Comparison of tetracycline and tigecycline binding to ribosomes mapped by dimethylsulphate and drug-directed Fe2+ cleavage of 16S rRNA

Gesine Bauer1, Christian Berens1, Steven J. Projan2 and Wolfgang Hillen1,*

1 Institut für Mikrobiologie, Biochemie und Genetik, Universität Erlangen, Erlangen, Germany; 2 Wyeth Research, Cambridge, MA, USA

Received 10 December 2003; returned 17 December 2003; revised 19 December 2003; accepted 21 December 2003


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: The new antibiotic tigecycline (9-t-butylglycylamido-minocycline; GAR-936) overcomes most of the known tetracycline resistance mechanisms. Here we analyse its mode of antibiotic action by probing 70S ribosomes of Escherichia coli with dimethylsulphate (DMS) and Fe2+-mediated cleavage to identify binding sites of tetracycline and tigecycline.

Methods: Fe2+-mediated cleavage makes use of the ability of Fe2+ to replace the Mg2+ ion complexed with tetracyclines. After addition of H2O2, Fe2+ generates short-lived, highly reactive hydroxyl radicals that can cleave RNA close to the tetracycline binding sites.

Results: We identified three prominent Fe2+-mediated cleavage sites in helices 29 and 34, and in the internal loop of helix 31 of 16S rRNA in the presence of tetracycline or tigecycline. Qualitatively, these sites are modified identically by both antibiotics, but quantitative differences observed in the cleavage intensities indicate that the drugs bind in slightly different orientations. These results are supported by DMS modification, mutational analysis of 16S rRNA and structural modelling of tigecycline at a tetracycline-binding site in the 30S ribosomal subunit.

Conclusions: Both derivatives bind to identical or overlapping sites and probably share the same mode of antibiotic action. The fact that tigecycline overcomes most of the known tetracycline resistance mechanisms is interpreted as a result of steric hindrance due to the large substituent at position 9.

Keywords: glycylcyclines, mode of action, tetracycline resistance


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tetracyclines inhibit protein biosynthesis by preventing the attachment of aminoacyl-tRNA to the ribosomal A-site. Owing to their broad-spectrum activity against pathogenic bacteria and the absence of major adverse side effects, they have been used extensively in therapy for human and animal infections and as growth promoters in agriculture. This has led to an increasing incidence of bacterial resistance, creating a demand for new tetracycline derivatives that can overcome resistance.1 The discovery of new derivatives, termed glycylcyclines, containing a glycylamido substitution at position 9 of different tetracycline derivatives represented significant progress,2 because they are active against a wide spectrum of Gram-negative and -positive bacteria, including resistant strains.3,4 One of these derivatives, tigecycline (9-t-butylglycylamido-minocycline), is currently undergoing clinical trials. Tigecycline shows antibiotic properties similar to tetracycline, but is not affected by tetracycline resistance mediated by efflux or ribosomal protection proteins.5 Data from filter binding assays indicate that tetracycline and two previously discovered glycylcycline derivatives, 9-(N,N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline (DMG-DMDOT) and 9-(N,N-dimethylglycylamido)-doxycycline (DMG-DOX), share a common ribosomal binding site, to which the glycylcyclines bind five times more effectively.6

The latest crystal structure data show up to six tetracycline binding sites on the 30S subunit of the ribosome,7,8 where site-1 is composed of a binding pocket formed by helix (h) 31 and h34 of 16S rRNA (helical numbering according to Mueller & Brimacombe9). Site-1 exhibits the highest degree of tetracycline occupancy.8 Various experiments provide additional biochemical evidence for tetracycline binding to site-1, e.g. enhanced dimethylsulphate (DMS) methylation at U1052 and C1054 in h3410 or inhibition of the RNA–RNA photocrosslink between h31 and h44 in the presence of tetracycline.11 The ribosomal protection proteins TetO and TetM may interfere with tetracycline binding to this high affinity site to confer resistance.12 In the presence of TetO, the enhanced DMS methylation induced by tetracycline at C1054 in h34 is abolished.13 If tigecycline shares this high-affinity binding site with tetracycline, the question arises of why TetM does not mediate tigecycline resistance.5 We compared rRNA interaction sites of tetracycline and tigecycline to analyse whether this discrepancy is the result of a different mechanism of action by tigecycline or a different binding mode. A detailed comparison of tetracycline and tigecycline binding sites on 16S rRNA required a new probing method, since only DMS modification in h34 correlates with biological activity of tetracycline derivatives.13,14 Other RNA probing methods like kethoxal or europium cleavage yielded no tetracycline-specific signals.10,15 Tetracycline-mediated Fe2+ cleavage has been used to identify the tetracycline binding site on Tet repressor and on the tetracycline efflux protein TetA.16,17 Since Fe2+ cleavage has also been employed to detect metal ion binding sites in group I intron RNA,18 we adapted this technique for the identification of tetracycline binding sites on rRNA in 70S ribosomes of Escherichia coli. We identified three cleavage sites induced by the presence of tetracycline or tigecycline. These sites are consistent with data obtained from DMS and mutational analysis of 16S rRNA.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Strains and plasmids

