From the Bristol-Myers Squibb Pharma Company, Wilmington, Delaware 19880
Received for publication, September 10, 2002, and in revised form, January 2, 2003
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ABSTRACT |
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Kinetic analysis of ribosomal peptidyltransferase
activity in a methanolic puromycin reaction with wild type and
drug-resistant 23 S RNA mutants was used to probe the structural basis
of catalysis and mechanism of resistance to antibiotics. 23 S RNA
mutants G2032A and G2447A are resistant to oxazolidinones both in
vitro and in vivo with the latter displaying a 5-fold
increase in the value of Km for initiator tRNA and
a 100-fold decrease in Vmax in puromycin
reaction. Comparison of the Ki values for oxazolidinones, chloramphenicol, and sparsomycin revealed partial cross-resistance between oxazolidinones and chloramphenicol; no cross-resistance was observed with sparsomycin, a known inhibitor of
the peptidyltransferase A-site. Inhibition of the mutants using a truncated CCA-Phe-X-Biotin fragment as a P-site substrate
is similar to that observed with the intact initiator tRNA, indicating that the inhibition is substrate-independent and that the
peptidyltransferase itself is the oxazolidinone target. Mapping of all
known mutations that confer resistance to these drugs onto the spatial
structure of the 50 S ribosomal subunit allows for docking of an
oxazolidinone into a proposed binding pocket. The model suggests that
oxazolidinones bind between the P- and A-loops, partially overlapping
with the peptidyltransferase P-site. Thus, kinetic, mutagenesis, and
structural data suggest that oxazolidinones interfere with initiator
fMet-tRNA binding to the P-site of the ribosomal
peptidyltransferase center.
Oxazolidinones, the only novel class of antibiotics identified in
the last two decades, are the focus of intensive discovery efforts
(1-9). Linezolid, an oxazolidinone, is approved for treatment of
infections caused by Gram-positive bacteria that are resistant to other
antibiotics. Emerging resistance to all known drugs, including the
"last resort" vancomycin family, poses a serious threat to the
public health worldwide. Understanding the mechanism of action of
oxazolidinones at the molecular level, therefore, has a great
importance for the development of the next generation of these novel
antibiotics and, ultimately, for the outcome of the ongoing battle
against drug-resistant pathogens.
Oxazolidinones impose their action at the initiation stage of
translation (4, 5), apparently via inhibition of preinitiation complex
formation (9). Our recent finding that oxazolidinones interfere with
binding of initiator tRNA to the ribosomal P-site (1), thus inhibiting
formation of the first peptide bond, prompted a search for similarities
between oxazolidinones and known inhibitors of peptidyltransferase. To
address this and other mechanistic questions, we have studied catalytic
properties of oxazolidinone-resistant ribosomes and compared the
mechanism of oxazolidinone inhibition with the action of known
peptidyltransferase inhibitors, such as chloramphenicol and
sparsomycin. To further define the oxazolidinone binding site, we have
mapped resistant mutations onto the three-dimensional structure of the
ribosomal 50 S subunit to reveal an inhibitor binding pocket and docked
an oxazolidinone inhibitor therein to show its fitness.
Reagents and Materials--
Puromycin, chloramphenicol,
and tRNA Preparation of Oxazolidinone-resistant 23 S RNA Mutant
Ribosomes--
Oxazolidinone-resistant mutant strains carrying a
single mutated copy of ribosomal 23 S RNA were selected and
characterized as reported in Refs. 2 and 33 using plasmids with
site-directed mutations (10) and a recently described single
plasmid-encoded rRNA operon system in Escherichia coli (11).
