From the
Pharmacia Corp., Kalamazoo, Michigan 49001,
¶ Center for Pharmaceutical Biotechnology, University of Illinois, Chicago, Illinois 60607
Received for publication, February 28, 2003
, and in revised form, April 8, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A great variety of antibiotics inhibit bacterial growth by binding to the ribosome and interfering with its functions in protein synthesis. The ribosome, whose size exceeds that of an average antibiotic by four orders of magnitude, presents multiple possibilities for the antibiotic binding (1, 2). Furthermore, in the course of translation, the ribosome cycles through various conformational states and interacts with a number of ligands and effectors, including tRNAs and translation factors, which affect the spatial structure of the ribosome (3). It is conceivable that the affinity of antibiotics for specific sites in the ribosome might depend on the conformational state of the particle.
Oxazolidinones are the first new class of antibiotics introduced into medical practice in 25 years. This class of protein synthesis inhibitors is extremely effective against Gram-positive bacteria (4). Several studies suggest that the ribosome is the primary target of action of these compounds (59); however, interaction of oxazolidinones with the ribosome clearly differs from other antibiotics because these drugs are active against isolates that have developed resistance to other ribosomal antibiotics (10). Despite intense interest, the exact site of oxazolidinone action remains to be elucidated. Although the location of resistance mutations point to the peptidyltransferase center of the large ribosomal subunit as the possible drug target (1114), cross-linking and footprinting of ribosomes in vitro had suggested interaction of oxazolidinones with different regions of the ribosome including the nucleotides U-2113, A-2114, U-2118, A-2119, and C-2153 in 23 S rRNA located in the vicinity of the ribosomal E-site (15) and at a considerable distance from the peptidyltransferase. In addition, these studies conducted with isolated ribosomes had pointed to possible interactions of oxazolidinones with A-864 in the 16 S rRNA of the small ribosomal subunit. Lack of a clear understanding of the oxazolidinone binding site on the ribosome and reliance on indirect approaches led to the proposal of diverse models of oxazolidinone action (7, 8, 9, 12, 1519).
One of the major limitations in studies of oxazolidinone action has been the low binding affinity of these compounds to isolated 70 S ribosomes or 50 S ribosomal subunits, e.g. 200 µM for eperezolid, one of the reference oxazolidinones (20). The low affinity binding to isolated ribosomes or ribosomal subunits limits the utility of standard footprinting techniques that are routinely used to identify potential direct interactions with rRNA (21, 22). In contrast to the low affinity of the drug for the "static" isolated ribosome, these drugs are potent inhibitors of cell-free translation, where eperezolid exhibits an IC50 of 2.5 µM in an Escherichia coli system (7, 23). Thus, the site of drug binding in purified non-translating ribosomes may not accurately represent the site responsible for the antibiotic activity. It is possible that the relevant site may transiently appear only in the dynamic structure of the ribosome engaged in protein synthesis.
To bridge the information gained from biochemical experiments in vitro and genetic studies in vivo and to identify the site of oxazolidinone action in intact bacteria, we have used photo-induced cross-linking, a technique that is commonly used in vitro. However, in contrast to the previous studies, we have cross-linked a radioactive probe to its putative target in intact, actively growing bacteria. We reasoned that this approach applied to living cells would localize drug binding in vivo in the site relevant for its antibacterial activity. Moreover, we anticipated that this approach would succeed even if the relevant binding site were formed only transiently in the translating ribosome.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bacterial StrainsThe linezolid-sensitive clinical isolate Staphylococcus aureus ATCC 29213 was used for the cross-linking experiments. S. aureus RN4220 NCTC8325-4 r, m+ (restriction, minus; modification, plus; Ref. 27) was used for the generation of insertional mutants.
