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Antimicrobial Agents and Chemotherapy, April 2006, p. 1489-1496, Vol. 50, No. 4
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.4.1489-1496.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Binding of Neomycin-Class Aminoglycoside Antibiotics to Mutant Ribosomes with Alterations in the A Site of 16S rRNA

Institut für Medizinische Mikrobiologie, Universität Zürich, CH-8006 Zürich, Switzerland,¹ 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, France²

Received 21 September 2005/ Returned for modification 15 November 2005/ Accepted 17 January 2006

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.

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