Crystal structure of paromomycin docked into the eubacterial ribosomal decoding a site

Background: Aminoglycoside antibiotics interfere with translation in both gram-positive and gram-negative bacteria by binding to the tRNA decoding A site of the 16S ribosomal RNA. Results: Crystals of complexes between oligoribonucleotides incorporating the sequence of the ribosomal A site of Escherichia coli and the aminoglycoside paromomycin have been solved at 2.5 Angstrom resolution. Each RNA fragment contains two A sites inserted between Watson-Crick pairs. The paromomycin molecules interact in an enlarged deep groove created by two bulging and one unpaired adenines. In both sites, hydroxyl and ammonium side chains of the antibiotic form 13 direct hydrogen bonds to bases and backbone atoms of the A site. In the best-defined site, 8 water molecules mediate 12 other hydrogen bonds between the RNA and the antibiotics. Ring I of paromomycin stacks over base G(1491) and forms pseudo-Watson-Crick contacts with A(1408). Both the hydroxyl group and one ammonium group of ring il form direct and water-mediated hydrogen bonds to the U(1495)oU(1406) pair. The bulging conformation of the two adenines A(1492) and A(1493) is stabilized by hydrogen bonds between phosphate oxygens and atoms of rings I and II. The hydrophilic sites of the bulging A(1492) and A(1493) contact the shallow groove of G=C pairs in a symmetrical complex. Conclusions: Water molecules participate in the binding specificity by exploiting the antibiotic hydration shell and the typical RNA water hydration patterns. The observed contacts rationalize the protection, mutation, and resistance data. The crystal packing mimics the intermolecular contacts induced by aminoglycoside binding in the ribosome.