Binding of Neomycin-Class Aminoglycoside Antibiotics to Mutant Ribosomes with Alterations in the A Site of 16S rRNA
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《抗菌试剂及化学方法》
1.Institut für Medizinische Mikrobiologie, Universit?t Zürich, CH-8006 Zürich, Switzerland,2.Institut de Biologie Moléculaire et Cellulaire du CNRS, URP Architecture et Réactivité de l'ARN, Université Louis Pasteur, 15 Rue René Descartes, F-67084 Strasbourg Cedex, France
ABSTRACT
Aminoglycoside antibiotics that bind to the aminoacyl-tRNA site (A site) of the ribosome are composed of a common neamine core in which a glycopyranosyl ring is attached to position 4 of a 2-deoxystreptamine moiety. The core is further substituted by one (ribostamycin), two (neomycin and paromomycin), or three (lividomycin A) additional sugars attached to position 5 of the 2-deoxystreptamine. To study the role of rings III, IV, and V in aminoglycoside binding, we used isogenic Mycobacterium smegmatis rrnB mutants carrying homogeneous populations of mutant ribosomes with alterations in the 16S rRNA A site. MICs were determined to investigate drug-ribosome interactions, and the results were compared with that of the previously published crystal structure of paromomycin bound to the ribosomal A site. Our analysis demonstrates that the stacking interaction between ring I and G1491 is largely sequence independent, that rings III and IV each increase the strength of drug binding to the ribosome, that ring IV of the 6'-NH3+ aminoglycosides compensates for loss of interactions between ring II and U1495 and between ring III and G1491, that the aminoglycosides rely on pseudo-base pairing between ring I and A1408 for binding independently of the number of sugar rings attached to the neamine core, that addition of ring V to the 6'-OH 4,5-aminoglycoside paromomycin does not alter the mode of binding, and that alteration of the U1406 · U1495 wobble base pair to the Watson-Crick interaction pair 1406C-1495G yields ribosomal drug susceptibilities to 4,5-aminoglycosides comparable to those seen with the wild-type A site.
INTRODUCTION
Aminoglycosides form a large family of water-soluble, polycationic amino sugars which are used as broad-spectrum antibacterial agents (14, 15). Common to all aminoglycosides is the neamine core. The neamine core is composed of a six-membered cyclitol (2-deoxystreptamine; ring II) glycosidically linked to a glucopyranosyl (ring I). Aminoglycosides target the ribosome by direct interaction with rRNA and they affect protein synthesis by inducing codon misreading and by inhibiting translocation of the tRNA-mRNA complex from the A to the P site (4, 15, 18, 28).
Helix 44 is located in the 1400 to 1500 region of 16S rRNA (Escherichia coli numbering is used throughout this article) and has been identified as the site of codon-anticodon interaction (A site) by a number of studies (3, 17, 19, 32). Although A-site-bound tRNA and aminoglycosides protect the same nucleotides in the decoding region, namely A1408, A1492, A1493, and G1494, aminoglycosides do not act by sterically hindering the binding of the tRNA to the ribosome. Instead, they interfere with the decoding process by inducing conformational alterations of the ribosomal A site (2, 19). Upon drug binding, nucleotides A1492 and A1493 flip out from helix 44; this change in conformation is normally only observed after the formation of a cognate tRNA-mRNA complex (2, 19, 20). Bulging out of A1492 and A1493 is most likely an essential step in aminoglycoside action, as it explains the miscoding properties of these drugs (4).
The neamine core of the aminoglycosides is essential for drug activity (4, 6). Nuclear magnetic resonance studies have revealed that rings I and II of the 4,5-aminoglycosides are sufficient to confer specific binding to a model A site RNA (6). A1408 and base pairs C1409-G1491 and U1406 · U1495 represent important elements of the drug binding pocket (Fig. 1). Ring I forms two direct hydrogen bonds to the Watson-Crick side of A1408 and is involved in a stacking interaction with G1491. Ring II participates in a network of hydrogen bond contacts of which the interactions between the 1-amino group and O-4 of U1495 and between the 3-amino group and N-7 of G1494 are sequence specific.
The neamine core makes additional direct or water-bridged hydrogen bond contacts to the phosphate backbone of A1492 and A1493 (2, 29, 30). Additional sugars (rings III, IV, and V) are attached to position 5 of the 2-deoxystreptamine moiety. The contacts of ring III are characterized by two direct hydrogen bonds, with N-7 of G1491 and with N-4 of C1407, linking the two RNA strands of helix 44 together. Ring IV is mainly involved in non-sequence-specific electrostatic interactions with the phosphate backbone, but the exact positioning of this ring remains to be established (2, 27). Recently, the structures of complexes between oligonucleotides containing the A site and neamine, neomycin, and lividomycin A (8) have been solved (Fig. 1).
