Sites Of Rnaaminoglycoside Interactions

Aminoglycoside antibiotics, as discussed in the preceding section, interact with RNA. Ribosome is the target for this class [10-12,148], and the antibiotics exert their antibacterial effects by binding to ribosomal RNA (rRNA) in the A site, causing misreading of the genetic code and inhibiting the process of translocation [149,150] (see above). Aminoglycoside antibiotics also interact with a variety of other biologically relevant RNA molecules. These include mRNA from human immunodeficiency virus (HIV-1), transactivation response element (TAR) [151], group I self-splicing introns [152], the hammerhead region of rRNA [153,154,155], and the hepatitis delta virus (HDV) ribozyme [156]. Next, we discuss recent developments in the binding of aminoglycoside antibiotics to RNA.

A. Binding of the Neomycin Class of Aminoglycosides to the A Site of 16S rRNA

Aminoglycoside antibiotics exert their antibacterial activity by interfering with protein biosynthesis. The common binding site for the structurally related aminoglyco-sides neamine (4), ribostamycin (28), paromomycin (25), and neomycin B (23) is localized in the A site of the prokaryotic 165 ribosomal RNA (rRNA) in the 305 subunit (Fig. 1a) [13-16,157]. These 2-deoxystreptamine-containing aminoglycoside antibiotics show a characteristic miscoding pattern that is distinct from that of streptomycin 73 [158], having a streptidine moiety in place of the 2-deoxystreptamine in its structure. They affect the codon-anticodon interaction in the A site of the 305 subunit by binding to the same site on rRNA, causing codon misreading and inhibiting translocation [149,150]. A specific interaction between the tRNA anticodons and mRNA codons is required for an accurate translation in the decoding region, which is formed by two short conserved sequences near the 3' end of 165 rRNA (the nucleotide 1400-1500 region, Fig. 1b).

Figure 1 (a) Secondary structure of the 165 rRNA from E. coli. (b) The sequence of the decoding region [arrow in (a)]. (c) The sequence of a 27-nucleotide RNA used to characterize aminoglycoside-RNA interaction, with the highly conserved A-site portion of the 165 boxed. (d) Structures of aminoglycoside antibiotics.

Figure 1 (a) Secondary structure of the 165 rRNA from E. coli. (b) The sequence of the decoding region [arrow in (a)]. (c) The sequence of a 27-nucleotide RNA used to characterize aminoglycoside-RNA interaction, with the highly conserved A-site portion of the 165 boxed. (d) Structures of aminoglycoside antibiotics.

To investigate the interactions between paromomycin and rRNA, Puglisi and colleagues [15] designed a small RNA fragment that mimics the structure of the A site of E. coli 165 rRNA. From these investigations, it appears that the universally conserved C1407 • G1494 base pair, A1408, and A1493 are required for the binding of pa-romomycin to the wild-type rRNA. An asymmetrical internal loop produced by a universally conserved adenine residue at position 1492 must also be present. In addition, base pairing is required in the lower stem for aminoglycoside binding.

Positions 1406 and 1495 are universally conserved uridines that form a U • U pair in the ribosome. Paromomycin typically binds to the U1406 • U1495 base pair. It binds to two binding sites, the A1406 • U1495 and U1406 • G1495 base pairs, but binds only weakly to the U1406 • A1495 base pair variant. Quantitative footprinting of oligonucleotide variants identified critical nucleotides for binding of paromomycin to the A-site oligonucleotide. These include the C1407 • G1494 base pair, the A • U base pair at positions 1410 and 1490, and nucleotides A1408, A1493, and U1495.

Puglisi and coworkers further characterized the structure and binding of paromomycin to the A-site rRNA by using NMR spectroscopy to determine the solution structure of the RNA-paromomycin complex (Fig. 2) [142]. Some salient features of this structure are as follows.

1. Paromomycin binds in the major groove of the A-site rRNA, within the internal loop (see Fig. 1). Distortion of the RNA backbone by the presence of the bulged nucleotide A1492 and noncanonical A1408 • A1493 base pair leads to the formation of a distinct binding pocket for paromomycin.

2. Rings I and II of paromomycin adopt chair conformations, and their equatorial amino and hydroxy moieties make specific contacts with RNA, stabilizing the RNA-antibiotic complex.

Figure 2 The RNA-paromomycin NMR structure. Paromomycin is shown by the capped-stick presentation. A portion of the RNA structure at the aminoglycoside binding site is shown as a surface.

3. The two amino groups at positions 1 and 3 of the 2-deoxystreptamine moiety (ring II), found in all aminoglycosides that bind to the A site, make hydrogen bonds to U1495 and G1494, respectively, and are essential for specific binding of these antibiotics to the rRNA.

4. Ring I stacks above the base moiety of G1491 such that its hydroxyl groups at positions 3' and 4' are directed toward the phosphate moieties of A1493 and A1492, respectively, but they are not essential for antibiotic function.

5. Rings III and IV weakly contribute to specific binding, but the amino and hydroxyl groups of ring IV may have weak electrostatic interaction with the phosphate backbone of U1406, C1407, and U1490.

6. The A1408 • A1493 pair is essential for antibiotic binding in prokaryotic ri-bosomes and leads to formation of the specific binding pocket for ring I, whereas in the case of eukaryotic ribosomes having G1408 in place of A1408, a base pair of equivalent geometry cannot be formed, and paromomycin binds weakly to the G1408 • A1493 base pair.

This RNA-paromomycin structure also explains a number of features that help show why modification of the aminoglycoside antibiotic by the resistance enzymes prevents the modified drug from binding to the ribosomal site. These features are as follows.

