and TEM-1 -lactamase is shown
in Fig. 5. The most significant
differences between the two structures are 1) the N-terminal helix of
TEM-1 is replaced by an extended chain in sPBP 5', 2) the disordered
region between residues 74 and 90 in sPBP 5' is visible in TEM-1 as a
small helix with connecting loops, 3) the extended loop (colored
blue in Fig. 4, of sPBP 5' has a slightly different
conformation compared with its counterpart in TEM-1, the so-called
loop, and 4) in sPBP 5' the connection between helices
8 and
9
contains a large
hairpin, whereas the equivalent connection in
TEM-1, between helices
9 and
10, is direct.
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A comparable degree of similarity with Class A -lactamases was also
observed in the recently determined structure of the Streptomyces K15 D,D-transpeptidase
(40). In fact, the K15 PBP and sPBP 5' are close structural relatives.
Domain 1 of sPBP 5' can be superimposed onto the K15 PBP structure with
an r.m.s. deviation of 1.2 Å between common main chain atoms (212 residues). The main differences of note are that the part of the K15
PBP structure equivalent to the disordered region in sPBP 5' (residues 74-90) is ordered and that the
7-
8 hairpin is considerably
longer in the K15 PBP structure (
2c-
2d), generating a
four-stranded
sheet on the surface of the K15 PBP. Unlike most
other PBPs, the K15 PBP does not contain a traditional hydrophobic
transmembrane anchor. Thus, it has been proposed that the
2c-
2d
hairpin promotes the association of the K15 PBP with the cell membrane
(40), which may explain its longer length.
The active site residues of sPBP 5' and TEM-1 -lactamase were
aligned by the program LSQKAB (26) (Fig.
6). Ser-44, Lys-47, Ser-110, Asn-112, and
Lys-213 are all spatially conserved with the corresponding residues in
TEM-1
-lactamase, again emphasizing the extensive homology between
these two proteins. Although the extended
-like loop at the lower
region of the active site of sPBP 5' is similarly positioned to the
loop of TEM-1, there are significant conformational differences between
the two. This is important because in Class A
-lactamases, the
loop contains two residues, Glu-166 and Asn-170, that are critical for
the rapid hydrolysis of the acyl-enzyme complex (41, 42). By analogy, the
-like loop in PBP 5 (residues 147-157) may also contain similar residues responsible for deacylation, thus explaining the high
-lactamase activity observed in wild-type PBP 5 (see
"Discussion").
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DISCUSSION |
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Here we report the structure of a soluble, mutant form of PBP 5'
solved to 2.3-Å resolution by x-ray crystallography. The structure of
sPBP 5' reveals two domains, a penicillin-binding domain and a domain
of unknown function. The fold of the penicillin-binding domain is
highly similar to Class A -lactamases, further emphasizing the close
evolutionary relationship between these two classes of
penicillin-interacting proteins.
Catalytic Function--
PBP 5 is a serine-based
D-alanine carboxypeptidase that is functionally similar to
the well studied family of serine proteases. Although both PBP 5 and
the serine proteases proceed through formation of an acyl-enzyme
intermediate followed by deacylation of the acylated enzyme, they have
evolved distinctly different catalytic mechanisms. The close structural
similarity of sPBP 5' with Class A -lactamases suggests a common
mechanism of acylation by
-lactam antibiotics for the two classes of
enzymes. In the light of the structure of PBP 5', the potential roles
in catalysis of the conserved active site residues of PBP 5 can now be discussed.
Acylation of PBP 5 with -Lactam Antibiotics and Peptide
Substrates--
The acylation reaction comprises four steps: formation
of the non-covalent complex, nucleophilic attack, tetrahedral
intermediate formation, and collapse to the acyl-enzyme. Mutational
evidence has shown that, at least for
-lactam antibiotics, a
positive charge at position 213 is crucial for formation of the
acyl-enzyme complex (33), suggesting that Lys-213 (of the KTG triad)
interacts with the carboxylate group on both
-lactam antibiotics and
peptide substrates. Interestingly, the equivalent residue in
-lactamases, Lys-234, has been proposed to stabilize the transition
state (43, 44). Since in our structure the
-amino group of Lys-213
interacts via potential hydrogen bonds with the hydroxyl group of
Ser-110 and the carbonyl group of Asn-107, an alternative role of this residue may be simply to maintain the correct active site architecture rather than to interact with substrate directly. It is interesting to
note that mutation of Lys-213 to arginine has no effect on penicillin
binding and hydrolysis yet abolishes CPase activity (33). Such a
mutation would increase the distance between two key components of the
active site,
9 and the C-terminal end of
4, by approximately 1 Å. This may lower the binding affinity for peptide substrates, which
presumably have a larger binding footprint without affecting the
smaller binding site for penicillin.