E. coli DH5{alpha}19 was routinely used as plasmid host. E. coli TA527 [{Delta}rrnE F ara {Delta}lac thi {Delta}(rrsB-gltT-rrlB)101 {Delta}(rrsH-ileV-alaV-rrlH) 103 {Delta}(rrsG-gltW-rrlG)30::lacZ+ {Delta}(rrsA-ileT-alaT-rrlA)34 {Delta}(rrsD-ileU-alaU-rrlD) 25::cat+ {Delta}(rrsC-gltU-rrlC)15::cat+ ilv+]20 was used to measure the MIC of tetracycline and tigecycline of mutated 16S rRNA in the absence of wild-type background. For viability, E. coli TA527 contains the pSC101 derivative pHK-rrnC+ bearing the entire rrnC operon from E. coli. pKK3535, a pBR322 derivative that carries the 7.5 kb BamHI fragment from {lambda}rifd, containing the entire rrnB operon from E. coli,21 was taken as a wild-type rRNA control. pKK1058C is a pKK3535 derivative bearing a point mutation of G->C at base 1058 of the 16S rRNA.22 The plasmid pKK966U was constructed by site-directed two-step PCR mutagenesis, whereby only part of the 16S rRNA was amplified using the primers 2220f_BglII (5'-AAG AAG ATC TGG AGG AAT ACC G-3'), 2916r_BsrGI (5'-GGT GTG TAC AAG GCC CGG G-3') and 2489r-966U (5'-CGC GTT GAA TCG AAT TAA ACC-3'), and plasmid pKK3535 as template. The PCR fragment obtained was digested with BglII and BsrGI, and cloned into the likewise digested pKK3535. Finally, the entire 16S rRNA was sequenced to ensure that only the desired and no additional mutations were present.

MIC determination

The activities of the antibiotics were determined by the agar dilution method following the recommendations of the NCCLS.23 The inocula of E. coli TA527 cells, which were adjusted to 107 cfu/mL, were applied to the surfaces of Mueller–Hinton agar plates to give 104 cfu/spot. This was confirmed by plating the corresponding number of cells on Mueller–Hinton agar plates containing no antibiotics. The plates were incubated at 36°C for 20 h in ambient air. MIC tests were performed three times.

Preparation of 70S ribosomes

Cultures from E. coli MRE600 were grown at 37°C to an OD600 of 0.5 in LB broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl), harvested and resuspended in ribosomal buffer 1 (20 mM Tris–HCl, pH 7.5, 100 mM NH4Cl, 10 mM MgCl2, 10 mM ß-mercaptoethanol). The isolation of 70S ribosomes via ultracentrifugation was performed as described previously.24

DMS modifications

Chemical modification of the 70S ribosome with DMS was carried out as described previously.25 For the DMS modification assays, 10 pmol of ribosomes in ribosomal buffer 2 [20 mM Tris–HCl, pH 7.5, 100 mM NH4Cl, 6 mM MgCl2, 3 mM dithiothreitol (DTT)] were incubated at 37°C for 30 min, followed by incubation at room temperature for 10 min in the absence or presence of antibiotics (final volume 49 µL). The DMS reaction was started by addition of 1 µL DMS (1:50 dilution in 96% ethanol) and incubation at room temperature for 5 min. The reaction was stopped with 1 µL of ß-mercaptoethanol (1:5 dilution in water).

