Structural basis for ribosomal MLSBK antibiotics resistance using mutated large ribosomal subunits

Site-directed mutagenesis has been done in Haloarcula marismortui using the strategy that Khorana and coworkers devised for deleting the bacteriorhodopsin gene from Halobacterium halobium (M. P. Krebs, R. Mollaaghababa, and H. G. Khorana, Proc. Natl. Acad. Sci. U.S.A. 90:1987--91, 1993). Experiments exploiting their approach have revealed that the chromosome of wild type H. marismortui contains three ribosomal RNA operons (rrnA, rrnB, and rrnC) not the two originally reported. A strain of H. marismortui has been prepared that lacks the rrnB operon, and another from which both the rrnB and rrnC operons have been deleted. In rich media, both mutant strains grow at about the rate as wild type H. marismortui. G2099 in 23S rRNA gene of the single operon strain has been changed to A, and a deletion mutation has been introduced into the gene for ribosomal protein L22 of the wild-type organism. Crystal structures of H. marismortui large ribosomal subunits containing the mutation G2099A (A2058 in E. coli) complexed with the antibiotics erythromycin, azithromycin, clindamycin, virginiamycin S and telithromycin explain the structural basis of the resistance of eubacterial ribosomes containing the A2058G mutation to these antibiotics. Azithromycin binds almost identically to G2099A mutant and wild type subunits, and the erythromycin affinity increases more than 104 fold to G2099A mutants, implying that desolvation of the N2 of G2099 accounts for their low affinity for macrolides. All macrolides bind similarly to the H. marismortui subunit but differ significantly from the complex structures proposed for the D. radioidurans subunit. The synergy in the binding of streptogramins A and B appears to result from a reorientation of the base of A2103 (A2062, E. coli) that is seen to stack between them. The structure of large subunit containing a three residue deletion mutant of L22 shows a change in the L22 structure and tunnel shape that sheds light on mechanism of its macrolide resistance phenotype.