Cross-linking ExperimentsS. aureus cultures were grown overnight with shaking in Mueller Hinton broth (Difco) at 37 °C. Cells were diluted 1:100 and grown for 34 h at 37 °C. When the optical density reached an A600 of 0.6, 11.4 ml of the resulting culture were centrifuged at 2000 x g for 2 min. The cells were resuspended in 170 µl of fresh Mueller Hinton medium containing either 20 µl of 2% Me2SO or non-radioactive competitor antibiotics in the same volume of Me2SO followed by 10 µl of the radioactive photoactive oxazolidinone 125I-XL (
210 µCi/sample representing 12 µM final concentration). After a 30-min incubation at 37 °C in the dark, the suspension was exposed in open tubes to UV light using a Stratalinker 1800 (Stratagene) set to an energy exposure of 180,000 µJ (2.1 min, 254 nm). The cells were sedimented in microcentrifuge tubes, washed with Dulbecco's phosphate-buffered saline (Invitrogen), and resuspended in 180 µl of lysis buffer containing 10 mM Tris-HCl (pH 7.4), 30 mM NH4Cl, 30 mM MgCl2, and 5 µg/ml lysostaphin. After a 15-min incubation at 37 °C, the debris of the lysed cells was sedimented at 18,000 x g for 15 min at 4 °C. The resulting supernatant was used for extraction of total RNA or for the isolation of ribosomes after ultracentrifugation at 450,000 x g for 30 min in a Beckman TLA-100 ultracentrifuge (4 °C, TLA100 rotor).
Isolation of Total RNAAn aliquot (180 µl) of cell lysate supernatant was diluted with buffer containing 10 mM Tris-HCl (pH 7.6), 6 mM EDTA, and 0.5% SDS to a volume of 250 µl, and RNA was extracted with phenol. The extracted RNA was precipitated by the addition of 400 µl of ethanol, and the pellet was washed with 70% ethanol, dried in vacuo, and either used directly for electrophoresis or subjected to primer extension or RNase H analysis. Electrophoresis of RNA was carried out either on 1% agarose gels or on 10% denaturing polyacrylamide gels. Gels were stained with ethidium bromide, photographed, dried, and exposed to BioMax film (Eastman Kodak Co.).
Identification of 23 S rRNA Bases Cross-linked to Oxazolidinones For the preliminary mapping of 23 S rRNA cross-links, total RNA prepared from the cross-linked sample was subjected to RNase H analysis (28, 29) essentially as described by Dontsova et al. (30). In brief, 4 µg of total RNA was combined in 20 µl of water with 6 pmol of individual deoxyribooligonucleotides or pairs of deoxyribooligonucleotides complementary to sites in domains V and VI of S. aureus 23 S rRNA. The solution was incubated for 30 s at 90 °C and then cooled over 10 min to 50 °C. A 2.5-µl aliquot of 10x reaction buffer (150 mM HEPES-KOH (pH 7.8), 500 mM NH4Cl, 10 mM MgCl2, 1 mM dithiothreitol) was added followed by the addition of 1 µl of RNase H (Seikagaku America) in 2.5 µl of the 1x reaction buffer. The reactions were incubated for 30 min at 55 °C, and RNA was precipitated by the addition of 100 µl of 0.3 M sodium acetate (pH 5.5) and 300 µl of ethanol. RNA was recovered by centrifuging for 10 min at 21,000 x g at 4 °C, resuspending in 7 µl of formamide loading dyes, heating for 30 s at 90 °C, and loading the sample onto a 6% denaturing polyacrylamide gel. The gels (20 cm x 20 cm x 1 mm) were electrophoresed at 20 W for 1.5 h, stained with ethidium bromide, photographed, dried, and exposed to a Phosphor-Imager screen (Molecular Dynamics).
For precise mapping of the cross-linked nucleotides, the amount of 127I-XL (in this case non-radioactive) added to S. aureus cells was increased to 2 mM. Oligonucleotide primers were 5'-terminally labeled, annealed to RNA, and extended with reverse transcriptase as described (31).
The following oligonucleotides were used for RNase H mapping and primer extension (numbers in parentheses correspond to the 23 S rRNA complementary sequence, E. coli numbering): SaL 2550, GCCGACATCGAGGTGCC (24942510); SaL 2720, GTCCATCCCGGTCCTCTCGTAC (26552676); SaL 2230, TAGTATCCCACCAGCGTCTC (21572176); SaL2340, ACCTTTGAGCGCCTCCG (22752291); EcL 2563, TCGCGTACCACTTTA (25632577).