RNA sequence determinants for aminoglycoside binding to the ribosomal A site have mostly been defined using model RNA oligonucleotides (6, 7, 9, 13, 16, 26). Data on in vivo mutagenesis of the ribosomal A site are scarce and compromised by limitations in Escherichia coli, which is frequently used as the model in ribosomal genetics. In particular, several rRNA nucleotides involved in forming the drug binding site could not be subjected to in vivo mutagenesis due to nonviability of the mutations in E. coli, e.g., U1495A, U1495C, G1491A, and C1409G (5, 25). These limitations were overcome with the development of the Mycobacterium smegmatis rrnB system (21-24, 27). To analyze the contributions of rings III, IV, and V to the binding of 4,5-aminoglycosides, we extended our experiments using M. smegmatis rrnB strains carrying a large set of mutations within the decoding site.
MATERIALS AND METHODS
Bacterial strains and DNA techniques. The conditions for selective plating and RecA-mediated homologous recombination to obtain mutant strains of M. smegmatis rrnB (for a list of mutants, see Table 1 and Fig. 2) were described previously (23). After mutant selection the strains were cultured on LB agar plates. Nucleic acid sequencing was used to verify that the point mutations had been introduced into the single functional rRNA operon of M. smegmatis rrnB. Taq cycle sequencing was done with fluorescently labeled nucleotides according to the manufacturer's instructions (Applied Biosystems).
Determination of the MICs. Strains of M. smegmatis rrnB were cultured on LB agar plates. Single-colony cultures, grown in LB medium supplemented with 0.05% Tween 80, were used for MIC tests in a microtiter plate format; at least three independent clones were analyzed per mutation. To 200 μl of starting cultures with an optical density at 600 nm of 0.025, the antibiotics to be tested were added in twofold series of dilutions ranging from 0.25 to 1,024 μg/ml. Paromomycin (P-9297), lividomycin (L-4518), neomycin (N-6386), and ribostamycin (R-2255) were obtained from Sigma; neamine (158966) was from ICN Biomedicals. The MIC is defined as the drug concentration at which the growth of the cultures is completely inhibited after incubation at 37°C. Cells were incubated for a period corresponding to 24 generations; the wild type has a generation time of 3 h, and the generation time of the mutants investigated was in the range of 3 to 6 h (U1495A, 8 h).
RESULTS
Experimental system. The 4,5-disubstituted 2-deoxystreptamines investigated share close structural similarities. The common core of the 4,5-aminoglycosides is composed of a 2-deoxystreptamine and a glycopyranosyl ring. The chemical group linked to the 6' position of ring I separates the 4,5-aminoglycosides into two subclasses: the 6'-amino subclass (neamine, ribostamycin, and neomycin) and the 6'-hydroxy subclass (paromomycin and lividomycin). The diversity within these two subclasses is based on the number of sugar moieties attached to position 5 of ring II: ribostamycin consists of neamine with one additional sugar attached to ring II; addition of a fourth ring to ring III of ribostamycin gives rise to neomycin. Except for the 6' position, paromomycin is identical to neomycin; an additional fifth ring attached to paromomycin gives rise to lividomycin (for a list of drug structures, see Fig. 3 and 4). The number of charges and the nature of the substituent at position 6' are identical for neamine and ribostamycin and for paromomycin and lividomycin; in both cases, however, the number of rings varies.
Mutagenesis in M. smegmatis rrnB results in isogenic strains carrying homogeneous populations of mutant ribosomes (for a list of mutant strains, see Table 1 and Fig. 2). MICs were determined to investigate ribosomal drug susceptibility. Data interpretation for neamine, ribostamycin, neomycin, paromomycin, and lividomycin was based on available X-ray structural information (2, 8, 29).
The drug susceptibility measurements given in Table 2 (see also comparison graphs in Fig. 3 and 4) reveal that in wild-type M. smegmatis rrnB the activity of the 4,5-aminoglycosides correlates with the number of sugar rings attached to the neamine core. Addition of a third or fourth ring increases the activity of the drug by approximately eightfold each. However, the presence of an additional fifth ring, as in lividomycin, does not markedly increase drug activity.
The pseudo-base pair between ring I and A1408 was recently defined as a critical interaction for aminoglycoside binding (22, 29, 31). Independent of the number of rings attached to the neamine core, disruption of the pseudo-base pair by the A1408G alteration prevents the binding of all 4,5-aminoglycosides with a 6'-NH3+ group (see Table 2). In contrast, for the 6'-OH aminoglycosides (paromomycin and lividomycin) the hydroxyl group at position 6 of ring I is likely to become an H-bond acceptor for N-1 or N-2 of A1408G, as discussed previously (22, 29, 31). Thus, these drugs can bind to the target site in presence of A1408G (see Table 2).