Acylation starts by nucleophilic attack of the active site Ser-44 (22)
on the amide bond of the peptide substrate or -lactam ring. In
serine proteases, the nucleophilicity of the serine residue is
activated by the so-called "charge-relay" system involving a
histidine and aspartic acid residue (45). In our structure it is
difficult to assess which of the surrounding residues enhance the
nucleophilicity of Ser-44. One possibility is that the amino group of
Lys-47, which is within hydrogen-bonding distance of the Ser-44
hydroxyl, acts as a general base. For this to occur, however, the
active site environment must promote the deprotonated state of the
-NH2 group, requiring a reduction in its
pKa of approximately 3 pH units. This might be
achieved via its hydrogen-bonding interactions with Asn-112 and the
carbonyl groups of His-151 and Ser-110 (Fig. 4). An alternative
possibility is that the nucleophilicity of Ser-44 may be enhanced by
the dipole moment of the helix
2. The role of the helix dipole has
recently been suggested to increase the nucleophilicity of the active
site cysteine in
-ketoacyl-acyl carrier protein synthase III, a
bacterial enzyme that catalyzes a Claisen condensation in type II fatty
acid synthesis (46).
It is interestingly to note that one characterized mutation, D175N,
which abolishes CPase activity and decreases acylation by 50-fold (19),
probably acts indirectly by perturbing the precise arrangement of the
active site rather than having a direct role in catalysis as previously
suggested. Our structure shows that the effect of this mutation is to
break a hydrogen bond between the side chains of Asp-175 and Tyr-52,
located at the C-terminal end of helix 2, more than 20 Å from
Ser-44. That such a subtle change has such a large impact on activity
is evidence that the precise positioning of the N-terminal end of
2,
which contains Ser-44 and Lys-47, is critical for catalysis.
The developing negative charge on the carbonyl oxygen of the
tetrahedral intermediate is likely stabilized by an oxyanion hole
similar to that proposed for many other enzymes, including serine
proteases, -lactamases, and other PBPs. In PBP 5' the oxyanion hole
is potentially formed by backbone amide groups of residues 214 and 216 from
9 and Ser-44 on
2. Arg-198 is positioned above the active
site and may also help stabilize the intermediate.
Hydrolysis of the Acyl-Enzyme Complex--
The close structural
overlap of PBP 5' with Class A -lactamases suggests that the
relatively high deacylation rate of the
-lactam-bound PBP 5 may be
mediated by the loop equivalent to the
loop in Class A
-lactamases. In the latter, two residues on this loop, Glu-166 and
Asn-170, are intimately involved in the rapid deacylation of the
acyl-enzyme complex (42, 47). In particular, Glu-166 is believed to
activate a conserved water molecule that attacks the carbonyl of the
acyl-enzyme (41, 48). By analogy, residues in the
-like loop of PBP
5 may also position a catalytic water molecule that attacks the
acyl-enzyme complex. Candidate residues from the
-like loop include
His-151 and Asp-154, both of which are conserved in other CPases (Fig.
3) and one of which, His-151, is close to the active site (Fig. 4).
However, in our structure of the deacylation-defective PBP 5', neither of these side chains is sufficiently close to the active site to
interact directly with substrate or potential catalytic water molecules
(Fig. 4). An alternative mechanism for hydrolysis of the acyl-enzyme
complex is that the backbone carbonyl moiety of His-151, which in our
structure is hydrogen-bonded to Lys-47 (Fig. 4), participates in
deacylation simply by favorably orienting a hydrolytic water molecule.
This is supported by the spatial equivalence of carbonyl group of
His-151 in PBP 5' with the carboxyl group of Glu-166 in TEM-1 (Fig.
6).