Isolation and Identification of Cross-linked ProteinsRibosomal pellets were resuspended in 70 µl of ribosome buffer (10 mM Tris-HCl, 30 mM NH4Cl, 30 mM MgCl2 (pH 7.4)). Two volumes of glacial acetic acid were added, and the tubes were mixed for 15 min at 4 °C before the precipitated RNA was removed by centrifugation at 18,000 x g for 15 min. The resulting supernatant was brought to 80% acetone and stored overnight at20 °C. The acetone pellet was recovered by centrifugation at 18,000 x g, washed with cold acetone to remove residual acetic acid, and lyophilized (32).
Two-dimensional gel electrophoresis was carried out as described by Adams (33). Lyophilized protein pellets were resuspended in 35 µl of buffer containing 9 M urea, 4% Nonidet P-40, 1% dithiothreitol, and 2% pH 3.510 ampholytes. Samples were applied to isoelectric focusing tube gels and focused for 23,000 volt-hours. Second-dimension gels were 1020% non-linear gradients run in Tris-glycine-SDS buffer (Bio-Rad). Gels were silver-stained (34), dried, and exposed to Kodak Biomax film.
High performance liquid chromatography separation of the extracted ribosomal proteins was carried out on a Vydac 218TPT C18 column according to a modified procedure of Cooperman et al. (35). The elution (1 ml/min) started with 75% A (water, 0.1% trifluoroacetic acid, v/v), 25% B (acetonitrile, 0.1% trifluoroacetic acid, v/v). Buffer B proportion was increased from 25 to 80% over a 100-min linear gradient.
Protein IdentificationThe specifically cross-linked proteins were excised from the gels, reduced, alkylated, and digested in situ with modified porcine trypsin (Promega) using a DigestPro robot (LC Packings). Nano liquid chromatography (nanoLC) tandem mass spectrometry analysis (nanoLC-MS/MS) was performed on a Micromass Q-Tof instrument equipped with a Z-spray ion source. Nanospray MS/MS data was used to identify proteins by comparing the experimental data with predicted data derived from protein and DNA databases. Tandem MS data was searched against the NCBInr and HGS protein databases using MASCOT (Matrix Science) programs maintained on an in-house server.
Targeted Disruption of LepA GeneThe LepA knock-out strain of S. aureus (RN4220) was constructed by insertional mutagenesis using an E. coli vector construct that is incapable of replication in S. aureus. A 310-bp region at the 5' end of the S. aureus LepA gene (yqeQ) was PCR-amplified from the genomic DNA using a pair of primers, CTCGATTATAGCACATATTGAC and TTTGTGCTTCGATACCTTGAG, and cloned into the pCR2.1 vector (Invitrogen) to create pCR-yqeQ-N310 plasmid. An ermB gene that confers erythromycin resistance in S. aureus was cloned into the NotI site of the pCR-yqeQ-N310 plasmid, and the resulting construct was electroporated into S. aureus (36). Genomic DNA was isolated from several erythromycin-resistant colonies, and yqeQ gene disruption was confirmed by PCR and Southern blot analysis. The lack of LepA expression in the constructed strain was confirmed by Western blot analysis using LepA-specific rabbit antibodies (Covance).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
For cross-linking in vivo, exponentially growing S. aureus cells were incubated with the 125I-XL photoprobe with or without competitor compounds for 30 min (optimal under these conditions) to allow drug penetration into the cell and binding to the target. To determine the specificity of the interaction, parallel incubations contained a 20-fold excess of non-photoactive competitors, active PNU-100592(S), or the control, inactive enantiomer PNU-107112(R) (Fig. 1). After these incubations, 125I-XL was cross-linked to its target by brief exposure to UV light (see "Experimental Procedures").
Extraction and electrophoresis of RNA from S. aureus cells that had been incubated with 125I-XL demonstrated that 23 S rRNA and tRNA were labeled selectively by the photoprobe (Fig. 2). Electrophoresis of the RNA on 1% agarose gels (Fig. 2A) clearly resolved 23 S, 16 S, and 5 S RNA. The 125I probe was cross-linked only to 23 S rRNA; no radioactive drug was seen associated with 16 S rRNA even upon prolonged exposure of the dried gels to film. Cross-linking of 125I-XL to 23 S rRNA likely occurred from the site of its inhibitory action because the yield of the cross-link was significantly reduced in the presence of an active drug PNU-100592(S) but not by its inactive enantiomer PNU-107112(R). (Fig. 2, lanes S and R). Separation of the extracted RNA by polyacrylamide electrophoresis (Fig. 2B) demonstrated that although no drug cross-linked to 5S rRNA, some amount of 125I-XL (much smaller than seen associated with 23 S rRNA) also cross-linked to RNA molecules co-migrating with tRNA. Specific cross-linking of biologically active 125I-XL to 23 S rRNA indicated that in their binding site in the large ribosomal subunit oxazolidinones form close contacts with certain residues of 23 S rRNA and possibly ribosome-bound tRNA.