Interactions with base pair C1409-G1491. The Watson-Crick base pair C1409-G1491 produces a seat in the pocket at the lower end of the drug binding site, enabling ring I to stack on G1491. In addition, N-7 of G1491 forms a hydrogen bond with the O-5" of ring III (2, 29, 31). The MIC data and the calculation of relative resistances given in Table 2 demonstrate that, within the 6'-NH3+ subclass, ribostamycin is always affected the most by alterations of C1409-G1491. While ribosomal susceptibility to neamine and neomycin decreased only moderately upon mutation of the C1409-G1491 base pair (relative resistances between 1 and 16 μg/ml), significant resistance is conferred to ribostamycin (relative resistances between 8 and 128 μg/ml). In particular, transversion mutations (G1491 to C or U and C1409 to G) have a profound effect on ribostamycin susceptibility. Compared to neomycin and as reported previously (21), the 6'-OH paromomycin is much more dependent on proper interaction with the C1409-G1491 base pair. The importance of this interaction is not alleviated by addition of a further amino sugar, as in lividomycin (Table 2).
The double mutations A1408G/C1409G and A1408G/G1491U confer high-level drug resistance to all 4,5-aminoglycosides tested. This is to be expected for the 6'-NH3+ subclass, due to disruption of the pseudo-base pair interaction of ring I with A1408 (21, 22). For the 6'-OH subclass this finding indicates that the effects of different mutations are more than additive, in particular for the combined A1408G/C1409G alteration, since the relative resistance of the combined A1408G/C1409G alteration is significantly higher than that of the single mutations A1408G and C1409G alone (Table 2).
Interactions with base pair U1406 · U1495. The wobble base pair U1406 · U1495 forms the binding pocket at the upper stem. One water-mediated contact between O-6 of ring II and O-4 of U1406 is observed in the 4,5-disubstituted 2-deoxystreptamines (8, 29). Mutation of U1406 to adenine does not affect drug susceptibility; alteration of U1406 to cytosine marginally influences drug susceptibility (relative resistances of 4 to 16 μg/ml). The U1406C/A1408G double mutation results in high-level resistance to all 4,5-aminoglycosides studied, indicating that for the 6'-OH subclass the effects of the individual mutations are more than additive.
N-1 of ring II makes an additional direct hydrogen bond contact with O-4 of U1495. The transition mutation U1495C exchanges the O-4 with an ammonium group, rendering the interaction of cytosine with N-1 of the 4,5-aminoglycosides unfavorable (electrostatic repulsion). However, the correct insertion of ring I into the binding pocket and the pseudo-base pair interaction between O-5' and N-6' of ring I with A1408 still allow binding of the 6'-NH3+ subclass to the ribosome. In contrast, the 6'-OH aminoglycosides are more affected by the U1495C alteration. The U1495A alteration is expected to prevent contacts between N-1 of ring II and nucleotide 1495 even more than a U-to-C mutation owing to the size of the adenine ring.
With the exception of neomycin, the U1495A alteration as well as the double mutation U1406C/U1495A confer high-level drug resistance to all 4,5-aminoglycosides tested, including neamine, ribostamycin, paromomycin, and lividomycin. Apparently, the interaction of the 6'-NH3+ ring I with both A1408 and ring IV is required to partly compensate for this major conformational change in the ribosomal A site. Unexpectedly, introducing the U1495G alteration into the U1406C mutant, thus replacing the U · U wobble base pair with a 1406C-1495G Watson-Crick interaction, nearly completely restores ribosomal susceptibility to the 4,5-aminoglycoside antibiotics.
DISCUSSION
Using a well-defined in vivo system, we investigated the role of rings III, IV, and V in the binding of 4,5-aminoglycosides to their ribosomal target. Site-directed mutagenesis of the ribosomal A site in a genetic model where manipulation of rRNA results in cells carrying homogeneous populations of mutant ribosomes (21-24) allowed a detailed investigation of drug-target interactions. The critical role of the 6' group of ring I in the aminoglycoside-ribosome interaction has previously been investigated by mutagenesis studies and will not be addressed here (22). In this study we focused on the role of rings III, IV, and V in drug binding.
In line with previous measurements of dissociation constants (7) ring III increases drug potency by a factor of 8, as determined by MIC measurements (Table 2). Ring III makes sequence-specific contacts with RNA, i.e., hydrogen bonding between 5"-OH and N-7 of G1491 and between 2"-OH and N-4 of C1407 (2, 29). Addition of ring IV increases the positive net charge, and it contacts the ribosomal A site at the backbone of helix 44 (2, 29). The strength of these electrostatic contacts raises drug activity eightfold as determined by MIC measurements of neomycin and ribostamycin. Overall, in our in vivo system neomycin is 64-fold more active than neamine. This finding is in good agreement with the dissociation constants reported for neomycin and neamine (0.5 μM versus 100 μM) (7). Addition of ring V affects the antibacterial activity and susceptibility of mutant ribosomes little, if at all (compare lividomycin to paromomycin), indicating that ring V makes only weak contacts with the A site in a non-sequence-specific manner, most likely with the RNA backbone, as seen in the crystal structure (8).
