Activation for catalysis of penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus by bacterial cell wall

Article abstract of DOI:10.1021/ja0434376

Methicillin-resistant Staphylococcus aureus (MRSA) has acquired a unique penicillin-binding protein (PBP), PBP 2a, which has rendered the organism resistant to the action of all available β-lactam antibiotics. The X-ray structure of PBP 2a shows the active site in a closed conformation, consistent with resistance to inhibition by β-lactam antibiotics. However, it is known that PBP 2a avidly cross-links the S. aureus cell wall, which is its physiological function. It is shown herein that synthetic fragments of the bacterial cell wall bind in a saturable manner to PBP 2a and cause a conformational change in the protein that makes the active site more accessible to binding to a β-lactam antibiotic. These observations and measurements point to a novel strategy by nature to keep the active site of PBP 2a sheltered from the inhibitory activity of the antibiotics, yet it becomes available to the polymeric cell wall by a requisite conformational change for the critical cell wall cross-linking reaction. Copyright

Full text of DOI:10.1021/ja0434376

Published on Web 02/01/2005
Activation for Catalysis of Penicillin-Binding Protein 2a from
Methicillin-Resistant Staphylococcus aureus by Bacterial Cell Wall
Cosimo Fuda, Dusan Hesek, Mijoon Lee, Ken-ichiro Morio, Thomas Nowak, and
Shahriar Mobashery*
Department of Chemistry and Biochemistry, UniVersity of Notre Dame, Notre Dame, Indiana 46556
Received October 29, 2004; E-mail: mobashery@nd.edu
Table 1. Kinetic Parameters for Interactions of Nitrocefin (1) with
PBP 2a in the Absence and Presence of Compounds 4, 5, and 6
Methicillin-resistant Staphylococcus aureus (“MRSA”) is a
leading cause of bacterial infections and a global scourge.1,2 The
recently emerged vancomycin-resistant variants of MRSA are
among the most challenging bacterial strains for chemotherapeutic
intervention.3-6 MRSA strains are resistant to all available â-lactam
antibiotics by the virtue of their acquisition of the mecA gene, which
encodes penicillin-binding protein 2a (PBP 2a).2,7
1
1
1
compound (mM)
k
2 (s-
)
×
103
Ks
(µM)
k2/Ks (M-1s-
)
k ) ×
3 (s- 106
0
3.7 ( 0.3
192 ( 24
19 ( 3
7.2 ( 0.1
35 ( 2
4
0.5
1.0
1.5
2.0
3.0
15 ( 1
25 ( 5
30 ( 5
30 ( 1
35 ( 5
160 ( 30
110 ( 30
95 ( 10
80 ( 10
45 ( 10
90 ( 20
180 ( 50
295 ( 60
380 ( 45
â-Lactam antibiotics acylate a critical active-site serine in PBPs.
This reaction is depicted below for nitrocefin (1), a chromogenic
cephalosporin (a â-lactam; 1 f 2) used in our study. The acyl-
enzyme species (2) undergoes very slow hydrolysis (to give 3);
hence, its longevity deprives bacteria of the critical functions of
PBPs leading to the bactericidal activities of â-lactam antibiotics.
The S. aureus PBP 2a is refractory to the action of available
â-lactam antibiotics since it has a closed active site, according to
the X-ray structure.8 The lack of access to the active-site serine
manifests itself in high dissociation constants (Ks) for the preacy-
lation complex and small rate constants for acylation (k2).7 However,
PBP 2a continues to catalyze its physiological reaction, the cross-
linking of the S. aureus cell wall, unencumbered.1,2 In essence, PBP
2a resists modification by â-lactam antibiotics, but remains a
competent catalyst for its physiological reaction. The advantage of
such an enzyme for bacterial survival in the face of the challenge
by these antibiotics is self-evident, but how PBP 2a possesses such
differential effects toward its substrate and inhibitors is presently
not understood.
700 ( 130
100 ( 4
28 ( 1
5
6
0.5
1.0
1.5
2.0
3.0
25 ( 5
185 ( 30
160 ( 30
120 ( 30
100 ( 15
75 ( 10
140 ( 20
250 ( 55
460 ( 150
650 ( 130
1100 ( 200
40 ( 15
55 ( 15
65 ( 15
80 ( 20
80 ( 2
30 ( 2
0.5
1.0
1.5
2.0
25 ( 10
35 ( 5
60 ( 10
85 ( 10
175 ( 60
140 ( 30
115 ( 5
95 ( 10
130 ( 45
260 ( 55
440 ( 90
900 ( 85
60 ( 8
left this moiety out of the structures, as we did not want the
compounds to be turned over by PBP 2a. The synthetic compounds
4 and 5 are smaller fragments of compound 6.
We have used nitrocefin (1), a chromogenic â-lactam, as a
reporter molecule to evaluate the effects of the peptidoglycan
fragments on PBP 2a. The methodology for cloning, expression,
and purification to homogeneity of PBP 2a and determination of
k2, k3, and Ks with â-lactam antibiotics have been reported.7 Table
1 (first line) gives the kinetic parameters for interactions of
nitrocefin with PBP 2a. Enzyme acylation (k2) proceeds slowly (t1/2
of 3.1 min) and deacylation (k3) is exceedingly slow (t1/2 of 26.7
h). In the presence of the peptidoglycan fragments 4-6 certain
trends were established. As the concentration of a given pepti-
doglycan fragment was increased, the rate constant for acylation
(k2) of PBP 2a by nitrocefin was enhanced, and the value of the
dissociation constant (Ks) was attenuated in each case. These
observations indicated that in the presence of increasing concentra-
tions of the peptidoglycan fragments, the active site became more
available to the â-lactam antibiotic, as judged by both the enhanced
facility of enzyme acylation and by improvement of the stability
of the noncovalent preacylation complex of the antibiotic and PBP
2a. The rate constants for deacylation (k3) were also enhanced by
approximately 3-fold in the presence of the peptidogylcan frag-
ments.
PBPs utilize the polymeric bacterial cell wall, also known as
the peptidoglycan, as substrate. We reasoned that surface interac-
tions of the polymeric substrate outside the closed active site must
facilitate the opening of the active site to make the entry of the