|
To map the approximate site(s) of 125I-XL cross-linking to 23 S rRNA, RNase H mapping was utilized (28, 29, 37). Total RNA prepared from cross-linked S. aureus cells was incubated with oligodeoxyribonucleotides complementary to specific regions of 23 S rRNA (Fig. 3A). The RNA in the formed DNA/RNA heteroduplex was cleaved with RNase H, and the resulting fragments were resolved by denaturing gel electrophoresis and identified by autoradiography. As shown in Fig. 3B, when cross-linked 23 S rRNA was cleaved with RNase H in the presence of oligonucleotide SaL 2550, complementary to the rRNA segment 24942510 (E. coli numbering), an 400-nucleotide-long radiolabeled 23 S rRNA fragment representing the 3' end of 23 S rRNA was released. No label was found associated with the longer 5' segment. Digestion of 23 S rRNA with RNase H in the presence of two oligonucleotides, SaL 2550 and SaL 2720 (23 S rRNA positions 26552676), released a single radiolabeled rRNA fragment
170 nucleotides long corresponding to the rRNA segment between the two oligonucleotides. Thus, 125I-XL was cross-linked exclusively to the 24942676 segment of 23 S rRNA.
|
The precise location of the cross-link was mapped by primer extension. A single additional band at position 2603 was seen when the rRNA sample prepared from cells incubated with non-radioactive 127I-XL was used as a template (Fig. 3C), corresponding to the drug cross-link at position A-2602. As expected from the results with the radioactive probe, the intensity of this band decreased when the cross-linking was prevented by incubation in the presence of a 20-fold excess of PNU-100592(S) in vivo. Thus, the single rRNA base cross-linked by the photoprobe in actively growing bacteria was A-2602.
A-2602 is one of the central nucleotides in the ribosomal peptidyltransferase center (38). Thus, cross-linking of the oxazolidinone photoprobe to A-2602 places the site of the drug action in the peptidyltransferase center.
Oxazolidinone Cross-linking to Ribosome-associated Proteins in VivoTo investigate possible in vivo cross-linking of 125I-XL to ribosome-associated proteins, ribosomes were pelleted from the lysates of S. aureus cells cross-linked with the drug, and proteins were fractionated by SDS electrophoresis. SDS-PAGE analysis of the proteins in the ribosome pellet demonstrated two radiolabeled protein bands of 64 and 11 kDa that were selectively cross-linked (Fig. 4A). The 125I-XL cross-linking to these the proteins showed the same specificity as was seen for the 23 S RNA in these samples; cross-linking was reduced in the presence of the biologically active competitor eperezolid, PNU-100592(S), but not by its inactive enantiomer. The specifically cross-linked proteins were purified, and their identity was determined by nanoLC-MS/MS analysis as described below.
|
The 11-kDa cross-linked protein resolved as a single image on two-dimensional electrophoresis with an isoelectric point close to 11 (Fig. 4B). The radioactive spot corresponding to the specifically cross-linked 11 kDa protein did not precisely overlay a silver stained protein on the respective gels, most likely due to the effect of the cross-linked drug on protein mobility. However, when protein was eluted from the gel beneath the radioactive spot and digested with trypsin, five peptides were identified by nanoLC-MS/MS as corresponding to the tryptic fragments of the 50 S subunit protein L27. Fig. 5A shows the S. aureus L27 sequence; the tryptic peptides identified are in bold. No other proteins were identified in this portion of the two-dimensional gel. To further confirm that the probe had cross-linked L27, ribosomal proteins prepared from cells cross-linked with 125I-XL were subjected to high performance liquid chromatography purification. The fraction containing the 11-kDa radioactive protein was collected and analyzed by MS/MS. The identity of L27 was again confirmed by the detection of five tryptic fragments that covered 39% of the total sequence. L27 is the only ribosomal protein that is suspected to reach very close to the active site of the peptidyltransferase center of the bacterial ribosome (see "Discussion"). Therefore, cross-linking of this protein by the oxazolidinone probe is compatible with the 23 S rRNA cross-link and with the general location of the site of the drug action within the ribosomal peptidyltransferase center.