For each pair of aminoglycosides with similar properties of overall charge and substituent at the 6' position, one can divide the effect of mutations into two categories: mutations for which the ratio of relative resistance is identical and those for which this ratio is different. In the neamine-ribostamycin pair, for instance, the C1409G mutation (and to a lesser extent G1491A or G1491C) confers higher relative resistance to ribostamycin than to neamine.
To investigate the ring I-G1491 interaction isolated from other interactions by additional rings, e.g., between 5"-OH of ring III and N-7 of G1491, we chose a reductionist approach, i.e., we determined MICs to neamine in a series of mutants with alterations in the C1409-G1491 base pair. These studies revealed that transitions affect drug binding weakly; transversions have a small albeit significant effect. We interpret these findings as evidence that the ring I-G1491 stacking interaction is largely sequence independent. Within the 6'-NH3+ subclass, ribostamycin is most affected by alterations of the C1409-G1491 base pair. This finding is rationalized by the X-ray structural data available for paromomycin (2, 27). In contrast to ribostamycin, with its ring III-G1491 interaction, neamine interacts with G1491 merely by non-sequence-specific stacking interaction with its ring I. Compared to that of ribostamycin, ring IV of neomycin apparently is able to compensate for the loss of sequence-specific interaction between ring III and G1491 and/or introduction of steric clashes as mediated by mutagenesis of C1409-G1491.
A thorough interpretation of the MIC measurements obtained upon mutagenesis of the U1406 · U1495 wobble base pair is in part limited by the low susceptibility of wild-type ribosomes to neamine. Most of the mutations, i.e., U1406C, U1406A, and U1495C, only moderately affect ribosomal susceptibility to the 6'-NH3+ aminoglycosides, if at all. The U1495A alteration confers significant resistance to ribostamycin (relative resistance, >128 μg/ml) but only moderately affects susceptibility to neomycin (relative resistance, 16 μg/ml).
The loss of ribosomal susceptibility to neamine and ribostamycin in the U1495A mutant can be rationalized by the loss of interaction between the 1-amino group of ring II and the O-4 of U1495 in the U1495A mutant and by less favorable binding of ring II. Apparently, ring IV of neomycin is able to partly compensate for this interaction. The U1406C · U1495A alteration confers high-level drug resistance to all 4,5-aminoglycosides except neomycin, which is only moderately affected. As discussed previously (22), the U1406C · U1495A alteration will result in a major change in base pair geometry precluding an appropriate positioning of ring II near the 1406-1495 pair and consequently poor insertion of ring I in the drug binding pocket.
Alteration of U1495A to G in the U1406C · U1495A double mutant replaces the natural U1406 · U1495 wobble base pair with a U1406C-U1495G Watson-Crick base pair and restores ribosomal susceptibility to all 4,5-aminoglycosides of the 6'-NH3+ and the 6'-OH subclasses investigated. This observation contrasts with the U1406-U1495A alteration, which confers significant resistance to most 4,5-aminoglycosides. Presumably, the U1406C-U1495G alteration has a conformation and geometry similar to that of the U1406-U1495A alteration. How then to explain this difference in drug susceptibility?
One rationale would refer to the different net charge provided by the functional group at position C-6 of U1495A (amino group, positive charge) and U1495G (keto group, negative charge). The keto group at C-6 of 1495G, together with the N-7 of 1495G, could possibly interact with the ring II N-1 ammonium group in a fashion similar to that of O-4 of the pyrimidine U1495 or, more generally, be involved in a network of electrostatic interactions between the positively charged aminoglycosides and their target RNAs (10, 11). The Hoogsteen sites of guanines are often involved in binding positive ions such as potassium ions (1).
Figure 5 shows the superposition of a 1406C-1495G base pair on the U1406 · U1495 pair. The distances between a typical aminoglycoside (N-1 and O-6 of ring II) and the Hoogsteen sites of 1495G are 3.0 ? (O-6) and 4.2 ? (N-7), respectively. It can also be seen how the amino group N-4 of 1406C may replace the observed water molecule between U1406 and the aminoglycoside such that a contact between N-4 of 1406C and O-6 of ring II can be made. Interestingly, mutants in which a single uracil is replaced with an adenine display disparate effects. Thus, the U1406-U1495A mutation leads to resistance to aminoglycosides, while the U1406A-U1495 mutant shows susceptibility to the neomycin class of aminoglycosides but less susceptibility to the kanamycin class (20).
Our data indicate that binding of 4,5-disubstituted aminoglycosides to the ribosomal A site depends on proper interaction of ring I with nucleotides A1408 and G1491 and is further stabilized by additional contacts, e.g., with U1495. Taken together, these data on site-directed mutagenesis of the drug target site provide a detailed analysis of the importance of the different binding contacts in the drug-ribosome interaction.
ACKNOWLEDGMENTS
This study was supported by grants from the Swiss National Science Foundation to E.C.B. (3200-BO-100780, NRP 49). B.F. is supported by a grant from the European Commission (QLRT-2001-000892); E.W. acknowledges support from the Institut Universitaire de France.