Further evidence that the -like loop in PBP 5' may indeed be
involved in deacylation comes from studies of deacylation-defective forms of PBP 5. In the G105D mutant, Gly-105 (small) is replaced by
aspartate (charged and bulky), which may disrupt the packing of helix
4 with the adjacent 74-90 loop and cause the apparent flexibility
of the latter observed in our crystal structure. In both Class A
-lactamases and the K15 D,D-transpeptidase,
the equivalent loop is ordered and packs against the counterparts of
helices
4,
5, and the
-like loop in sPBP 5'. A second
deacylation-defective PBP 5 is obtained through modification of Cys-115
by para-chloromercuribenzoate in the wild-type enzyme (18).
This residue is located on helix
5 and packs into the space between
2 and
4. Introduction of such a bulky group would doubtless
perturb this close packing and may also affect the conformation of the
adjacent 74-90 loop. Thus it appears that a decrease in deacylation
may result from changes in the specific region of the structure
containing
4,
5, the 74-90 loop, and the
-like loop. In the
structure of wild-type PBP 5, it will be interesting to note whether
the 74-90 loop is more ordered and indeed whether the conformation of
the
-like loop is altered as a result.
Relationship of sPBP 5' to -Lactamases--
The structural
homology between the Streptomyces R61
D,D-peptidase and PC1
-lactamase was noted
as early as 1986 (11, 12), but the similarity between sPBP 5' and TEM-1
-lactamase is even more pronounced. A comparable degree of
similarity to Class A
-lactamases also has been observed with the
Streptomyces K15 D,D-transpeptidase
(40). It has been suggested that each of the different classes of
-lactamases have evolved from various classes of PBPs (49). Our data
are entirely consistent with this hypothesis.
The main structural difference between the penicillin binding domain of
sPBP 5' and Class A -lactamases is in the sequence and conformation
of the
loop, perhaps suggesting that Class A
-lactamases evolved
directly from a PBP 5-like CPase. This would be logical because CPases
already had a relatively efficient hydrolysis mechanism for peptide
substrates. By incorporating two new amino acids on the
loop and by
removing residues specific for CPase activity,
-lactamases evolved a
highly efficient hydrolysis mechanism and at the same time altered
their specificity toward
-lactam antibiotics and away from peptide substrates.
Role of Domain 2--
The two-domain structure of PBP 5, in which
catalytic activity resides in domain 1, is consistent with previous
genetic studies showing that deletion of the protein after residue 263 results in an enzyme with near wild-type CPase activity and -lactam
antibiotic affinity (50). This, however, leaves the role of domain 2 unclear. One possibility is that this domain mediates interactions with other cell wall-synthesizing enzymes to recruit PBP 5 to areas of
active cell wall synthesis. Indeed, the all
structure of this
domain may facilitate such interactions through hydrogen bonding across
strands. In E. coli, the lytic transglycosylase, MltA,
interacts with several PBPs as well as the scaffolding protein, MipA
(8), suggesting the existence of a cell wall-synthesizing complex, of
which PBP 5 may be a part. Alternatively, domain 2 may function simply
as a linker to position the active site in domain 1 closer to the
peptidoglycan layer, where it can interact with cell wall peptides.
Conclusion--
By solving the crystal structure of a
deacylation-defective mutant, we have obtained the first structural
information for PBP 5. PBP 5 shares the same fold and has a common
active site architecture with the well studied Class A -lactamases.
Inspection of this structure suggests that Lys-47 may act as the
general base to promote the nucleophilicity of Ser-44 during acylation. In contrast, the mechanism of deacylation is less clear. However, solving the wild-type PBP 5 structure and comparing it to PBP 5' should
provide significant insight into how PBP 5 catalyzes hydrolysis of the
acyl-enzyme complex. These studies are in progress.
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FOOTNOTES |
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* 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.
The atomic coordinates and the structure factors (code 1hd8) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
** To whom correspondence should be addressed: Dept. of Pharmacology, CB#7365 Mary Ellen Jones Bldg., Chapel Hill, NC 27599-7365. E-mail: nicholas@med.unc.edu.
Published, JBC Papers in Press, August 30, 2000, DOI 10.1074/jbc.M004471200
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ABBREVIATIONS |
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The abbreviations used are: PBP, penicillin-binding protein; sPBP, soluble PBP; CPase, carboxypeptidase; MIR, multiple isomorphous replacement; r.m.s., root mean square.
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