Binding of neomycin-class aminoglycoside antibiotics to mutant ribosomes with alterations in the A site of 16S rRNA

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 DeltarrnB 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.

FIG. 1.

A: Superposition of neamine (gray ball and sticks) and ribostamycin (orange) molecules docked in the minimal A site of the ribostamycin crystal structure (orange). B: Superposition of the lividomycin A (magenta ball and sticks) and paromomycin (cyan ball and sticks) molecules docked in the minimal A site of the paromomycin crystal structure (cyan). C: Superposition of the neomycin (yellow ball and sticks) and paromomycin (cyan ball and sticks) molecules docked in the minimal A site of the paromomycin crystal structure (cyan). Data on crystal structures are from references and .

FIG. 2.

Secondary structure of the bacterial A site (E. coli numbering). The introduced mutations are indicated by the arrows, and the highly conserved A site is boxed. Reprinted from reference .

FIG. 3.

Resistance profile of 4,5-disubstituted 2-deoxystreptamines with a 6′-NH₃⁺ group (neomycin subclass) represented as comparison graphs. Each axis of the comparison graph represents the influence of the corresponding introduced mutation (highlighted in bold). The relative resistances conferred by each of the mutations are plotted in log scale, with the smallest value in the innermost circle. Points lying on the outermost circle indicate that the corresponding mutations result in a relative resistance outside the measurable range. Neamine and ribostamycin bind weakly to wild-type ribosomes, and thus, quantitative analysis is limited. To reflect this limitation the values outside the measurable range are depicted by a hatched area.

FIG. 4.

Resistance profile of 4,5-disubstituted 2-deoxystreptamines with a 6′-OH group (paromomycin subclass) represented as comparison graphs. Each axis of the comparison graph represents the influence of the corresponding introduced mutation (highlighted in bold). The relative resistances conferred by each of the mutations are plotted in log scale, with the smallest value in the innermost circle. Points lying on the outermost circle indicate that the corresponding mutations result in a relative resistance outside the measurable range.

FIG. 5.

Manual superposition of a 1495G-1406C interaction on a U1495 · U1406 base pair in the crystal structure of paromomycin (29). The N-1 and O-6 atoms of ring II which contact positions 1495 and 1406 are indicated, as well as the N-7 and O-6 of G1495 and N-4 of C1406. Shown in gray is U1495 · U1406 with water molecules bound.