Cells from 4 liters of exponentially growing wild type or mutant
E. coli culture (A590 = 0.6) were
pelleted by centrifugation in a Sorvall SLA3000 rotor at 9110 rpm for
15 min at 4 °C. Cell pellets were resuspended in an equal
volume of buffer A (20 mM HEPES-KOH (pH 7.6 at
0 °C), 6 mM MgCl2, 30 mM
NH4Cl, 4 mM Synthesis of Initiator
fMet-tRNA Puromycin Peptidyltransferase Reaction--
The in
vitro peptidyltransferase assay was carried out in a
45-µl mixture containing the following components: 36.7 mM Tris-HCl (pH 7.4 at 23 °C), 10 mM
MgCl2, 266.7 mM KCl, 0.01 µM of
E. coli 70 S ribosomes, 0.01-0.4 µM
f-[35S]Met-tRNA Fragment Peptidyltransferase Reaction--
Reactions were
performed at room temperature and contained the following components in
a 60-µl volume: 36.7 mM Tris-HCl (pH 7.4 at 23 °C), 10 mM MgCl2, 266.7 mM KCl, 0.15 µM of E. coli MRE600 70 S ribosomes, various
concentrations of CCA-Phe-X-Biotin, 50 µM
[3H]puromycin (0.5 Ci/mmol), 33.3% MeOH, and 0.0083%
Me2SO. The reaction mixtures were stopped by addition of
0.5 volume of 7 M guanidine-HCl with 20 mM EDTA
(pH 8.0) at different incubation time points. The quenched reactions
were then transferred to a SAM96Biotin capture plate
prewetted with 2 M NaCl. After incubation for 10 min at
room temperature, free [3H]puromycin was removed by
filtration and subsequent washes with 2 M NaCl. The bound
Biotin-phe-[3H]puromycin was measured using a Packard
Topcount-NTXTM by adding 15 µl of Microscint-20.
Mutant Km and Vmax
Determination--
Assuming that the ribosomal peptidyltransferase has
an ordered mechanism of action (12), in which the P-site substrate, A,
and the A-site substrate, B, associate with the ribosome, E, in
an obligate order: the A-site substrate can only bind to the preformed
P-substrate-ribosome complex, as shown in Reaction 1,
IC50 and Ki Determination--
To
determine Ki values in the puromycin and fragment
reactions, initial rates of the reaction in the presence of different
concentrations of inhibitors were evaluated by quantifying product at a
single time point (1). Incubation times for wild type and different
mutants have been selected based on linearity of time courses for
different peptidyltransferases. The puromycin reaction was performed
using 50 µM puromycin, 0.2 µM
fMet-tRNA
For sparsomycin, a competitive inhibitor of A-site (13-15), Equation 2
was used.
Modeling of Oxazolidinone Binding Site--
Nucleotides
in Haloarcula marismortui 23 S RNA for the
oxazolidinone-resistant mutations reported for Halobacterium
halobium and E. coli in Refs. 2, 20, and 21 have
been assigned by alignment of the corresponding 23 S sequences: G2032
(2073), C2057 (2098), G2058 (2099), C2062 (2104), G2447 (2482), C2453
(2487), U2499 (2535), C2500 (2536), A2502 (2538), and U2503 (2539),
with H. marismortui numbering given in parentheses. Mutated
nucleotides as well as A- and P-loop regions have been mapped onto the
50 S crystal structure using WebLab ViewerLite 3.5 software.
Oxazolidinone cross-linking positions reported by Matassova et
al. (22) could not be visualized in the 2.4-Å electron density
map (23); thus, the docking procedure was predominantly based on steric
considerations as well as on mutant inhibition data. The oxazolidinone
inhibitor XA043 was modeled into a potential binding pocket on the
ribosome using the x-ray coordinates with Protein Data Bank entry code 1FFK and Sybyl molecular modeling software. The inhibitor was built and
minimized in the gas phase before manually docking it into the pocket
near residues where oxazolidinone-resistant mutations occur and the
active site K+ resides.