Fe2+-mediated hydroxyl radical cleavage reactions

Fe2+-mediated hydroxyl radical cleavage of the 70S ribosome was carried out as described previously.18 Four microlitres of ribosomes (5 pmol) and 1 µL of a 10x antibiotic stock solution in TAKA7 (50 mM Tris–HCl, pH 7.5, 70 mM NH4Cl, 30 mM KCl, 7 mM MgCl2)26 were incubated with 2 µL of 5x native cleavage buffer (25 mM MOPS–KOH, pH 7.0, 3 mM MgCl2, 400 µM spermidine) and incubated for 30 min at 37°C, followed by 10 min incubation at room temperature. One microlitre of 1.25 mM FeCl2 was added to the reaction tube, mixed by centrifugation and incubated for 1 min before adding 1 µL of 6.25 mM sodium ascorbate. After 1 min, 1 µL of 6.25 mM H2O2 was added to initiate the reaction, and rapidly mixed. The final concentrations were 125 µM for Fe2+ and 625 µM for both sodium ascorbate and H2O2. The cleavage reaction was stopped after 1 min by the addition of thiourea to a final concentration of 100 mM.

Mg2+ competition of Fe2+ cleavage

In the Mg2+ competition experiments, MgCl2 was added as a 10x stock solution of the final Mg2+ concentration to the 1.25 mM FeCl2 solution. The Fe2+/Mg2+ mixture was then pipetted into the reaction tube and the cleavage reaction continued as above.

Extraction of rRNA

The RNA was extracted by treatment of the ribosomes with 200 µL of ribosomal extraction buffer (0.3 M sodium acetate, 0.5% SDS, 5 mM EDTA) at room temperature followed by phenolization (3x) and isoamylalcohol/chloroform (1:24) treatment. Thereafter, the RNA was ethanol precipitated, resuspended and residual phenol was eliminated by diethylether treatment. After a final ethanol precipitation, the RNA was resuspended in 10 µL of Millipore water.

Primer extension and gel electrophoresis

RNA (2.5 µL) was mixed with 1 µL of 4.5x hybridization buffer (225 mM HEPES–KOH, pH 7.0, 450 mM KCl) and 1 µL of 5' 32P-labelled primer (0.25 pmol; 100 000 cpm). The hybridization was carried out by heating the mixture for 1 min at 96°C followed by 15 min incubation at room temperature. The extension reaction was started by the addition of 5x first-strand buffer (50 mM Tris–HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2), 10 mM DTT, 167 µM dNTP mix, 3 U RNAguard RNase inhibitor and 10 U SuperscriptII RnaseH Reverse Transcriptase (Invitrogen), and carried out in a final volume of 15 µL at 42°C for 50 min. To stop the reaction, the RNA was degraded by addition of 2 µL of 1 M NaOH, followed by a 15 min incubation at 42°C. After neutralization by adding 2 µL of 1 M HCl, the DNA was precipitated with ethanol. The DNA was resuspended in 10 µL of loading buffer (0.3% each of Bromophenol Blue and xylene cyanol, 10 mM EDTA, pH 7.5, and 97.5% deionized formamide) and electrophoresed in denaturating 8–15% polyacrylamide (PAA) sequencing gels. Modifications were identified and quantified by phosphoimager analysis (FUJIFILM Bas-1500; TINA200, Raytest, Straubenhardt, Germany). The positions of the modifications were mapped by dideoxynucleotide sequencing using pKK3535 or pKK1058C as a template.

Superposition figure

Superposition of tetracycline with DMG-DMDOT was carried out with VMD 1.8.1 structure viewer.27 VMD was developed by the Theoretical and Computational Biophysics Group in the Beckman Institute for Advanced Science and Technology at the University of Illinois at Urbana-Champaign (http://www.ks.uiuc.edu/Research/vmd/). Coordinates were taken from the RCSB protein data bank files 1I97 and 1ORK.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
DMS modification of 16S rRNA

DMS modification experiments were carried out in the presence of tetracycline and tigecycline to test whether they show the same binding sites. 70S ribosomes from E. coli MRE600 were treated with DMS in the presence of tetracycline or tigecycline at concentrations ranging from 0.1 to 300 µM. The ribosomal RNA was isolated and used as a template for primer extension analysis. Protection from DMS methylation at A892 in h27 (see Figure 1) was only detected for tetracycline, but not for tigecycline (Figure 2a). Quantification showed that the intensity of the signal corresponding to A892 steadily decreased at tetracycline concentrations from 10 up to 300 µM, yielding a two-fold reduction compared with the control, while tigecycline did not affect signal intensity (Figure 2b).