peptidoglycan possible. This interaction would be absent with the
small-molecule â-lactam antibiotics. Availability of three analogues
of the bacterial peptidoglycan fragments (compounds 4-6) in our
lab, each prepared in multistep syntheses,9,10 provided an op-
portunity to explore this possibility. Compound 6 has four residues
of alternating N-acetylglucosamine (NAG) and N-acetylmuramic
acid (NAM). The NAM residues have been appended with the
pentapeptide (NAM-L-Ala-γ-D-Glu-L-Lys-D-Ala-D-Ala) seen in
many bacteria. In some S. aureus, the γ-D-Glu, is substituted with
γ-D-Gln (we have prepared derivatives with this change in structure,
compounds 7 and 8, which behave the same in interactions with
PBP 2a; Supporting Information). The peptidoglycan of S. aureus
has a pentaglycyl moiety, the site of the cross-linking reaction of
PBP 2a, appended to the side chain of L-Lys. We have deliberately
These trends on kinetic parameters argue for a change in protein
conformation from that seen in the X-ray structure for the closed
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C O M M U N I C A T I O N S
favorable entropic factors in interactions of cell wall with many
PBPs, the effective concentrations of the cell wall components that
the PBPs experience on the bacterial cytoplasmic membrane are
high.9,11 As such, there might not have been compelling reasons
for evolution of enhanced affinity for the cell wall by many of
these enzymes. This is true for many important physiological
processes. Regardless, it is likely that the Kd value for a much larger
fragment of peptidoglycan, one that would approach a polymeric
character or one with the pentaglycyl moiety appended to the L-Lys
residue, would be lower than those measured for compounds 4-6.
This would make the process more efficient, such that the many
cross-linking events of the cell wall could proceed unencumbered
within the 20-30 min required for the doubling of the population
of S. aureus growing under favorable conditions.
What has been presented above describes a model for activation
of PBP 2a for its catalytic function consistent with interactions of
the cell wall, its polymeric substrate, outside the active site. Lee et
al. have presented X-ray structural evidence for another PBP that
indicates that for the cross-linking reaction, the enzyme is capable
of sequestering sequentially two portions of the cell wall in grooves
on the surface of the enzyme where, at the closest point, the two
grooves are within 20 Å of each other.12 We believe that similar
binding sites for the peptidoglycan exist for PBP 2a. The binding
of the first peptidoglycan piece to the surface outside the active
site would appear to stimulate the opening of the active site to allow
catalysis to commence.
It has not escaped us that such stimulatory activity of the
peptidoglycan on PBP 2a may be exploited in sensitizing the protein
toward inhibition by the existing â-lactam antibiotics. Such a
strategy would entail identifying a suitable mixture of a pepti-
doglycan fragment and a â-lactam antibiotic that would work in
concert in reversing the deleterious â-lactam-resistant phenotype
of MRSA.
Figure 1. (A) Far-UV circular dichroic spectra of compound 4 by itself
(4.0 mM, s), of PBP 2a by itself (3 µM, b), and of PBP 2a (3 µM) in the
presence of 4 at 1.0 mM (2), 2.0 mM ([), 3.0 mM (9), and 4.0 mM (O).
The lines connect the data points and were not fitted to any specific model.
(B) Change in molar ellipticity of PBP 2a at 222 nm as a function of the
concentrations of compound 4.
active site to one that makes the active site more accessible to
nitrocefin in the presence of the peptidoglycan fragments. We
reasoned that a change in protein conformation should be detectable
by circular dichroic (CD) measurements. As shown in Figure 1,
compound 4 does not have any far-UV chromophore in the range
of wavelengths for the study of the protein. The CD spectrum of
PBP 2a (Figure 1A) shows two strong minima at 208 and 222 nm
corresponding to R-helices typically seen in this family of proteins.
On addition of compound 4 to the solution of PBP 2a, the protein
undergoes a conformation change consistent with a decrease in the
degree of helicity. This process is saturable (Figure 1B) with respect
to the analogue of the cell wall fragment. The change in the
conformation is likely not limited to mere decrease in helicity, but
a more definitive analysis of the structural consequences of binding
of the peptidoglycan fragments to PBP 2a should await X-ray
analyses of these complexes. The results of the CD studies with
compounds 5 and 6 paralleled those with 4 (see Supporting
Information).
Double reciprocal plots of the concentrations of peptidoglycan
fragments versus k2 values furnished the dissociation constants for
the peptidoglycan fragments (Kd). The Kd values for compounds
4, 5, and 6 were 1.0 ( 0.4, 2.0 ( 0.8, and 2.8 ( 1.0 mM,
respectively. In light of the saturation seen in the CD experiments
(Figure 1B), a similar analysis was carried out by CD for
compounds 4 and 5 (the available quantity of 6, which was
synthesized in 37 steps, was not sufficient for this analysis). This
analysis furnished Kd values of 1.1 ( 0.2 and 1.4 ( 0.2 mM for
compounds 4 and 5, respectively. The two independent methods
furnish similar dissociation constants for the noncovalent complex
of the peptidoglycan fragments and PBP 2a.
Acknowledgment. This work was supported by the National
Institutes of Health.
Supporting Information Available: Experimental procedures,
including those for kinetics and synthetic protocols and characterization
of all new compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
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