Crystal structure of the bacterial ribosomal decoding site complexed with a synthetic doubly functionalized paromomycin derivative: a new specific binding mode to an a-minor motif enhances in vitro antibacterial activity

The crystal structure of the complex between oligonucleotide containing the bacterial ribosomal decoding site (A site) and the synthetic paromomycin analogue 1, which contains the gamma-amino-alpha-hydroxybutyryl (L-haba) group at position N1 of ring II (2-DOS ring), and an ether chain with an O-phenethylaminoethyl group at position C2' of ring III, is reported. Interestingly, next to the paromomycin analogue 1 specifically bound to the A site, a second molecule of 1 with a different conformation is observed at the crystal packing interface which mimics the A-minor interaction between two bulged-out adenines from the A site and the codon-anticodon stem of the mRNA-tRNA complex. Improved antibacterial activity supports the conclusion that analogue 1 might affect protein synthesis on the ribosome in two different ways: 1) specific binding to the A site forces maintenance of the "on" state with two bulged out adenines, and 2) a new binding mode of 1 to an A-minor motif which stabilizes complex formation between the ribosome and the mRNA-tRNA complex regardless of whether the codon-anticodon stem is of the cognate or near-cognate type.