Structural origins of gentamicin antibiotic action
1998; Springer Nature; Volume: 17; Issue: 22 Linguagem: Inglês
10.1093/emboj/17.22.6437
ISSN1460-2075
Autores Tópico(s)Bacterial Genetics and Biotechnology
ResumoArticle16 November 1998free access Structural origins of gentamicin antibiotic action Satoko Yoshizawa Satoko Yoshizawa Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, 94305-5400 USA Search for more papers by this author Dominique Fourmy Dominique Fourmy Present address: Laboratoire de RMN, CNRS ICSN, 1 Avenue de la terrasse, 91190 Gif-sur-Yvette, France Search for more papers by this author Joseph D. Puglisi Corresponding Author Joseph D. Puglisi Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, 94305-5400 USA Search for more papers by this author Satoko Yoshizawa Satoko Yoshizawa Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, 94305-5400 USA Search for more papers by this author Dominique Fourmy Dominique Fourmy Present address: Laboratoire de RMN, CNRS ICSN, 1 Avenue de la terrasse, 91190 Gif-sur-Yvette, France Search for more papers by this author Joseph D. Puglisi Corresponding Author Joseph D. Puglisi Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, 94305-5400 USA Search for more papers by this author Author Information Satoko Yoshizawa1, Dominique Fourmy2 and Joseph D. Puglisi 1 1Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, 94305-5400 USA 2Present address: Laboratoire de RMN, CNRS ICSN, 1 Avenue de la terrasse, 91190 Gif-sur-Yvette, France *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:6437-6448https://doi.org/10.1093/emboj/17.22.6437 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Aminoglycoside antibiotics that bind to the ribosomal A site cause misreading of the genetic code and inhibit translocation. The clinically important aminoglycoside, gentamicin C, is a mixture of three components. Binding of each gentamicin component to the ribosome and to a model RNA oligonucleotide was studied biochemically and the structure of the RNA complexed to gentamicin C1a was solved using magnetic resonance nuclear spectroscopy. Gentamicin C1a binds in the major groove of the RNA. Rings I and II of gentamicin direct specific RNA-drug interactions. Ring III of gentamicin, which distinguishes this subclass of aminoglycosides, also directs specific RNA interactions with conserved base pairs. The structure leads to a general model for specific ribosome recognition by aminoglycoside antibiotics and a possible mechanism for translational inhibition and miscoding. This study provides a structural rationale for chemical synthesis of novel aminoglycosides. Introduction The ribosome is the target of many clinically important antibiotics. These compounds, which include aminoglycosides, tetracyclines and macrolides, interfere with essential steps of protein synthesis. The RNA components of ribosomes are central to their catalytic function, and functional sites are highly conserved among organisms (Noller, 1991). Most antibiotics that bind to the ribosome have been shown to interact with ribosomal RNA (Moazed and Noller, 1987a,b; Woodcock et al., 1991). The complex three-dimensional folds of RNA represent specific targets for small molecule drug recognition, yet the high conservation of functional sites across species suggests problems of toxicity. Aminoglycosides are the best characterized class of antibiotics that bind directly to ribosomal RNA (Figure 1). Aminoglycosides cause decreases in translational accuracy and inhibit translocation of the ribosome (Davies et al., 1965; Davies and Davis, 1968). Aminoglycoside antibiotics bind to a conserved sequence of rRNA that is near the site of codon-anticodon recognition in the aminoacyl-tRNA site (A site) of 30S subunits (Figure 2). Aminoglycoside binding stabilizes the tRNA-mRNA interaction in the A site by decreasing tRNA dissociation rates, which interferes with proofreading steps that ensure translational fidelity (Karimi and Ehrenberg, 1994). Besides their medical importance, aminoglycoside antibiotics have provided insights into ribosome function. Figure 1.Structures of the aminoglycoside antibiotics that