Binding of neomycin-class aminoglycoside antibiotics to mutant ribosomes with alterations in the A site of 16S rRNA.
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.
Institut für Medizinische Mikrobiologie, Universität Zürich, Gloriastr. 30/32, CH-8006 Zürich, Switzerland. shobbie@immv.unizh.ch
Sven N
SN
Hobbie
Peter
P
Pfister
Christian
C
Bruell
Peter
P
Sander
Boris
B
François
Eric
E
Westhof
Erik C
EC
Böttger
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001665:Binding Sites; D016297:Mutagenesis, Site-Directed; D009355:Neomycin / Q000737:chemistry / Q000378:metabolism*; D012336:RNA, Ribosomal, 16S / Q000737:chemistry*; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship
D000900:Anti-Bacterial Agents; D012336:RNA, Ribosomal, 16S; D009355:Neomycin