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Figure 1. Sites of interaction of tetracycline (Tc) and tigecycline (TGC) with the 16S rRNA. The secondary structure of the E. coli 16S rRNA32 is shown schematically. Located within the grey boxes and shown in more detail in the enlarged sections are bases that: (i) display altered reactivity towards DMS probing in the presence of tetracycline,10 tigecycline or tRNA;33 (ii) when mutated lead to a weak resistance against tetracycline22 and tigecycline; (iii) become crosslinked to tetracycline;30 (iv) RNA–RNA crosslinks affected by tetracycline;11 or (v) show Fe2+-mediated cleavage in the presence of tetracycline and tigecycline.

 


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Figure 2. Effects of tetracycline and tigecycline on DMS modification of bases in 16S rRNA. E. coli 70S ribosomes were incubated with different amounts of tetracycline and tigecycline and methylated with DMS as described in the Materials and methods section. Sites of modification were detected by primer extension and analysed by electrophoresis on denaturating 10% PAA gels, shown in (a) and (c). In both (a) and (c): lanes A and C, dideoxy sequencing lanes; lane R, unmodified RNA; lane D, DMS-modified RNA in the absence of antibiotics; lane T, unmodified RNA in the presence of 300 µM tetracycline; and lanes T10–300, DMS modification in the presence of 10, 30, 100 and 300 µM tetracycline. The following abbreviations are different in (a) and (c). In (a): lane G, unmodified RNA in the presence of 300 µM tigecycline; lanes G10–300, DMS modification in the presence of 10–300 µM tigecycline. In (c): lane G, unmodified RNA in the presence of 10 µM tigecycline; lanes G0.1–10, DMS modification in the presence of 0.1–10 µM tigecycline. Stars indicate protection of A892 (a) and enhancement of C1054 (c). The effects of DMS modifications on rRNA in the presence of rising amounts of tetracycline or tigecycline were quantified in a phosphoimager [shown in (b) and (d)] and compared with the control (lane D, DMS-modified RNA in the absence of antibiotics). Quantification was adjusted for loading differences by standardization to regions unaffected by tetracycline or tigecycline.

 
Enhanced methylation at C1054 in h34 (see Figure 1) was observed for tetracycline and tigecycline (Figure 2c). Quantification of the signal demonstrated an increase in intensity from 10 up to 100 µM of tetracycline, to a maximum of 1.3-fold compared with the control. In contrast, just 1 µM of tigecycline induced the maximal 1.4-fold increase in signal intensity (Figure 2d).

Tetracycline-directed Fe2+-mediated hydroxyl radical cleavage patterns

Fe2+ can replace Mg2+ chelated to tetracyclines forming a tetracycline–Fe2+ complex, which is an inducer of TetR.28 We thus assume that tetracycline–Fe2+ is isostructural to tetracycline–Mg2+. We used the Fe2+-chelated complexes of tetracycline or tigecycline to map their binding sites on 16S rRNA by Fe2+ cleavage. After addition of H2O2, the bases and the RNA backbone are cleaved in the close proximity of bound tetracycline derivatives. We probed 70S ribosomes isolated from E. coli MRE600 with 125 µM Fe2+, 625 µM sodium ascorbate and 625 µM H2O2 in the presence of 1–100 µM tetracycline or tigecycline. The rRNA was isolated and used as template for primer extension analysis. The primers chosen were spaced approximately every 250 nucleotides on the 16S rRNA. Three prominent regions of cleavage, seen as strong stops of reverse transcription, were observed. Sequence reactions run in parallel revealed one cleavage site in the internal loop of h31, one in h34 and one in h29 (compare Figure 1; helical numbering according to Mueller & Brimacombe9). Figure 3 shows sections of 10% denaturating polyacrylamide gels with cleavage sites.