bind in the A site of 16S rRNA. Comparison of the gentamicin components (4-6 ring II-ring I, ring II-ring III linkages) and the neomycin group (4-5 ring II-ring I, ring II-ring III linkages) of aminoglycosides. The neomycin group includes paromomycin, neomycin, ribostamycin and neamine. Ribostamycin contains all rings except ring IV while neamine lacks both rings III and IV. Download figure Download PowerPoint Figure 2.The secondary structure of an oligonucleotide that corresponds to E.coli 16S rRNA in the region of the A site. The natural sequence is boxed. Download figure Download PowerPoint Though chemically distinct, related aminoglycoside antibiotics all bind in the ribosomal A site (Figure 1). Aminoglycosides are positively charged at biological pH (Botto and Coxon, 1983), which contributes to RNA binding. A comparison of the antibiotics suggests chemical groups that are essential for aminoglycoside function (Benveniste and Davies, 1973). Rings I and II are the most common moieties of the aminoglycosides, although their substitution patterns may differ. The N1 and N3 amino groups of ring II (2-deoxystreptamine) are common to all aminoglycosides, as are hydrogen-bond donors at the 2′ and 6′ positions of ring I. The linkage of ring III to 2-deoxystreptamine can vary. In the neomycin class, including aminoglycosides such as ribostamycin and paromomycin, ring III is connected to position 5 of ring II (4,5-disubstituted ring II), whereas in the kanamycin class, including the gentamicins, ring III is linked to position 6 of ring II (4,6-disubstituted ring II). Rings III of the kanamycin class aminoglycosides share common chemical groups at the 2′′ and 3′′ positions. Among aminoglycosides, the total number of rings can also vary from two to four. A large group of enzymes covalently modifies aminoglycosides to yield resistance (Shaw et al., 1993). These modifications, which include acetylation of amino groups and phosphorylation and adenylation of hydroxyl groups, occur primarily on rings I and II. The interaction between the aminoglycoside antibiotic paromomycin and the A site was previously characterized biochemically using an RNA molecule (27 nucleotides) containing the target site for these antibiotics (Recht et al., 1996) (Figure 2). The solution structure of this RNA molecule alone and that of the complex with paromomycin were solved by nuclear magnetic resonance (NMR) (Fourmy et al., 1996, 1998b). The antibiotic binds in the major groove of the RNA within a pocket created by an A1408·A1493 base pair and a single bulged adenine (A1492). Specific interactions occur between chemical groups of rings I and II of paromomycin and the conserved nucleotides in the RNA. The related aminoglycosides, neomycin, ribostamycin and neamine, that have a common core of rings I and II, bind in a qualitatively similar manner to the A-site RNA as paromomycin (Fourmy et al., 1998a). These studies explained the molecular basis for the interaction of 4,5-disubstituted ring II (neomycin class) aminoglycosides and 16S rRNA. All clinically useful aminoglycosides contain a 4,6-disubstituted ring II (kanamycin class). Gentamicin C is a representative of this class of aminoglycosides and is a mixture of three components, gentamicin C1a, C2 and C1, that have different patterns of methylation at the 6′ position of ring I (Figure 1). In addition, ring I of the gentamicin C components lacks the 3′ and 4′ hydroxyl groups compared with ring I of paromomycin. These 3′, 4′ and 6′ hydroxyl groups are involved in the interaction with A-site RNA in the paromomycin-A-site RNA structure. The different chemical structures of gentamicin C allow us to test the roles of ring I substituents and different ring II-ring III linkages on aminoglycoside-RNA affinity. To understand better how aminoglycoside antibiotics bind to ribosomal RNA and interfere with translation, and to understand the superiority of the 4,6-disubstituted class as drugs, we have characterized the interaction of gentamicin C components with the ribosome and with the model A-site RNA oligonucleotide using biochemical and biophysical methods. Furthermore, we present