We thank T. Janui for expert technical assistance, A. Vasella for helpful discussions, and A. Makovec for typing the manuscript.
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ABSTRACT
Aminoglycoside antibiotics that bind to the aminoacyl-tRNA site (A site) of the ribosome are composed of a common neamine core in which a glycopyranosyl ring is attached to position 4 of a 2-deoxystreptamine moiety. The core is further substituted by one (ribostamycin), two (neomycin and paromomycin), or three (lividomycin A) additional sugars attached to position 5 of the 2-deoxystreptamine. To study the role of rings III, IV, and V in aminoglycoside binding, we used isogenic Mycobacterium smegmatis rrnB mutants carrying homogeneous populations of mutant ribosomes with alterations in the 16S rRNA A site. MICs were determined to investigate drug-ribosome interactions, and the results were compared with that of the previously published crystal structure of paromomycin bound to the ribosomal A site. Our analysis demonstrates that the stacking interaction between ring I and G1491 is largely sequence independent, that rings III and IV each increase the strength of drug binding to the ribosome, that ring IV of the 6'-NH3+ aminoglycosides compensates for loss of interactions between ring II and U1495 and between ring III and G1491, that the aminoglycosides rely on pseudo-base pairing between ring I and A1408 for binding independently of the number of sugar rings attached to the neamine core, that addition of ring V to the 6'-OH 4,5-aminoglycoside paromomycin does not alter the mode of binding, and that alteration of the U1406 · U1495 wobble base pair to the Watson-Crick interaction pair 1406C-1495G yields ribosomal drug susceptibilities to 4,5-aminoglycosides comparable to those seen with the wild-type A site.
INTRODUCTION
Aminoglycosides form a large family of water-soluble, polycationic amino sugars which are used as broad-spectrum antibacterial agents (14, 15). Common to all aminoglycosides is the neamine core. The neamine core is composed of a six-membered cyclitol (2-deoxystreptamine; ring II) glycosidically linked to a glucopyranosyl (ring I). Aminoglycosides target the ribosome by direct interaction with rRNA and they affect protein synthesis by inducing codon misreading and by inhibiting translocation of the tRNA-mRNA complex from the A to the P site (4, 15, 18, 28).
Helix 44 is located in the 1400 to 1500 region of 16S rRNA (Escherichia coli numbering is used throughout this article) and has been identified as the site of codon-anticodon interaction (A site) by a number of studies (3, 17, 19, 32). Although A-site-bound tRNA and aminoglycosides protect the same nucleotides in the decoding region, namely A1408, A1492, A1493, and G1494, aminoglycosides do not act by sterically hindering the binding of the tRNA to the ribosome. Instead, they interfere with the decoding process by inducing conformational alterations of the ribosomal A site (2, 19). Upon drug binding, nucleotides A1492 and A1493 flip out from helix 44; this change in conformation is normally only observed after the formation of a cognate tRNA-mRNA complex (2, 19, 20). Bulging out of A1492 and A1493 is most likely an essential step in aminoglycoside action, as it explains the miscoding properties of these drugs (4).
The neamine core of the aminoglycosides is essential for drug activity (4, 6). Nuclear magnetic resonance studies have revealed that rings I and II of the 4,5-aminoglycosides are sufficient to confer specific binding to a model A site RNA (6). A1408 and base pairs C1409-G1491 and U1406 · U1495 represent important elements of the drug binding pocket (Fig. 1). Ring I forms two direct hydrogen bonds to the Watson-Crick side of A1408 and is involved in a stacking interaction with G1491. Ring II participates in a network of hydrogen bond contacts of which the interactions between the 1-amino group and O-4 of U1495 and between the 3-amino group and N-7 of G1494 are sequence specific.
The neamine core makes additional direct or water-bridged hydrogen bond contacts to the phosphate backbone of A1492 and A1493 (2, 29, 30). Additional sugars (rings III, IV, and V) are attached to position 5 of the 2-deoxystreptamine moiety. The contacts of ring III are characterized by two direct hydrogen bonds, with N-7 of G1491 and with N-4 of C1407, linking the two RNA strands of helix 44 together. Ring IV is mainly involved in non-sequence-specific electrostatic interactions with the phosphate backbone, but the exact positioning of this ring remains to be established (2, 27). Recently, the structures of complexes between oligonucleotides containing the A site and neamine, neomycin, and lividomycin A (8) have been solved (Fig. 1).