|
Analysis of the 64-kDa cross-linked protein by two-dimensional electrophoresis is shown in Fig. 4C. The gel beneath the radioactive spot was excised, digested with trypsin, and analyzed by nanoLC-MS/MS as described under "Experimental Procedures." Eleven tryptic fragments were identified (shown in bold in Fig. 5B) that matched the S. aureus protein LepA. No other proteins were identified in this region of the gel. LepA belongs to the GTPase family of and exhibits significant homology to the translation factors EF-G and EF-Tu, indicating its possible involvement in translation and association with the ribosome. Antibodies generated against LepA showed the presence of the protein in both the ribosomal pellet and the post-ribosomal supernatant (Fig. 4D, left panel). Notably, however, the protein cross-linked by 125I-XL was detected only in the ribosomal pellet (Fig. 4D, right panel), suggesting that association of LepA with the ribosome was required for cross-linking by the drug.
LepA Is Not the Target of Oxazolidinone ActionTo investigate whether LepA represented a target of oxazolidinone action, we constructed a S. aureus strain in which the LepA gene (yqeQ) was disrupted by insertional mutagenesis (see "Experimental Procedures"). LepA gene disruption was confirmed by PCR and Southern blotting; the lack of LepA expression in the engineered strain was also verified by Western blotting using LepA-specific antibodies. The LepA knockout strain exhibited growth characteristics comparable with those for the wild type. When the 125I-XL was cross-linked to its target in the LepA knockout strain, the 64-kDa cross-linked band was absent, confirming that LepA was a target for oxazolidinone cross-linking. However, the 23 S rRNA, tRNA, and protein L27 cross-links remained unaffected as did the minimum inhibitory concentration (MIC) for the active oxazolidinones used in this study (data not shown). Thus, although LepA bound to the ribosome was cross-linked specifically by 125I-XL, 125LepA is not required for oxazolidinone action and does not appear to be a direct target of the drug action.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Only specific RNA and protein molecules were cross-linked when the biologically active oxazolidinone azido-derivative, XL, was activated by UV irradiation after its accumulation in the bacterial cells. The cellular macromolecules contacted by the drug in the putative site of action included 23 S rRNA (A-2602), tRNA, and the two proteins L27 and LepA. The relevance of these contacts is supported by the high selectivity of the interaction; only one of the three ribosomal RNAs (23 S rRNA) and only one of more than 50 different ribosomal proteins (L27) was cross-linked by the drug in the living cell. It is highly likely that 125I-XL was cross-linked to these macromolecules from the site of oxazolidinone action since the biologically active oxazolidinone, eperezolid, was able to compete with 125I-XL for the binding site, whereas its inactive isomer was without effect. This conclusion is further supported by the 75% reduction of all cross-links in two strains of S. aureus carrying oxazolidinone-resistant mutations G2447U and G2576U in 23 S rRNA (not shown).
Cross-linking of the photoactive oxazolidinone in vivo to A-2602, one of the central nucleotides in the ribosomal peptidyltransferase center, is the key result that defines ribosomal peptidyltransferase as the main site of drug action. Fig. 6 shows the position of A-2602 in relationship to the known linezolid resistance mutations identified in Gram-positive (green), Gram-negative (gold), or archaeal (blue) ribosomes (1114). The site of the drug cross-link and the resistance mutations are located in close vicinity to each other and are all clustered within or nearby the central loop of domain V of 23 S rRNA, the core of the catalytic peptidyltransferase center of the ribosome. This proximity of this site of cross-linking of the drug in vivo to the mutations producing oxazolidinone resistance is particularly evident in the crystallographic structure of the large ribosomal subunit (Fig. 6B). The spatial arrangement of the resistance mutations versus A-2602 cross-linked by the azido group attached from the C-ring side of the oxazolidinone indicates a possible general orientation of the drug molecule within its binding site, with the pharmacophoric oxazolidinone group protruding toward the active site of the peptidyltransferase center and the extended (C-ring) appendage reaching toward A-2602.