the NMR structure of a gentamicin C1a-A-site oligonucleotide complex. Results Binding of gentamicin components to the 30S ribosomal subunit The three components of gentamicin C were purified from the mixture and the binding of each component of gentamicin C to 30S subunits was assayed by chemical probing with dimethyl sulfate (DMS) at pH 7.2. Each component of gentamicin C protects the same bases of 16S rRNA from modification, although the protections are observed at different concentrations (Figure 3A). A weak footprint is observed at G1494(N7) and A1408(N1) in the presence of 1 μM gentamicin C1a and 10 μM gentamicin C2. The intensity of the gentamicin C1 footprint is weaker and the concentration of antibiotic required to observe the footprint is higher (100 μM). The same bases of 16S rRNA were previously shown to be protected by binding of the gentamicin C mixture to 30S subunits (Moazed and Noller, 1987a). The difference in the affinity of gentamicin C1a and C1 explains their relative inhibitory effects on translation in vitro (Benveniste and Davies, 1973). Figure 3.(A) Autoradiograph of DMS probing reactions on 30S ribosomal subunits. Lane 1 is a control reaction with no DMS added. Lane 2 is a DMS probing reaction in the absence of gentamicin. Subunits were present at a concentration of 100 nM in all reactions. Lanes 3-5 are reactions in the presence of 1, 10 and 100 μM gentamicin C1, respectively. Lanes 6-8 are reactions in the presence of 1, 10 and 100 μM gentamicin C2, respectively. Lanes 9-11 are reactions in the presence of 1, 10 and 100 μM gentamicin C1a, respectively. Bands corresponding to nucleotides G1494 and A1408 are indicated. (B) Autoradiograph of DMS probing reactions on 3′ end-labeled 27 nt RNA. In all reactions, the oligonucleotide is present at a concentration of 5 nM. DMS probing reactions were carried out on ice. Lane 1 is a control reaction with no DMS added. Lane 2 is a DMS probing reaction in the absence of gentamicin. Lanes 3-8 are reactions in the presence of 0.005, 0.01, 0.05, 0.1, 0.5 and 1 μM gentamicin C1a, respectively. (C) Graph showing the reactivity to DMS at G1494(N7) at 25°C as a function of increasing gentamicin C1a (●) and C2 (□) concentration. Reactivity between lanes was normalized using the reactivity of the tetraloop G as the standard. (D) Graph showing the reactivity to DMS at G1494(N7) at 0°C as a function of increasing gentamicin C1a (●), C2 (□) and C1 (▵) concentration. Reactivity between lanes was normalized using the reactivity of the tetraloop G as the standard. Download figure Download PowerPoint The three components of gentamicin C bind to the 30S ribosomal subunit at the same binding site but with different affinities; gentamicin C1a binds to 30S subunits with slightly higher affinity than C2, whereas C1 binds with the lowest affinity compared the others. A 27 nucleotide A-site oligomer (Figure 2) was shown previously to mimic aminoglycoside antibiotic interaction with the ribosome and to be a convenient tool to measure the Kds of aminoglycoside-rRNA complexes (Recht et al., 1996; Fourmy et al., 1998a). The same oligonucleotide was used here to study the gentamicin interaction with the A site. Binding of gentamicin components to an A-site model oligonucleotide The interaction of each gentamicin component with the A-site oligonucleotide was assayed by chemical probing with DMS at pH 7.0. It was shown previously that in the presence of 10 μM paromomycin, residues G1405, A1408 and G1494 were strongly protected from chemical modification by DMS whereas G1491 and G1497 were weakly protected, with an estimated Kd of 0.2 μM (25°C) for paromomycin binding (Recht et al., 1996). Here, a similar footprint on the A-site oligonucleotide was observed for G1494 and G1405 with gentamicin C1a (Figure 3) and C2 with an observed Kd of 2 μM (Figure 3). For gentamicin C1 a weak footprint at G1405(N7) was observed on the A-site oligonucleotide at 100 μM or 1 mM consistent with its weaker affinity for the 30S ribosomal subunit. The weakness of the protection prevents any precise determination of the Kd of gentamicin C1 for the A-site RNA at room temperature. To