Catalytic Properties of 23 S RNA Mutants in Puromycin
Reaction--
Km and Vmax
values for both A- and P-site substrates in the puromycin reaction for
the drug-resistant 23 S RNA mutants were determined and compared with
the wild type ribosomes. For mutant G2447A (position 2482 in H. marismortui), significant changes in both Km
and Vmax values are observed for both substrates (Fig. 1, A and B).
The values of Km for initiator tRNA and puromycin
were increased by 5- and 2-fold, respectively, and Vmax decreased 100-fold (Table
I). Erythromycin-resistant mutant A2058G
(2099 in H. marismortui), which is weakly resistant to oxazolidinones in vivo (2, 33), exhibited a 3-fold
increase in Km value for initiator tRNA with no
change for that of puromycin. Oxazolidinone- and
chloramphenicol-resistant and erythromycin-hypersensitive mutant G2032A
(2973) has Km and Vmax values
that are similar to the wild type. Thus, two of the three studied
oxazolidinone-resistant mutants (G2447A and A2058G) had decreased
affinity for the initiator tRNA (P-site) substrate with none of the
mutations affecting the interaction with the A-site substrate
puromycin.
Effect of Peptidyltransferase Inhibitors upon Activity of 23 S RNA
Mutants in Puromycin and Fragment Reactions--
To define the
oxazolidinone binding site, we studied inhibition of
oxazolidinone-resistant mutants in two different substrate systems: in the puromycin reaction that employs initiator
fMet-tRNA
Inhibition profiles of mutant peptidyltransferases in the puromycin
reaction with the oxazolidinones, XA043 and linezolid, are shown in
Fig. 2, A and B,
respectively. As expected based on their high levels of resistance
in vivo (2, 33), oxazolidinone-resistant mutants G2447A and
G2032A are not inhibited by oxazolidinones in vitro. The
effect of chloramphenicol on peptidyltransferase mutants is shown in
Fig. 2C. Oxazolidinone-resistant mutant G2447A is not
inhibited by chloramphenicol either, although it is highly sensitive to
the A-site inhibitor sparsomycin. In contrast, oxazolidinone-resistant mutant G2032A shows rather a moderate in vitro resistance
against chloramphenicol, indicated by less than 4-fold decrease in
affinity for this inhibitor. Inhibition of mutant peptidyltransferases with the A-site inhibitor sparsomycin is shown in Fig. 2D.
An inhibition constant of ~0.4 µM determined in this
work for sparsomycin was in good agreement with the
Ki value of ~1 µM reported previously for this antibiotic with a pentamer fragment substrate by
Harris and Pestka (34). Oxazolidinone-resistant mutants are either more
sensitive to inhibition by sparsomycin than the wild type (A2058G and
G2447A) or 2-fold less sensitive (G2032A), indicating little
cross-resistance.
When the fragment P-site substrate was used in place of initiator
fMet-tRNA, the cross-resistance to oxazolidinones (Fig. 3, A and B) and
chloramphenicol (Fig. 3C) for mutant G2447A remains with a
somewhat lower level of resistance to oxazolidinone XA043. It is
noteworthy that an opposite effect, a stimulation of the fragment
reaction, was observed for this mutant in the presence of linezolid.
The effects of inhibitors on the reactions catalyzed by mutant G2032A
were affected by which P-site substrate was used: initiator tRNA leads
to less inhibition than the truncated P-site substrate. At the same
time, mutations had insignificant, less than 2-fold, effect on the
magnitude of inhibition with sparsomycin (Fig. 3D).
To compare inhibitors with different mechanisms of action,
Ki values were calculated (summarized in Table
II). Mutants A2058G and G2032A are
~3-4-fold less sensitive toward linezolid in comparison with XA043.
Both of these mutants are less affected by chloramphenicol than wild
type. Mutant G2447A was not inhibited by chloramphenicol at all, but
its sensitivity to oxazolidinones decreased by more than 100-fold. A
weakly resistant to oxazolidinones mutant (A2058G) (2, 33) is sensitive
toward oxazolidinones in a fragment reaction, while displaying a
moderate in vitro resistance when whole initiator tRNA was
used as a substrate (Figs. 2 and 3, compare A and
B, respectively). None of the mutations studied displayed a
significant level of resistance to an A-site inhibitor sparsomycin.