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Figure 3. Fe2+-mediated hydroxyl radical cleavage of the 16S rRNA. E. coli 70S ribosomes were incubated with different amounts of tetracycline and tigecycline, and treated with Fe2+/H2O2 as described in the Materials and methods section. Cleavage sites were detected by primer extension and analysed by electrophoresis on a denaturating 10% PAA gel. (a, b) Cleavage sites and quantification of signals in the internal loop of h31 (tetracycline binding site-1); (c, d) cleavage sites and quantification of signals in h34 (tetracycline binding site-1); (e, f) cleavage sites and quantification of signals in h29 (tetracycline binding site-4). Lanes A, C, G and U, dideoxy sequencing lanes; lane R, unmodified RNA; lane H, control in which Fe2+ was omitted; lane F, Fe2+/H2O2-cleaved RNA in the absence of antibiotics; lane T, unmodified RNA in the presence of 100 µM tetracycline and H2O2; lanes T1–100, Fe2+/H2O2 cleavage in the presence of 1–100 µM tetracycline; lane G, unmodified RNA in the presence of 100 µM tigecycline and H2O2; lanes G1–100, Fe2+/H2O2 cleavage in the presence of 1–100 µM tigecycline. Lines left of the sequence indicate regions of Fe2+-mediated hydroxyl radical cleavage. The cleavage of 16S rRNA in the presence of rising amounts of tetracycline (T1–T100) or tigecycline (G1–G100) was quantified in a phosphoimager [plots shown in (b), (d) and (f)] and compared with the control (lane F, cleaved rRNA in the absence of antibiotics). Quantification was adjusted for loading differences by standardization to regions unaffected by tetracycline or tigecycline.

 
Cleavage products in the internal loop of h31 were mapped to region A964–A969 (Figure 3a). Signal quantification yielded a continuous increase in signal intensity for tetracycline concentrations from 1 to 100 µM, whereas saturation was reached in the presence of 30 µM tigecycline (Figure 3b).

Cleavage products in h34 were mapped to nucleotides C1195–A1197 (Figure 3c). Cleavage product quantification yielded an increase in signal intensity with 1–30 µM tetracycline. Saturation was reached with 30 µM tetracycline. No concentration-dependent change in signal intensity was detected for tigecycline. In addition, the overall effect for tigecycline was smaller than that for tetracycline (Figure 3d).

Cleavage products in h29 were mapped to region A1339–U1341 (Figure 3e). Quantification showed that the signal strength increases from 1 to 100 µM tetracycline and from 1 to 10 µM tigecycline (Figure 3f). The signal intensity for 10 or 100 µM tetracycline resembled that for 1 or 10 µM tigecycline, respectively.

Mg2+ competition of Fe2+ cleavage

To show that Fe2+ and Mg2+ interact with the same sites, we performed a Mg2+ competition experiment. The cleavage reaction was carried out in the presence of increasing amounts of MgCl2 (1.5–20 mM) leading to Fe2+:Mg2+ ratios ranging from 1:12 to 1:160, since Fe2+ shows a 100-fold higher affinity to tetracycline than Mg2+.28 The results of a representative Mg2+ competition are shown for the internal loop of h31 (A964–A969) in Figure 4a. Quantification shows that the extent of cleavage by Fe2+ was reduced for tetracycline- and tigecycline-directed reactions, reaching intensities close to the background at 20 mM Mg2+ (Figure 4b).



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Figure 4. Mg2+ competition of Fe2+ cleavage in 16S rRNA. 70S ribosomes were treated with 125 µM Fe2+ and rising amounts of Mg2+ (1.5–20 mM) in the presence of 100 µM tetracycline (T100) and tigecycline (G100), respectively. Fe2+ cleavage was carried out as described in the Materials and methods section. Cleavage sites were determined by primer extension and analysed by electrophoresis on a denaturating 10% PAA. (a) Cleavage sites in h31. Lanes A and U, dideoxy sequencing lanes; lane R, unmodified RNA; lane H, control in which Fe2+ was omitted in the presence of 5 mM Mg2+; lane F, Fe2+/H2O2-cleaved RNA in the absence of antibiotics (5 mM Mg2+); lanes Fe2+:Mg2+, Fe2+:Mg2+ ratios from 1:12 to 1:160 in the presence of 100 µM tetracycline and tigecycline, respectively. (b) Phosphoimager quantification of the Mg2+ competition adjusted for loading differences by standardization to regions unaffected by tetracycline or tigecycline. Abbreviations as in (a).