compare the Kds of different gentamicins, the same experiments were performed at 4°C (Figure 3). Gentamicin C1a and C2 bind to the A-site RNA with similar affinities and Kds of 0.01 and 0.025 μM were observed, respectively. Gentamicin C1 binds to the A-site RNA with lower affinity compared with the other two species and a Kd of 0.5 μM was observed. The high affinity gentamicin C1a-A-site RNA complex was further studied by high resolution NMR, and its solution structure is presented below. Structure determination of the gentamicin C1a-RNA complex Specific binding of gentamicin C1a to the A-site RNA was characterized by monitoring the chemical shift changes of imino proton RNA resonances as a function of antibiotic concentration as previously described (Recht et al., 1996; Fourmy et al., 1998a). A 1:1 complex was formed between the A-site RNA and gentamicin C1a, consistent with the Kd of 2 μM at 25°C and the RNA concentration of 3 mM. Upon addition of gentamicin C1a, the imino protons of U1490 and G1491 are shifted downfield by 0.4 and 0.6 p.p.m., respectively. The proton resonances of the RNA-gentamicin C1a complex were assigned using a non-labeled RNA and a 13C-15N-labeled RNA. Gentamicin C1a was not isotopically labeled. The free gentamicin C1a proton resonances were assigned as well. A total of 379 Nuclear Overhauser Effect (NOE) derived distance restraints, of which 46 were intermolecular RNA-gentamicin distance restraints (Figure 4), and 111 dihedral restraints were determined (Tables I and II). Structures of the complex were calculated using a simulated annealing protocol. A randomized array of atoms corresponding to the RNA was heated to 1000 K, and bonding, distance and dihedral restraints, and a repulsive quartic potential were gradually increased to full value over 40 ps of molecular dynamics. The molecules were then cooled to 300 K during 10 ps and subjected to a final energy minimization step that included an attractive Lennard-Jones potential. No electrostatic term was included in the target function. In the first cycle, ∼90% of the final experimental restraints were used to calculate structures de novo. Thirty-eight of the 148 de novo structures converged to a low energy conformation, based on restraint violation energy. There were differences >100 kcal/mol in restraint violation energy between converged and unconverged structures. The 38 converged structures were then subjected to a second round of simulated annealing with the final set of restraints. Structural statistics for the 38 final stimulated structures are listed in Table I. Figure 4.2D plane at the chemical shift of C1404 C6 (139.0 p.p.m.) from a 3D 13C HMQC-NOESY experiment performed on the A-site RNA-gentamicin C1a complex at 35°C with a mixing time of 200 ms. Intermolecular NOEs between the proton H6 of C1404 with ring III of gentamicin C1a are indicated. Download figure Download PowerPoint Table 1. Structural statistics and atomic r.m.s. deviations a (SA)r versus SA versus (SA)r Final forcing energies Distance and dihedral restraints 11.4 ± 0.3 11.1 R.m.s.d. from experimental distance restraints (Å)b All (379) 0.0249 ± 0.0002 0.0248 RNA (326) 0.0252 ± 0.0002 0.0250 gentamicin C1a (7) 0.0241 ± 0.0062 0.0243 RNA-gentamicin C1a (46) 0.0228 ± 0.0013 0.0227 R.m.s.d. from experimental dihedral restraints (degrees) (111) 0.0127 ± 0.0016 0.0118 Deviations from idealized geometry bonds (Å) 0.0260 ± 0.0001 0.0260 angle (degrees) 0.0621 ± 0.0001 0.0620 impropers (degrees) 0.0485 ± 0.0025 0.0495 Heavy-atom r.m.s.d. All RNA + gentamicin C1a 0.76 0.89 Ordered RNA + gentamicin C1ac 0.58 0.72 gentamicin C1a 0.23 0.24 gentamicin C1a ring I 0.19 0.16 gentamicin C1a ring II 0.03 0.02 gentamicin C1a ring III 0.09 0.09 a refers to the final 38 simulated annealing structures, SA to the average structure obtained by taking the average coordinates of the 38 simulated annealing structures best-fitted to one another, and (SA)r to the average structure after restrained energy minimization. b The 38 final structures did not contain distance violations of >0.2 Å or dihedral violations of >10°. Numbers in parentheses refer to number of