The structural basis of macrolide-ribosome binding assessed using mutagenesis of 23S rRNA positions 2058 and 2059.
Macrolides are a diverse group of antibiotics that inhibit bacterial growth by binding within the peptide tunnel of the 50S ribosomal subunit. There is good agreement about the architecture of the macrolide site from different crystallography studies of bacterial and archaeal 50S subunits. These structures show plainly that 23S rRNA nucleotides A2058 and A2059 are located accessibly on the surface of the tunnel wall where they act as key contact sites for macrolide binding. However, the molecular details of how macrolides fit into this site remain a matter of contention. Here, we have generated an isogenic set of single and dual substitutions at A2058 and A2059 in Mycobacterium smegmatis to investigate the effects of the rRNA mutations on macrolide binding. Resistances conferred to a comprehensive array of 11 macrolide compounds are used to assess models of macrolide binding predicted from the crystal structures. The data indicate that all macrolides and their derivatives bind at the same site in the tunnel with their C5 amino sugar in a similar orientation. Our data are compatible with the lactone rings of 14-membered and 16-membered macrolides adopting different conformations, enabling the latter compounds to avoid a steric clash with 2058G. This difference, together with interactions conveyed via substituents that are specific to certain ketolide and macrolide sub-classes, influences the binding to the large ribosomal subunit. Our genetic data show no support for a derivatized-macrolide binding site that has been proposed to be located further down the tunnel.
Pfister
Peter
P
Institut für Medizinische Mikrobiologie, Universität Zürich, Gloriastrasse 30-32, CH-8028 Zürich, Switzerland.
Jenni
Simon
S
Poehlsgaard
Jacob
J
Thomas
Ashley
A
Douthwaite
Stephen
S
Ban
Nenad
N
Böttger
Erik C
EC
D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001665:Binding Sites; D003198:Computer Simulation; D024881:Drug Resistance, Bacterial; D018942:Macrolides / Q000378:metabolism*; D016296:Mutagenesis; D020102:Mycobacterium smegmatis / Q000187:drug effects* / Q000235:genetics*; D012338:RNA, Ribosomal, 23S / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D018942:Macrolides; D012338:RNA, Ribosomal, 23S