|
The site of oxazolidinone action in the ribosomal peptidyltransferase center is also supported by the polypeptides cross-linked by 125I-XL in the living cell. The data conclusively show an interaction of 125I-XL with the large ribosomal subunit protein, L27. Existing biochemical evidence suggests a close association of protein L27 with the peptidyltransferase center. The 3' end of azido derivatives of aminoacyl- and peptidyl-tRNA bound in the E. coli ribosomal peptidyltransferase active site cross-link to L27 in vitro (3941). Biochemical and genetic studies indicate that this interaction may engage the N terminus of the protein2; by inference, one would expect that the oxazolidinone cross-link also engages the N-terminal part of L27. Indeed, cleavage of the cross-linked L27 at Lys 57 with endoproteinase Glu-C demonstrated that the 125I cross-link was associated with the N terminus of the L27 protein (data not shown). L27 was previously shown to be cross-linked in vitro by several other antibiotics that affect ribosomal peptidyltransferase activity (4246). This observation suggests that the oxazolidinone binding site is located in the vicinity of the sites impacted by other peptidyltransferase-targeted drugs. However, the mode of oxazolidinone binding must be unique since the majority of ribosomal mutations conferring resistance to other ribosomal antibiotics does not affect cell sensitivity to oxazolidinones (10).
Although first high resolution structure of the 50 S ribosomal subunit of Haloarcula marismortui x-ray studies of showed no proteins in the peptidyltransferase active site of the archaeal ribosome (47), Archaea lack the L27 homolog. The crystal structure of the large ribosomal subunit from the eubacterium Deinococcus radiodurans shows a close proximity of L27 to the peptidyltransferase center (48). The globular C-terminal part of L27 is "buried" under the central protuberance of the large ribosomal subunit, whereas the unstructured N-terminal region emerges at the subunit interface and may protrude in the direction of the peptidyltransferase center. Though the precise location of the N terminus of L27 in the solved structure is ambiguous, the highly flexible nature of the N-terminal protein "tail" is consistent with the notion that it might reach close to the drug binding site in the peptidyltransferase center (48). An interference with L27 function might also be expected to affect ribosome assembly and the fidelity of translation (49), functions that are also inhibited by oxazolidinones (19, 50). We were not able to test, however, whether L27 is required for drug interaction since the L27 knockout appears to be lethal in S. aureus.
Another protein cross-linked by 125I-XL in vivo is LepA. The function of LepA in the cell is unclear. This protein, which was originally found associated with the cell membrane fraction (39), exhibits considerable homology to the translation factor GTPases, and thus, its interaction with the ribosome is likely (51). The protein is distributed between the ribosomal pellet and free (supernatant) fractions (Fig. 4D). Importantly, only the ribosome-associated fraction of LepA was cross-linked by 125I-XL, suggesting that cross-linking occurs from the ribosome-associated drug. This is supported by the observation that rRNA mutations conferring resistance to oxazolidinones significantly decrease 125I-XL cross-linking to LepA in parallel to reductions in the other cross-links (not shown). Despite cross-linking of photoactive oxazolidinone to the LepA protein, inactivation of the LepA gene did not confer any appreciable resistance to oxazolidinones, suggesting that LepA was cross-linked essentially as a bystander rather than as the target of drug action. It is also possible that the function of LepA may be replaced by a redundant translation factor in the knockout mutant. However, an important functional implication of this cross-linking result is that this presents the first biochemical indication of LepA involvement in protein synthesis. The results demonstrate that a portion of this protein, while bound to the ribosome in vivo, must extend to the site of oxazolidinone binding in the ribosomal peptidyltransferase center.