 
Mutational analysis of 16S rRNA

As well as comparing cleavage patterns, we also examined the influence of mutations in 16S rRNA for both derivatives. E. coli TA527 carries deletions spanning the 16S and 23S coding regions in all seven chromosomal rRNA operons. A single E. coli rRNA operon (rrnC) carried on the plasmid pHK-rrnC+ (kanamycin resistant) supplies 16S and 23S rRNA to the cell.20 This strain allowed the analysis of plasmid-encoded 16S rRNA mutations on tetracycline susceptibility without wild-type background. Plasmid pKK3535, a pBR322 derivative (ampicillin resistant) that carries the ribosomal E. coli operon rrnB,21 was used as a wild-type control. pKK1058C is a derivative of pKK3535 containing the 16S rRNA mutation G1058->C.22 Plasmid pKK966U was constructed in this study by site-directed mutagenesis of pKK3535 to introduce a G->U transversion at base 966 in h31. E. coli TA527 pHK-rrnC+ was transformed with pKK3535, pKK1058C or pKK966U, and plated on selective medium containing ampicillin to replace pHK-rrnC+. The loss of pHK-rrnC+ was confirmed by counter-selection for kanamycin susceptibility. We determined MICs for the strains TA527 pKK3535, TA527 pKK1058C and TA527 pKK966U of tetracycline and tigecycline (see the Materials and methods section for details). The results are summarized in Table 1. E. coli TA527 containing pKK1058C was eight-fold more resistant to tetracycline and to tigecycline compared with the same strain carrying pKK3535. The mutation encoded by pKK966U confers a fourfold increase in MIC of tetracycline and tigecycline as compared with TA527 pKK3535.


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Table 1. MICs (mg/L) of tetracycline and tigecycline for E. coli TA527 containing different plasmids
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Three tetracycline-mediated Fe2+ cleavage sites were obtained in the rRNA. They are all in good accordance with published data from genetics, biochemistry and crystallography (compare Table 2). The affected bases not only overlap with bases within a distance of 10 Å to tetracycline in the crystal structures,7,8 but also all cleavages affect sites of functional importance. The Fe2+ cleavage sites in h31 and h34 map to tetracycline binding site-1,7,8 which is functionally important for antibiotic activity, since tetracycline-resistant Helicobacter pylori strains contain the triple mutation A965U/G966U/A967C in h31.29 We introduced the U965/U966/C967 sequence in E. coli (G966->U) and detected a four-fold increase in MIC of both tetracycline and tigecycline. In addition, a mutation of G1058->C in h34 confers tetracycline resistance.22 Since this mutation does not affect directly the tetracycline binding site, it was suggested that distortion of the G1058–U1199 base pair might lead to a conformational change of the neighbouring tetracycline binding pocket.8 This base exchange mediates an eight-fold increased resistance to tigecycline. These findings establish the functional importance of this site for tigecycline activity.


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Table 2. Fe2+-mediated and tetracycline- or tigecycline-directed cleavage sites on 16S rRNA and the corresponding crystallography and biochemical data
 
Tetracycline binding to h27/h11, designated either site-27 or site-5,8 was not detectable by Fe2+ cleavage. This is not unexpected, as Brodersen et al.7 did not see a magnesium ion complexed with tetracycline at site-2. Protection from DMS modification at A892 in h27 was observed only in the presence of tetracycline, and not with tigecycline. Since protection at this position seems to be related to the presence of a pseudoaxial OH group at position 6,14 which is lacking in tigecycline, this result is also consistent with previous data.

The third Fe2+ cleavage region is located in h29, close to tetracycline binding site-4. Oehler et al.30 observed tetracycline photocrosslinking to this region. 30S subunits containing crosslinked tetracycline showed a decreased translational activity when reconstituted with 50S subunits. Evidence for the physiological relevance of this site was also provided by the observation that a {Delta}942 mutation in h29 led to an increased MIC of tetracycline in H. pylori.29 We establish here that this site is also important for tigecycline binding.