1. Disruption of the antibiotic binding pocket, formed by a base pair at positions 1409 and 1491, leads to aminoglycoside resistance in prokaryotes.

2. Enzymatic N1-methylation of A1408 is known to impair binding of the antibiotic to rRNA [128], a base that is a hydrogen bond acceptor in the A1408 • A1493 base pair. This modification gives resistance to kanamycins by disrupting the critical hydrogen bonding interactions between these two bases, which are required for ami-noglycoside binding.

3. Enzymatic modification by acetylation of the amino group at position 3 of the 2-deoxystreptamine moiety would disrupt the essential hydrogen bonding between this amino group and G1494, as well as specific interactions with the A1493 phosphate. Any modification in ring I, which is tightly ensconced in the a1408-A1493 pocket (e.g., such as the common 3'-phosphorylation or 6'-acetylation of amino-glycoside) prevents binding to rRNA by steric hindrance or undesired electrostatic interaction.

Subsequently, the structure of the A-site region of rRNA in the absence of aminoglycoside was determined by Fourmy et al. by NMR spectroscopy [159]. Comparison of this structure with the paromomycin-rRNA complex indicated a local conformational change in the A-site RNA upon paromomycin binding. In the absence of paromomycin, the asymmetrical internal loop is closed by a Watson-Crick base pair (C1407 • G1494) and by two noncanonical base pairs (U1406 • U1495 and A1408 • A1493). The nucleic base A1492 stacks below A1493 and is intercalated between the upper and lower stems. Paromomycin binding stabilizes the conformation of A1492 and A1493, which are less well defined in the free RNA. It also changes the hydrogen bonding pattern of the A1408 • A1493 base pair such that hydrogen bonding increases from 1 in the free form to 2 in the bound form. Comparison of these free and bound conformations reveals that the two universally conserved residues, A1492 and A1493, are displaced toward the minor groove of the rRNA helix in the paromomycin-rRNA complex such that their N1-positions point into the minor groove of the A-site rRNA. These changes in the rRNA conformation, induced by the bound aminoglycoside, indicate a mechanism for the action of aminoglycoside antibiotics on translation, as elaborated shortly.

Further studies on binding of the neomycin class of aminoglycosides to the A site of 165 rRNA revealed that neomycin B (23) binds to the rRNA with affinity similar to that of paromomycin (25) in slow exchange with the free form (Fig. 1d) [160]. This suggests that the change of the hydroxyl group with the amino group at position 6' of ring I has little effect on aminoglycoside binding affinity. Other observations showed NMR chemical shift changes for ribostamycin (28) and neamine (4) similar to those collected for neomycin and paromomycin complexes, but with smaller magnitudes, indicating that rings I and II of the neomycin class of amino-glycosides are sufficient to direct their binding to a unique binding pocket on the model A-site RNA. The specificity of ribostamycin and neamine interactions with the A-site RNA also was demonstrated by footprinting on mutant oligonucleotides. The U1495 ^ A mutation resulted in significant decreases of binding affinity in pa-romomycin, ribostamycin, and neamine due to disruption of the ring II hydrogen bonding, indicating specific targeting to the A site of at least ring II of these ami-noglycoside antibiotics. In a similar study, several functionalities in the major groove of the A-site RNA, including G1405 (N7), G1491 (N7), G1494 (N7), A1408 (N7), A1493 (N7), A1408 (N1), A1492 (N1), and A1493 (N1), as well as the pro-fl phosphate oxygens of A1492 and A1493 in 165 rRNA (Fig. 1c) were identified as essential for a high-affinity paromomycin binding to the 165 rRNA from E. coli (161).

Wong and colleagues investigated the specificity of ring IV of neomycin (Fig. 1) in binding to the A-site RNA [162]. They used the A-site RNA sequence previously employed by Puglisi and coworkers [15], which mimics the A site of the prokaryotic 165 ribosomal RNA as a binding target for antibiotic, to design a series of neomycin B derivatives modified in the idose ring (Fig. 3). The sequence of RNA with U1495 mutated to A (Fig. 3) was used as a negative control for in vitro binding studies because U1495 is a critical base for specific aminoglycoside binding, and replacement of this base with A leads to complete loss of specific binding [15,16].

The binding results showed similar specificity for neamine (4) and ribostamycin (28), but threefold lower affinity was observed for 28. Addition of an uncharged idose moiety to 28 to generate 129 does not change the affinity or specificity of binding, indicating the importance of the charged amino groups of the idose ring for binding. A 40-fold higher activity for compound 130, compared to 129, and further improvement of affinity in neomycin B (23) compared to that of 130, prove that the amino groups of idose ring are necessary for specific binding in vitro and that they cannot be replaced by the amino groups attached to flexible linkers. The basis for this assertion is that the addition of an amino group to ribostamycin via a flexible linker to furnish 127 increases the affinity to some extent, but specificity remains unaffected. Addition of one more amino group to generate 128 results in diminishing returns on the affinity without affecting the specificity. However, the minimum inhibitory concentration (MIC) data of these compounds showed comparable antibacterial activity to that of neomycin B, indicating that the in vivo activity does not always correlate well with the in vitro binding results. These discrepancies reflect probable conformational difference of the ribosome in vivo.

Wong and coworkers in a recent investigation [163] studied the A-site-binding aminoglycosides, including the 4,5-disubstituted 2-deoxystreptamine derivatives (ne-omycin B, paromomycin, and ribostamycin) and the 4,6-disubstituted 2-deoxystrep-

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