RNA sequence determinants for aminoglycoside binding to the ribosomal A site have mostly been defined using model RNA oligonucleotides (6, 7, 9, 13, 16, 26). Data on in vivo mutagenesis of the ribosomal A site are scarce and compromised by limitations in Escherichia coli, which is frequently used as the model in ribosomal genetics. In particular, several rRNA nucleotides involved in forming the drug binding site could not be subjected to in vivo mutagenesis due to nonviability of the mutations in E. coli, e.g., U1495A, U1495C, G1491A, and C1409G (5, 25). These limitations were overcome with the development of the Mycobacterium smegmatis rrnB system (21-24, 27). To analyze the contributions of rings III, IV, and V to the binding of 4,5-aminoglycosides, we extended our experiments using M. smegmatis rrnB strains carrying a large set of mutations within the decoding site.
MATERIALS AND METHODS
Bacterial strains and DNA techniques. The conditions for selective plating and RecA-mediated homologous recombination to obtain mutant strains of M. smegmatis rrnB (for a list of mutants, see Table 1 and Fig. 2) were described previously (23). After mutant selection the strains were cultured on LB agar plates. Nucleic acid sequencing was used to verify that the point mutations had been introduced into the single functional rRNA operon of M. smegmatis rrnB. Taq cycle sequencing was done with fluorescently labeled nucleotides according to the manufacturer's instructions (Applied Biosystems).
Determination of the MICs. Strains of M. smegmatis rrnB were cultured on LB agar plates. Single-colony cultures, grown in LB medium supplemented with 0.05% Tween 80, were used for MIC tests in a microtiter plate format; at least three independent clones were analyzed per mutation. To 200 μl of starting cultures with an optical density at 600 nm of 0.025, the antibiotics to be tested were added in twofold series of dilutions ranging from 0.25 to 1,024 μg/ml. Paromomycin (P-9297), lividomycin (L-4518), neomycin (N-6386), and ribostamycin (R-2255) were obtained from Sigma; neamine (158966) was from ICN Biomedicals. The MIC is defined as the drug concentration at which the growth of the cultures is completely inhibited after incubation at 37°C. Cells were incubated for a period corresponding to 24 generations; the wild type has a generation time of 3 h, and the generation time of the mutants investigated was in the range of 3 to 6 h (U1495A, 8 h).
RESULTS
Experimental system. The 4,5-disubstituted 2-deoxystreptamines investigated share close structural similarities. The common core of the 4,5-aminoglycosides is composed of a 2-deoxystreptamine and a glycopyranosyl ring. The chemical group linked to the 6' position of ring I separates the 4,5-aminoglycosides into two subclasses: the 6'-amino subclass (neamine, ribostamycin, and neomycin) and the 6'-hydroxy subclass (paromomycin and lividomycin). The diversity within these two subclasses is based on the number of sugar moieties attached to position 5 of ring II: ribostamycin consists of neamine with one additional sugar attached to ring II; addition of a fourth ring to ring III of ribostamycin gives rise to neomycin. Except for the 6' position, paromomycin is identical to neomycin; an additional fifth ring attached to paromomycin gives rise to lividomycin (for a list of drug structures, see Fig. 3 and 4). The number of charges and the nature of the substituent at position 6' are identical for neamine and ribostamycin and for paromomycin and lividomycin; in both cases, however, the number of rings varies.
Mutagenesis in M. smegmatis rrnB results in isogenic strains carrying homogeneous populations of mutant ribosomes (for a list of mutant strains, see Table 1 and Fig. 2). MICs were determined to investigate ribosomal drug susceptibility. Data interpretation for neamine, ribostamycin, neomycin, paromomycin, and lividomycin was based on available X-ray structural information (2, 8, 29).
The drug susceptibility measurements given in Table 2 (see also comparison graphs in Fig. 3 and 4) reveal that in wild-type M. smegmatis rrnB the activity of the 4,5-aminoglycosides correlates with the number of sugar rings attached to the neamine core. Addition of a third or fourth ring increases the activity of the drug by approximately eightfold each. However, the presence of an additional fifth ring, as in lividomycin, does not markedly increase drug activity.
The pseudo-base pair between ring I and A1408 was recently defined as a critical interaction for aminoglycoside binding (22, 29, 31). Independent of the number of rings attached to the neamine core, disruption of the pseudo-base pair by the A1408G alteration prevents the binding of all 4,5-aminoglycosides with a 6'-NH3+ group (see Table 2). In contrast, for the 6'-OH aminoglycosides (paromomycin and lividomycin) the hydroxyl group at position 6 of ring I is likely to become an H-bond acceptor for N-1 or N-2 of A1408G, as discussed previously (22, 29, 31). Thus, these drugs can bind to the target site in presence of A1408G (see Table 2).
