Stepwise binding of tylosin and erythromycin to Escherichia coli ribosomes, characterized by kinetic and footprinting analysis.
Erythromycin and tylosin are 14- and 16-membered lactone ring macrolides, respectively. The current work shows by means of kinetic and chemical footprinting analysis that both antibiotics bind to Escherichia coli ribosomes in a two-step process. The first step established rapidly, involves a low-affinity binding site placed at the entrance of the exit tunnel in the large ribosomal subunit, where macrolides bind primarily through their hydrophobic portions. Subsequently, slow conformational changes mediated by the antibiotic hydrophilic portion push the drugs deeper into the tunnel, in a high-affinity site. Compared with erythromycin, tylosin shifts to the high-affinity site more rapidly, due to the interaction of the mycinose sugar of the drug with the loop of H35 in domain II of 23 S rRNA. Consistently, mutations of nucleosides U2609 and U754 implicated in the high-affinity site reduce the shift of tylosin to this site and destabilize, respectively, the final drug-ribosome complex. The weak interaction between tylosin and the ribosome is Mg2+ independent, unlike the tight binding. In contrast, both interactions between erythromycin and the ribosome are reduced by increasing concentrations of Mg2+ ions. Polyamines attenuate erythromycin affinity for the ribosome at both sequential steps of binding. In contrast, polyamines facilitate the initial binding of tylosin, but exert a detrimental, more pronounced, effect on the drug accommodation at its final position. Our results emphasize the role of the particular interactions that side chains of tylosin and erythromycin establish with 23 S rRNA, which govern the exact binding process of each drug and its response to the ionic environment.
Petropoulos
Alexandros D
AD
Laboratory of Biochemistry, School of Medicine, University of Patras, 26500 Patras, Greece.
Kouvela
Ekaterini C
EC
Dinos
George P
GP
Kalpaxis
Dimitrios L
DL
D000900:Anti-Bacterial Agents / Q000737:chemistry*; D004917:Erythromycin / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D009154:Mutation; D011073:Polyamines / Q000737:chemistry; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000737:chemistry* / Q000235:genetics; D012270:Ribosomes / Q000737:chemistry* / Q000235:genetics; D015645:Tylosin / Q000737:chemistry*
D000900:Anti-Bacterial Agents; D011073:Polyamines; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D004917:Erythromycin; D008274:Magnesium; D015645:Tylosin