Mutational studies of the ribosome by genetic means have been a
fruitful approach to investigation of the functional role of the
different regions of this remarkable ribozyme (24-26). However, the
biochemical effects of ribosomal mutations were hindered by an
inability to isolate mutant ribosomes free of wild type counterparts because of the presence of multiple copies of both 16 and 23 S RNA in
most eubacteria. Development of the single plasmid-encoded rRNA operon
system in E. coli (11) made possible engineering, isolation, and enzymatic characterization of mutant ribosomes in
vitro. Steady state experiments using purified drug-resistant ribosomal mutants have yielded meaningful catalytic parameters. These,
in turn, have allowed initial structure-function relationships at the
peptidyltransferase center to be determined, as well as possible
mechanisms of action and resistance to antibiotics at the level of the
catalyst itself.
We find that 23 S RNA G2032A and G2447A mutations result in decreased
affinity for the P-site substrate initiator fMet-tRNA and insensitivity
toward oxazolidinones and chloramphenicol. Nucleotide 2447 belongs to
the peptidyltransferase region that has been shown to be involved in
interactions with the acyl moiety of initiator tRNA (P-site) (27-30).
We also demonstrate that the affinity for the A-site substrate is not
affected by the mutations that confer resistance to oxazolidinones.
According to our inhibition results (Figs. 2 and 3), chloramphenicol,
an inhibitor of both P- and A-sites (16, 17), displays partial
cross-resistance with oxazolidinones, whereas a specific inhibitor of
the A-site, sparsomycin, does not. Thus, this suggests that
oxazolidinones inhibit the methanolic peptidyltransferase reaction
primarily via interference with the P-site substrate binding. In
addition, we have shown previously that oxazolidinones are competitive
with initiator tRNA (P-site substrate) inhibitors (1). The accumulated
evidence leads us to conclude that oxazolidinones are inhibitors of the
peptidyltransferase P-site.
Truncation of the P-site substrate did not eliminate in
vitro resistance toward oxazolidinones and chloramphenicol. This
can be interpreted in terms of the binding site for these drugs being located on the 50 S part of the P-site in a close proximity to the
CCA-fMet binding site. We suggest that mutations at positions G2447 and
G2032 interfere with the binding of the isoacceptor part of the
initiator tRNA and at the same time contribute to formation of the
oxazolidinone binding site. Mutant A2058G showed no resistance toward
oxazolidinones in the fragment reaction, whereas a moderate decrease in
affinity for oxazolidinone linezolid was observed in the pulomycin
reaction. This can be interpreted in terms of the possible structural
changes in the P-site induced by this mutation, in particular in
binding of the non-isoacceptor portion of the initiator tRNA. A
remarkable observation that a catalysis of the fragment reaction by
mutant G2447A is stimulated in the presence of linezolid, but inhibited
by XA043, suggests that inhibition with oxazolidinones might occur via
conformational change in the mutant active center that distorts the
substrate(s) binding site, leading to a dichotomous outcome depending
on the structure of the inhibitory molecule bound.
To further define the oxazolidinone binding site, mutations that lead
to oxazolidinone resistance (2, 20, 21, 33) were mapped onto the
crystal structure (23) of the peptidyltransferase 50 S subunit (Fig.
4). Although the mutated nucleotides are
relatively dispersed in the primary structure of 23 S rRNA, these
mutations map into a confined space between the P- and A-loops in the
three-dimensional structure, which partially overlaps with the P-site.