Other tetracycline binding sites identified by crystallography have not been related to antibiotic activity. Since the three regions undergoing tetracycline- and tigecycline-dependent Fe2+ cleavage cover physiologically relevant sites, and since they are modified identically by both drugs, we conclude that tetracycline and tigecycline share the same binding sites on 16S rRNA. Tigecycline seems to bind more effectively to these binding sites, since maximal signal intensities for Fe2+ cleavage and DMS methylation are reached at lower concentrations compared with tetracycline. This is consistent with data from Bergeron et al.,6 who detected a five times higher binding affinity of the glycylcyclines DMG-DMDOT and DMG-DOX to the ribosome compared with tetracycline.

The qualitative differences in Fe2+ cleavage intensities indicate that tigecycline might bind these sites in another orientation, which is probably due to the large substituent at position 9. Such different orientations of tetracycline and another glycylcycline derivative, DMG-DMDOT, in complex with TetR has already been reported.31 We superimposed the co-crystal structures of DMG-DMDOT as seen in the TetR complex31 and tetracycline in the ribosomal tetracycline binding site-1,8 as shown in Figure 5. The bulky substituent at position 9 of DMG-DMDOT clashes with the phosphate backbone of G1053. It is therefore impossible for this molecule to bind site-1 identically to tetracycline. This difference must be even more pronounced for tigecycline, since it carries a t-butylglycylamido group, which is bulkier than the dimethylglycylamido substituent present in DMG-DMDOT. Since we demonstrated that tigecycline does bind to site-1, it must either be in a different orientation or the rRNA conformation must be altered to accommodate the bulkier drug.



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Figure 5. (a) Surface structure of tetracycline complexed with Mg2+ bound to ribosomal binding site-1 formed by h31 and h34.8 (b) Superposition of tetracycline bound to site-1 and the glycylcycline derivative DMG-DMDOT from the Orth et al.31 structure. The 9-(N,N-dimethylglycylamido) substituent of DMG-DMDOT overlaps with the backbone phosphate group of G1053 (highlighted by an arrow). C1054 is marked for orientation. Coordinates were taken from the RCSB protein data bank files 1I97 and 1ORK.

 

    Acknowledgements
 
We thank Dr Catherine Squires for strain TA527, Dr Jeremy Ross for the plasmids pKK3535 and pKK1058C, and Irina Alpeeva for constructing the plasmid pKK966U. We also thank Martin Köstner for help with structural modelling. This work was supported by the Fonds der chemischen Industrie through a personal grant to G.B.


    Footnotes
 
* Corresponding author. Tel: +49-9131-8528081; Fax: +49-9131-8528082; E-mail: whillen{at}biologie.uni-erlangen.de Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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3 . Boucher, H. W., Wennersten, C. B. & Eliopoulos, G. M. (2000). In vitro activities of the glycylcycline GAR-936 against Gram-positive bacteria. Antimicrobial Agents and Chemotherapy 44, 2225–9.[Abstract/Free Full Text]

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6 . Bergeron, J., Ammirati, M., Danley, D. et al. (1996). Glycylcyclines bind to the high-affinity tetracycline ribosomal binding site and evade Tet(M)- and Tet(O)-mediated ribosomal protection. Antimicrobial Agents and Chemotherapy 40, 2226–8.[Abstract]

7 . Brodersen, D. E., Clemons, W. M., Carter, A. P. et al. (2000). The structural basis for the action of the antibiotics tetracycline, pactamycin, and hygromycin B on the 30S ribosomal subunit. Cell 103, 1143–54.[ISI][Medline]

8 . Pioletti, M., Schluenzen, F., Harms, J. et al. (2001). Crystal structures of complexes of the small ribosomal subunit with tetracycline, edeine and IF3. EMBO Journal 20, 1829–39.[Abstract/Free Full Text]

9 . Mueller, F. & Brimacombe, R. (1997). A new model for the three-dimensional folding of Escherichia coli 16S rRNA. I. Fitting the RNA to a 3D electron microscopic map at 20 Å. Journal of Molecular Biology 271, 524–44.[CrossRef][ISI][Medline]

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