restraints. c RNA residues G1405 to A1410, U1490 to C1496 and all gentamicin C1a residues. Table 2. Gentamicin C1a-RNA intermolecular NOE restraints used for structure calculations RNA Gentamicin C1a C1404 H6 ring III (3′′Me) H5 ring III (3′′Me) H4 ring III (3′′Me) H3′ ring III (3′′Me) G1405 H8 ring III (3′′Me) H3′ ring III (3′′Me) U1406 H5 ring III (3′′Me, 4′′Me, 3′′, 2′′, 5′′ax, 5′′eq) C1407 H5 ring III (4′′Me, 5′′ax, 5′′eq) H4 ring III (1′′, 2′′, 4′′Me), ring II (6) G1491 H8 ring I (2′, 3′, 4′) H1 ring I (6′) H2′ ring I (3′) H3′ ring I (3′, 4′) A1492 H8 ring I (3′, 5′) A1493 H8 ring I (5′, 6′), ring II (2ax) H2′ ring II (2ax, 2eq) H3′ ring II (3) G1494 H1 ring III (1′′), ring II (2ax, 2eq, 6) H3′ ring II (2ax, 2eq) U1495 H5 ring II (1, 2ax, 2eq, 3), ring III (1′′) G1497 H1 ring III (3′′Me) The overall structure of the A-site RNA is well-defined. The 38 final structures where heavy atoms of the RNA and gentamicin C1a are superimposed is shown in Figure 5A. The atomic root mean squared deviation (r.m.s.d.) of the superimposed 38 final structures is 0.76 Å (Table I). Figure 5.(A) Best-fit superposition of 38 final simulated annealing structures of the A-site RNA-gentamicin C1a complex, viewed from the major groove side of the RNA. The heavy atoms have been superimposed. The RNA is shown in beige and gentamicin C1a is red. The three rings of gentamicin C1a are numbered as in Figure 1. (B) Single representative structure of A-site RNA-gentamicin C1a complex. All heavy atoms are displayed. The same colors as in (A) are used except that RNA phosphate groups are highlighted. (C) Best-fit superposition of gentamicin C1a of the 38 final structures of the A-site RNA-gentamicin C1a complex. Gentamicin C1a is in red and nitrogen atoms are highlighted in blue. Inter-ring hydrogen bonds are represented by dashed lines. Gentamicin C1a rings are labeled as in Figure 1. Download figure Download PowerPoint Structure of the gentamicin C1a-RNA complex The A-site RNA structure complexed to gentamicin C1a is formed by two A-form helical stems that close an asymmetric internal loop, which contains non-canonical pairings (Figure 5B). The upper stem is extended through a non-canonical U1406·U1495 base pair and a Watson-Crick C1407·G1494 base pair that close the internal loop. In the U1406·U1495 base pair the N3 and O4 of U1406 can form hydrogen bonds with O2 and N3 of U1495 U1406(O4)·U1495(N3), U1406(N3)·U1495(O2), U1406(N3)·U1495(O4) and U1406(O2)·U1495(N3) are 3.7 ± 0.1, 4.8 ± 0.1, 4.3 ± 0.1 and 5.3 ± 0.1 Å apart, respectively. The hydrogen bond between the U1406(O4) and the U1495(N3) position indicates formation of a similar hydrogen bond network as the one defined in the paromomycin complex (where the N3 and O4 of U1406 hydrogen bond with O2 and N3 of U1495). A water molecule bridging the positions U1406(N3) and the U1495(O2) positions could explain the longer distance observed here (4.8 ± 0.1 Å compared with 3.5 ± 0.3 Å for the paromomycin-RNA complex) (Fourmy et al., 1996). In the gentamicin C1a complex, A1408 and A1493 are stacked between the two stems and base paired (Figure 5B). Two families of A1408(N1)-A1493(N6) distances in the ensemble of structures are observed with the distances of 3.7 ± 0.6 Å and 4.6 ± 0.1 Å. In the former family, an A1408(N1)·A1493(N6) hydrogen bond can form. In the latter class, the A1408(N1)·A1493(N6) hydrogen bond is substituted by an A1408(N3)·A1493(N6) hydrogen bond and the average distance within these 15 structures is 3.8 ± 0.2 Å. Both hydrogen bonding schemes involve an antiparallel strand orientation of the two adenosines with anti-glycosidic torsion angles. In the free RNA, a single hydrogen bond [A1408(N1)·A1493(N6)] was identified (Fourmy et al., 1998b). In the paromomycin-RNA complex, A1408(N6)·A1493(N7) and A1408(N1)·A1493(N6) distances in the ensemble of structures were consistent with formation of two hydrogen bonds. The difference in the A1408·A1493 base pairing mode between the gentamicin and paromomycin complexes is difficult to interpret since fewer distance restraints in this region are available in the gentamicin-RNA complex. The A1408·A1493 pair is buckled in