As seen in Fig. 4A, the mutations are clustered near the
central channel of the ribosome that permits substrate entry to the
P-site. There is a cavity of sufficient size to accept the inhibitor
XA043 as shown in Fig. 4C, which contains a potassium ion
that might be required for catalytic activity (30). Little
reorganization of the nucleotide residues is necessary for inhibitor
access to the binding site. Although the binding site model is
preliminary and cannot predict the exact conformations of the binding
pocket and the inhibitor, especially in light of potential structural differences between archaeal and eubacterial ribosomes, the model offers an explanation of the inhibitory action against P-site activity
by oxazolidinones.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, and 0.1 mM
benzamidine hydrochloride) and centrifuged at 9110 rpm in SLA3000 rotor
for 10 min. The pellets were transferred into a mortar and ground with
prechilled alumina followed by the addition of an equal volume of
Buffer A supplemented with
-mercaptoethanol, phenylmethylsulfonyl
fluoride, and benzamidine hydrochloride. After incubation with
RNase-free DNase for 30 min. at 0 °C, alumina and cell debris were
removed by centrifugation at 15,000 for 15 min and 30,000 × g for 30 min, respectively. Supernatant was then layered
over an equal volume of 1.1 M sucrose cushion in Buffer B
(Buffer A + 0.5 M NH4Cl) and centrifuged at
100,000 × g for 15 h. After rinsing with Buffer
A, the resulting ribosome pellet was resuspended in 4 ml of Buffer A. The tight-coupled 70 S ribosomes were divided into portions, frozen in
liquid nitrogen, and stored at
80 °C.
the corresponding velocity equation would be given by Equation 1.
Determination of apparent Vmax and
Km parameters for fMet-tRNA
(Eq. 1)
V data into the corresponding rate equations.
Based on competitive behavior toward both initiator tRNA (P-site
substrate) and puromycin (A-site) (1), Equation 3 was used for
oxazolidinones.
(Eq. 2)
It was shown previously that chloramphenicol binds to a
different site on the ribosome from the A-site inhibitor sparsomycin (16) and also inhibits sparsomycin-induced binding of the fragment P-site substrate to the ribosomes (17). Competitive mixed or competitive behavior toward puromycin (A-site substrate) (18, 19) has
also been reported for this inhibitor. Based on these results, it was
assumed that chloramphenicol is a competitive inhibitor with respect to
both sites, and the data were fitted to the same equation (3) as for oxazolidinones.
(Eq. 3)
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (22K):
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Fig. 1.
Determination of catalytic
parameters for 23 S RNA mutant peptidyltransferases in puromycin
reaction. Determination of the Km values for
the P-site substrate was performed at fixed concentrations of puromycin
(150 µM) and 70 S ribosomes (0.010 µM)
under the concentration of initiator
f-[35S]Met-tRNA ) and
A2058G (
), G2032A (
), and G2447A (
) mutants. B,
double-reciprocal plot of puromycin reaction in the presence of
different concentrations of puromycin for wild type (
) and A2058G
(
), G2032A (
), and G2447A (
) mutants.
Catalytic properties of mutant peptidyltransferases in puromycin
reaction
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Fig. 2.
Inhibition of activity of
oxazolidinone-resistant peptidyltransferases in puromycin reaction with
various antibiotics. Antibiotics at various concentrations
were added to the 50-µl reaction mixtures containing 0.012 µM of the corresponding E. coli 70 S
ribosomes, 0.2 µM
f[35S]Met-tRNA , wild type;
, A2058G mutant;
,
G2032A mutant;
, G2447A mutant. A, inhibition with
oxazolidinone XA043; B, inhibition with oxazolidinone
linezolid; C, inhibition with chloramphenicol; D,
inhibition with sparsomycin.
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Fig. 3.