Cross-linking of photoactive oxazolidinone to a critical base, A-2602, in the peptidyltransferase center as well as clustering of oxazolidinone resistance mutations, all point to the peptidyl-transferase as the primary site of the drug action. Although binding of oxazolidinones to the ribosome and 50 S subunit can be observed in vitro (52), oxazolidinone binding in vitro demonstrates a 50100-fold weaker affinity than would be predicted from their in vivo inhibitory concentrations (20, 52). Because there is no evidence that oxazolidinones are concentrated within bacteria, these results suggest that the ribosome must adopt a specific conformation to create a high affinity oxazolidinone binding site. We were unable to show any specific (competed by eperezolid) cross-linking in isolated ribosomes (not shown).
"Opening" of a oxazolidinone binding site in the translating ribosome may occur after interaction of the ribosome with one of its ligands. Binding of several different ribosome-targeted antibiotics is known to be stimulated by simultaneous binding of co-ligands. For example, the affinity of sparsomycin significantly increases in the presence of peptidyl-tRNA (53), enhancing its ability to also cross-link to A-2602 (54). Moreover, erythromycin binding may be stimulated by specific nascent peptides (55). An additional ligand might either allosterically affect the conformation of nucleotides involved in oxazolidinone binding or directly participate in formation of the drug binding site. Consistent with this model, cross-linking of tRNA was observed in our studies, suggesting that occupation of one of the tRNA sites on the ribosome may facilitate drug binding. Several studies demonstrate that oxazolidinones may interfere with positioning of fMet-tRNA in the P-site of 70 S ribosomes, thereby preventing the initiation of translation (8, 16, 18). In agreement with this conclusion, the A-2602 base that is exclusively cross-linked by 125I-XL in the living cell has been implicated in the interaction of the 3' end of transfer RNA with the ribosome (56). Although the identity of the tRNA species cross-linked by oxazolidinones is not known, the inability of this antibiotic class to inhibit polysome elongation (7) suggests that fMet-tRNA is a likely candidate. Initial interaction of fMet-tRNA with the ribosome could facilitate drug binding, leading to interference with accommodation of fMet-tRNA in the ribosomal P-site.
In the "high affinity" conformation of the ribosome, induced either by binding of one of the ribosomal ligands (tRNA, translation factors) or by ribosome cycling through different conformational states during translation, the rRNA nucleotides involved in drug binding might adopt an orientation favoring their interaction with the drug. Binding of the antibiotic would then "lock" the ribosome in this conformation, thereby preventing subsequent structural transitions of rRNA required for protein synthesis. Such a mode of action is reminiscent of that proposed for pactamycin and several other ribosome-targeted antibiotics (1, 57). In this respect it is worth noting that the nucleotide cross-linked by 125I-XL in vivo, A-2602, appears to be the most flexible in the peptidyltransferase center, assuming a different conformation depending upon the state of the ribosome (47, 48, 58). Specific orientations of A-2602 were proposed to be critical for some activities of the peptidyltransferase center (58). One of the conformations of this nucleotide may favor oxazolidinone binding. A similar dependence of sparsomycin binding on A-2602 orientation was recently proposed (59).
Overall, the in vivo cross-linking approach used in this study provides strong evidence that a binding site for the oxazolidinone antibiotics is formed at or near the peptidyltransferase center in actively translating bacterial ribosomes. This is consistent with the location of the resistance mutations and with the biochemical studies that suggest that oxazolidinones interfere with first peptide bond formation (5, 7, 8, 16, 18). In its binding site in the ribosome, the oxazolidinones appear to be able to interact with ribosomal protein L27, ribosome-associated protein LepA, and tRNA. Such interactions could explain the additional reported effects of the oxazolidinones on ribosome formation and fidelity of translation. The information gained from these studies in actively growing bacteria should help unravel the mechanism of oxazolidinone action, whereas the approach itself can be used for studies of other currently used and newly developed anti-ribosomal drugs.
![]() |
FOOTNOTES |
---|
To whom correspondence should be addressed: Pharmacia Corp., 301 Henrietta St., Kalamazoo, MI 49001. Tel.: 269-833-9505; E-mail: jrcolca{at}pharmacia.com.
1 The abbreviations used are: 125I-XL, 125I-labeled PNU-259621; nanoLC-MS/MS, nano liquid chromatography tandem mass spectrometry (MS).
2 B. A. Maguire, A. D. Beniaminov, L. Xiong, A. S. Mankin, and R. A. Zimmermann, submitted for publication.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|