Interactions with base pair C1409-G1491. The Watson-Crick base pair C1409-G1491 produces a seat in the pocket at the lower end of the drug binding site, enabling ring I to stack on G1491. In addition, N-7 of G1491 forms a hydrogen bond with the O-5" of ring III (2, 29, 31). The MIC data and the calculation of relative resistances given in Table 2 demonstrate that, within the 6'-NH3+ subclass, ribostamycin is always affected the most by alterations of C1409-G1491. While ribosomal susceptibility to neamine and neomycin decreased only moderately upon mutation of the C1409-G1491 base pair (relative resistances between 1 and 16 μg/ml), significant resistance is conferred to ribostamycin (relative resistances between 8 and 128 μg/ml). In particular, transversion mutations (G1491 to C or U and C1409 to G) have a profound effect on ribostamycin susceptibility. Compared to neomycin and as reported previously (21), the 6'-OH paromomycin is much more dependent on proper interaction with the C1409-G1491 base pair. The importance of this interaction is not alleviated by addition of a further amino sugar, as in lividomycin (Table 2).
The double mutations A1408G/C1409G and A1408G/G1491U confer high-level drug resistance to all 4,5-aminoglycosides tested. This is to be expected for the 6'-NH3+ subclass, due to disruption of the pseudo-base pair interaction of ring I with A1408 (21, 22). For the 6'-OH subclass this finding indicates that the effects of different mutations are more than additive, in particular for the combined A1408G/C1409G alteration, since the relative resistance of the combined A1408G/C1409G alteration is significantly higher than that of the single mutations A1408G and C1409G alone (Table 2).
Interactions with base pair U1406 · U1495. The wobble base pair U1406 · U1495 forms the binding pocket at the upper stem. One water-mediated contact between O-6 of ring II and O-4 of U1406 is observed in the 4,5-disubstituted 2-deoxystreptamines (8, 29). Mutation of U1406 to adenine does not affect drug susceptibility; alteration of U1406 to cytosine marginally influences drug susceptibility (relative resistances of 4 to 16 μg/ml). The U1406C/A1408G double mutation results in high-level resistance to all 4,5-aminoglycosides studied, indicating that for the 6'-OH subclass the effects of the individual mutations are more than additive.
N-1 of ring II makes an additional direct hydrogen bond contact with O-4 of U1495. The transition mutation U1495C exchanges the O-4 with an ammonium group, rendering the interaction of cytosine with N-1 of the 4,5-aminoglycosides unfavorable (electrostatic repulsion). However, the correct insertion of ring I into the binding pocket and the pseudo-base pair interaction between O-5' and N-6' of ring I with A1408 still allow binding of the 6'-NH3+ subclass to the ribosome. In contrast, the 6'-OH aminoglycosides are more affected by the U1495C alteration. The U1495A alteration is expected to prevent contacts between N-1 of ring II and nucleotide 1495 even more than a U-to-C mutation owing to the size of the adenine ring.
With the exception of neomycin, the U1495A alteration as well as the double mutation U1406C/U1495A confer high-level drug resistance to all 4,5-aminoglycosides tested, including neamine, ribostamycin, paromomycin, and lividomycin. Apparently, the interaction of the 6'-NH3+ ring I with both A1408 and ring IV is required to partly compensate for this major conformational change in the ribosomal A site. Unexpectedly, introducing the U1495G alteration into the U1406C mutant, thus replacing the U · U wobble base pair with a 1406C-1495G Watson-Crick interaction, nearly completely restores ribosomal susceptibility to the 4,5-aminoglycoside antibiotics.
DISCUSSION
Using a well-defined in vivo system, we investigated the role of rings III, IV, and V in the binding of 4,5-aminoglycosides to their ribosomal target. Site-directed mutagenesis of the ribosomal A site in a genetic model where manipulation of rRNA results in cells carrying homogeneous populations of mutant ribosomes (21-24) allowed a detailed investigation of drug-target interactions. The critical role of the 6' group of ring I in the aminoglycoside-ribosome interaction has previously been investigated by mutagenesis studies and will not be addressed here (22). In this study we focused on the role of rings III, IV, and V in drug binding.
In line with previous measurements of dissociation constants (7) ring III increases drug potency by a factor of 8, as determined by MIC measurements (Table 2). Ring III makes sequence-specific contacts with RNA, i.e., hydrogen bonding between 5"-OH and N-7 of G1491 and between 2"-OH and N-4 of C1407 (2, 29). Addition of ring IV increases the positive net charge, and it contacts the ribosomal A site at the backbone of helix 44 (2, 29). The strength of these electrostatic contacts raises drug activity eightfold as determined by MIC measurements of neomycin and ribostamycin. Overall, in our in vivo system neomycin is 64-fold more active than neamine. This finding is in good agreement with the dissociation constants reported for neomycin and neamine (0.5 μM versus 100 μM) (7). Addition of ring V affects the antibacterial activity and susceptibility of mutant ribosomes little, if at all (compare lividomycin to paromomycin), indicating that ring V makes only weak contacts with the A site in a non-sequence-specific manner, most likely with the RNA backbone, as seen in the crystal structure (8).
For each pair of aminoglycosides with similar properties of overall charge and substituent at the 6' position, one can divide the effect of mutations into two categories: mutations for which the ratio of relative resistance is identical and those for which this ratio is different. In the neamine-ribostamycin pair, for instance, the C1409G mutation (and to a lesser extent G1491A or G1491C) confers higher relative resistance to ribostamycin than to neamine.