the ensemble of conformations, with A1493 at a 35° angle to the plane of A1408. Formation of the A1408·A1493 pair is consistent with the protection of A1408(N1) from methylation upon binding of the gentamicin C components to the ribosome (Figure 3). The reactivities of the N1 positions of A1492 and A1493 are unaffected by antibiotic binding, and these groups are solvent-accessible on the minor groove side in the antibiotic-RNA complex. The RNA backbone is distorted by the presence of the bulged nucleotide A1492 and the non-canonical A1408·A1493 pair. This distortion results in widening of the major groove (the distance between the C1404 phosphate and A1492 phosphate is 17.17 Å in the minimized average structure as opposed to the normal A form helix distance 10.5 Å) and leads to formation of a distinct binding pocket for gentamicin (Figure 6). Figure 6.Binding pocket of gentamicin in the A-site RNA. The Connolly surface of the RNA is represented by blue dots and the gentamicin C1a is red. The view is from the major groove of the RNA. The three rings of gentamicin C1a are numbered as in Figure 1. The base moiety of residue G1491 which is creating a platform for ring I binding is highlighted in yellow. Download figure Download PowerPoint Gentamicin C1a binds in the major groove of the A-site RNA within the internal loop (Figures 5A and 6). Gentamicin C1a is well defined in the ensemble of the 38 structures of the gentamicin-RNA complex (Figure 5C and Table I). When bound to the RNA, the three rings of gentamicin C1a fit into the major groove widened by the bulged A1492. Ring I (purposamine) is positioned near the A1408·A1493 pair and stacks above the base moiety of G1491 (Figures 6 and 7A). The orientation of the conserved 6′ hydrogen-bond donor, -NH2 in gentamicin C1a, is not well defined in the solution structure. The distance between the ring I C6′ and the pro-R oxygen of the A1493 phosphate (4.6 ± 0.2 Å) is consistent with a direct contact between the 6′-amino group of ring I and the phosphate group of A1493. The 6′ nitrogen is positioned within hydrogen bonding distance to A1493(N7) (3.2 ± 0.1 Å) and G1491(N3) (3.8 ± 0.3 Å). The amino group at the 2′ position of ring I could make contacts with the phosphate atom of A1493. Figure 7.RNA-gentamicin C1a contacts observed in the solution structure. (A) Stereo view of specific contacts made between rings I and II of gentamicin C1a and A-site RNA. The RNA is in beige, gentamicin C1a is red, and the view is looking into the major groove of the RNA. Important chemical groups are shown explicitly. The nitrogen atoms are highlighted in blue. Possible hydrogen bonding contacts are indicated by dashed lines. (B) Stereo view of specific contacts made between ring III of gentamicin C1a and A-site RNA. The same colors as in Figure 6A are used. Possible hydrogen bonding contacts are indicated by dashed lines. Download figure Download PowerPoint Ring II (2-deoxystreptamine) spans the U1406·U1495 and C1407·G1494 base pairs. The amino groups at positions 1 and 3 of ring II make hydrogen bonds to U1495(O4) and G1494(N7), respectively (Figure 7A). The amino group at position 3 may also make contact with the phosphate between A1493 and G1494. These ring II contacts were also observed in the paromomycin-RNA complex. Ring III (garosamine) is positioned towards the upper stem (Figures 4 and 7B). Chemical groups of ring III of gentamicin that are common among the kanamycin group antibiotics make specific contacts with universally conserved nucleotides of the A site of 16S rRNA (Figure 7B). The 2′′ hydroxyl group is within hydrogen bonding distance of G1405(O2) and U1406(O4). The nitrogen atom of the aminomethyl group at position 3′′′ of ring III forms a hydrogen bond with the G1405(N7) and may also contact the phosphate of G1405. The methyl group packs against the aromatic ring of C1404 and one face of the ribose of G1405. The 4′′′ hydroxyl forms a hydrogen bond with the phosphate between G1405 and U1406. Several intramolecular hydrogen bonds between the different rings of gentamicin C1a were identified (Figures 5C, 7A and 7B). The amino group at the 2′ position of