title: Minimum sequence requirements for the binding of paromomycin to the rRNA decoding site A.
abstract: We have recently introduced a computational methodology that combines molecular dynamics (MD) simulations, free-energy calculations, and in vitro binding assays to predict the minimum RNA structural requirements for selective, high-affinity RNA binding to small-molecule ligands. Here, we show that this methodology can be applied to the conformationally flexible aminoglycoside antibiotic paromomycin. A RNA consisting of an 11-mer:10-mer duplex that contains one 16S ribosome RNA decoding A-site bound to paromomycin was simulated for 4 ns. The methodology predicts that the 11-mer:10-mer duplex binds to paromomycin with high affinity, whereas smaller RNA duplexes lose complex stability and the ability to bind paromomycin. The predicted high-affinity binding to paromomycin of the 11-mer:10-mer duplex was confirmed experimentally (EC(50) = 0.28 microM), as well as the inability of smaller complexes to bind. Our simulations show good agreement with experiment for dynamic and structural properties of the isolated A-site, including hydrogen-bonding networks and RNA structural rearrangements upon ligand binding. The results suggest that MD simulations can supplement in vitro methods as a tool for predicting minimum RNA-binding motifs for both small, rigid ligands, and large, flexible ligands when structural information is available.
authors.affiliation: School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI 53705, USA.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Anderson
authors.forename: Sandro
authors.initials: S
authors.lastname: Mecozzi
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D019295:Computational Biology; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000378:metabolism; D010303:Paromomycin / Q000737:chemistry / Q000378:metabolism*; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000737:chemistry / Q000378:metabolism*; D017423:Sequence Analysis, RNA; D013816:Thermodynamics
chemical_list: D000900:Anti-Bacterial Agents; D009692:Nucleic Acid Heteroduplexes; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D010303:Paromomycin