Inhibition of activity of
oxazolidinone-resistant peptidyltransferases in fragment reaction with
various antibiotics. Antibiotics at various concentrations were
added to the 50-µl reaction mixtures containing 0.24 µM
of the corresponding E. coli 70 S ribosomes, 6 µM of the CCA-Phe-X-Biotin fragment, and 50 µM puromycin (0.5 Ci/mmol). , wild type;
A2058G
mutant;
, G2032A mutant;
, G2447A mutant. A,
inhibition with oxazolidinone XA043; B, inhibition with
oxazolidinone linezolid; C, inhibition with chloramphenicol;
D, inhibition with sparsomycin.
Ki values for peptidyltransferase inhibitors
in puromycin reaction
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 4.
A proposed binding site for
oxazolidinones on the 50 S ribosome. A, mapping of
oxazolidinone-resistant mutations onto 50 S ribosomal subunit. A- and
P-loop regions are shown in white and blue color,
respectively, with oxazolidinone-resistant mutations displayed in
yellow. The yellow arrow points to the area
occupied by residues, which upon mutation confer oxazolidinone
resistance: G2032 (2073), C2057 (2098), G2058 (2099), C2062 (2104),
G2447 (2482), C2453 (2487), U2499 (2535), C2500 (2536), A2502 (2538),
and U2503 (2539), with H. marismortui numbering given in
parentheses. B, oxazolidinone binding site on 50 S ribosomal
subunit. H. marismortui residues are labeled with
the corresponding E. coli residues in
parentheses. C, docking of oxazolidinone XA043
structure into the binding pocket. After docking the inhibitor XA043
into the putative binding pocket on the ribosome, an 8-Å sphere around
the inhibitor was extracted for display. The residues that upon
mutation confer oxazolidinone resistance are colored in
purple, and the other residues are colored in
green, XA043 is colored by atom type, and the
K+ cation is in orange. H. marismortui
residues are labeled with the corresponding E. coli residues in parentheses. The view is from the
center of the P-site helix (Fig. 4A) looking outward
radially.
The functional role of the nucleotide G2447 is of a particular interest due to the ongoing controversy about the mechanism of catalysis by ribosomal peptidyltransferase. The low catalytic efficiency and markedly decreased affinity for the P-site substrate of mutant G2447A suggests an important contribution to the catalysis by this nucleotide. At the same time, it raises questions about the proposed general acid-base mechanism hypothesis (30, 31). If nucleotide G2447 plays an important role in the pKa shift of the catalytic residue A2051, one would expect this nucleotide to be essential both in vivo and in vitro, which has not been observed (3, 10, 32). Thus, there is a clear need for further mechanistic studies of this essential ribozyme.
We conclude that oxazolidinones inhibit binding of the initiator tRNA
to the peptidyltransferase P-site on 50 S, preventing the formation of
the first peptide bond. One would also predict that clinical resistance
to this class of drugs would evolve by introducing mutations that would
alter the fine structure of the P-site, as was observed in the case of
two oxazolidinone-resistant mutants, selected in the laboratory. One
would also predict that frequency of resistance acquired through this
target-based mechanism should be quite low due to redundancy of rRNA
operons in bacteria. Thus, this key ribozyme, encoded by multiple rDNA
genes, presents an excellent drug discovery target for the development
of novel antibiotics, the clinical usefulness of which would not be
eroded by single point mutations.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Al E. Dahlberg and Dr. Jill Thompson for insightful discussions. We thank Dr. Steve T. Gregory for providing site-directed 23 S RNA mutants and Dr. Jonathan A. Mills for sharing oxazolidinone-resistant mutant strains. We appreciate the help of Utpal Patel with the computer analysis of the Ki values.
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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.
To whom correspondence should be addressed: Johnson & Johnson PRD, San Diego, CA 92121. Tel.: 858-320-3385; E-mail:
ebobkova@prdus.jnj.com.
§ Present address: Aventis Pharmaceuticals, Bridgewater, NJ 08807.
¶ Present address: Wyeth-Ayerst, Pearl River, NY 10965.
Present address: GlaxoSmithKline, Collegeville, PA 19426.
Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M209249200
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