ring I forms a hydrogen bond with the oxygen of the hydroxyl group at the position 5 of ring II. This hydroxyl group is also hydrogen bonded to the ring III oxygen. This internal network of hydrogen bonds could help to orient the three rings for binding of gentamicin to the RNA. Comparison of the gentamicin C1a-RNA and paromomycin-RNA complexes Superposition of the RNA-paromomycin and RNA-gentamicin C1a complexes demonstrates the similarities and differences of the two complexes. The core RNA (residues G1405·A1410; U1490·C1496) of the two structures were superimposed and the r.m.s.d. value found is 1.48 Å. Within the superimposed structures the r.m.s.d. for rings I and II of gentamicin C1a and paromomycin are 1.28 and 0.41 Å, respectively. The superposition of the two structures (Figure 8) clearly shows that ring III of gentamicin C1a interacts with the upper stem of the A-site RNA (spanning the U1406·U1495 and G1405·C1496 base pairs) as opposed to rings III and IV of paromomycin, which interact with the lower stem (Fourmy et al., 1996). Rings I and II in the two complexes are similarly oriented, while the different ring II-ring III linkages lead to different ring III positions. Ring III is linked to position 6 of ring II in gentamicin C1a and to position 5 in paromomycin (Figure 1). The chemical structures of ring I in gentamicin and paromomycin differ. Nevertheless, similar specific contacts are established between ring I of the aminoglycoside and the RNA through common hydrogen-bond donor groups (Figure 7A). Figure 8.Best-fit superposition of the paromomycin-RNA and gentamicin C1a-RNA complexes, viewed from the major groove side of the RNA. The heavy atoms of the core (nucleotides U1406 to A1410, and U1490 to U1495) of the RNA are superimposed. Only the core is represented. For the paromomycin-RNA complex, the RNA is represented in brown and the antibiotic in yellow. For the gentamicin C1a-RNA complex, the RNA is in tan and the gentamicin in red. Download figure Download PowerPoint The importance of the ring III-G1405 contacts in the binding of gentamicin C1a to the RNA was further investigated by mutagenesis. No binding of gentamicin C1a was observed by footprinting to a mutant RNA oligonucleotide in which the G1405·C1496 base pair was flipped to a C1405·G1496 base pair, whereas paromomycin binding was not affected by the mutation (Figure 9). These results agree with the lack of significant RNA chemical shift changes and the absence of intermolecular gentamicin-RNA NOEs upon formation of a 1:1 complex of gentamicin C1a with this mutant RNA (data not shown). Figure 9.(A) View of specific contacts made between ring III of gentamicin C1a and A-site RNA. The RNA is in tan, gentamicin C1a is red, and the view is into the major groove of the RNA. Possible hydrogen bonding contacts between G1405/C1496 base pair and ring III of gentamicin C1a are indicated by dashed lines. (B) Autoradiograph of DMS probing reactions on the 3′ end labeled 27 nucleotide RNA with G1405C/C1496G substitutions. In all reactions, the oligonucleotide is present at a concentration of 5 nM. Lanes 1 and 9 are control reactions with no DMS added. Lanes 3 and 10 are DMS probing reaction in the absence of antibiotics. DMS probing reactions were carried out at 25°C. Lanes 3-8 are reactions in the presence of 0.25, 0.5, 1, 5, 10 and 100 μM gentamicin C1a, respectively. Lanes 11-16 are reactions in the presence of 0.25, 0.5, 1, 5, 10 and 100 μM paromomycin, respectively. Download figure Download PowerPoint Discussion Binding of gentamicin C components to the A site Binding of each gentamicin C component to the A site was studied qualitatively on the 30S subunit and quantitatively on the A-site RNA oligonucleotide. Upon binding of each component, the same bases of the A-site RNA were protected against chemical modification by DMS, indicating a common ribosomal binding site. Quantitative analysis of the chemical footprinting of the A-site RNA showed that gentamicin C1a and C2 bind to the A site with similar affinities, C1a slightly higher than C2. Gentamicin C1 binds to A-site
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