To investigate the ring I-G1491 interaction isolated from other interactions by additional rings, e.g., between 5"-OH of ring III and N-7 of G1491, we chose a reductionist approach, i.e., we determined MICs to neamine in a series of mutants with alterations in the C1409-G1491 base pair. These studies revealed that transitions affect drug binding weakly; transversions have a small albeit significant effect. We interpret these findings as evidence that the ring I-G1491 stacking interaction is largely sequence independent. Within the 6'-NH3+ subclass, ribostamycin is most affected by alterations of the C1409-G1491 base pair. This finding is rationalized by the X-ray structural data available for paromomycin (2, 27). In contrast to ribostamycin, with its ring III-G1491 interaction, neamine interacts with G1491 merely by non-sequence-specific stacking interaction with its ring I. Compared to that of ribostamycin, ring IV of neomycin apparently is able to compensate for the loss of sequence-specific interaction between ring III and G1491 and/or introduction of steric clashes as mediated by mutagenesis of C1409-G1491.
A thorough interpretation of the MIC measurements obtained upon mutagenesis of the U1406 · U1495 wobble base pair is in part limited by the low susceptibility of wild-type ribosomes to neamine. Most of the mutations, i.e., U1406C, U1406A, and U1495C, only moderately affect ribosomal susceptibility to the 6'-NH3+ aminoglycosides, if at all. The U1495A alteration confers significant resistance to ribostamycin (relative resistance, >128 μg/ml) but only moderately affects susceptibility to neomycin (relative resistance, 16 μg/ml).
The loss of ribosomal susceptibility to neamine and ribostamycin in the U1495A mutant can be rationalized by the loss of interaction between the 1-amino group of ring II and the O-4 of U1495 in the U1495A mutant and by less favorable binding of ring II. Apparently, ring IV of neomycin is able to partly compensate for this interaction. The U1406C · U1495A alteration confers high-level drug resistance to all 4,5-aminoglycosides except neomycin, which is only moderately affected. As discussed previously (22), the U1406C · U1495A alteration will result in a major change in base pair geometry precluding an appropriate positioning of ring II near the 1406-1495 pair and consequently poor insertion of ring I in the drug binding pocket.
Alteration of U1495A to G in the U1406C · U1495A double mutant replaces the natural U1406 · U1495 wobble base pair with a U1406C-U1495G Watson-Crick base pair and restores ribosomal susceptibility to all 4,5-aminoglycosides of the 6'-NH3+ and the 6'-OH subclasses investigated. This observation contrasts with the U1406-U1495A alteration, which confers significant resistance to most 4,5-aminoglycosides. Presumably, the U1406C-U1495G alteration has a conformation and geometry similar to that of the U1406-U1495A alteration. How then to explain this difference in drug susceptibility?
One rationale would refer to the different net charge provided by the functional group at position C-6 of U1495A (amino group, positive charge) and U1495G (keto group, negative charge). The keto group at C-6 of 1495G, together with the N-7 of 1495G, could possibly interact with the ring II N-1 ammonium group in a fashion similar to that of O-4 of the pyrimidine U1495 or, more generally, be involved in a network of electrostatic interactions between the positively charged aminoglycosides and their target RNAs (10, 11). The Hoogsteen sites of guanines are often involved in binding positive ions such as potassium ions (1).
Figure 5 shows the superposition of a 1406C-1495G base pair on the U1406 · U1495 pair. The distances between a typical aminoglycoside (N-1 and O-6 of ring II) and the Hoogsteen sites of 1495G are 3.0 ? (O-6) and 4.2 ? (N-7), respectively. It can also be seen how the amino group N-4 of 1406C may replace the observed water molecule between U1406 and the aminoglycoside such that a contact between N-4 of 1406C and O-6 of ring II can be made. Interestingly, mutants in which a single uracil is replaced with an adenine display disparate effects. Thus, the U1406-U1495A mutation leads to resistance to aminoglycosides, while the U1406A-U1495 mutant shows susceptibility to the neomycin class of aminoglycosides but less susceptibility to the kanamycin class (20).
Our data indicate that binding of 4,5-disubstituted aminoglycosides to the ribosomal A site depends on proper interaction of ring I with nucleotides A1408 and G1491 and is further stabilized by additional contacts, e.g., with U1495. Taken together, these data on site-directed mutagenesis of the drug target site provide a detailed analysis of the importance of the different binding contacts in the drug-ribosome interaction.
ACKNOWLEDGMENTS
This study was supported by grants from the Swiss National Science Foundation to E.C.B. (3200-BO-100780, NRP 49). B.F. is supported by a grant from the European Commission (QLRT-2001-000892); E.W. acknowledges support from the Institut Universitaire de France.
We thank T. Janui for expert technical assistance, A. Vasella for helpful discussions, and A. Makovec for typing the manuscript.
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