title: Thermodynamics of aminoglycoside-rRNA recognition: the binding of neomycin-class aminoglycosides to the A site of 16S rRNA.
abstract: We use spectroscopic and calorimetric techniques to characterize the binding of the aminoglycoside antibiotics neomycin, paromomycin, and ribostamycin to a RNA oligonucleotide that models the A-site of Escherichia coli 16S rRNA. Our results reveal the following significant features: (i) Aminoglycoside binding enhances the thermal stability of the A-site RNA duplex, with the extent of this thermal enhancement decreasing with increasing pH and/or Na(+) concentration. (ii) The RNA binding enthalpies of the aminoglycosides become more exothermic (favorable) with increasing pH, an observation consistent with binding-linked protonation of one or more drug amino groups. (iii) Isothermal titration calorimetry (ITC) studies conducted as a function of buffer reveal that aminoglycoside binding to the host RNA is linked to the uptake of protons, with the number of linked protons being dependent on pH. Specifically, increasing the pH results in a corresponding increase in the number of linked protons. (iv) ITC studies conducted at 25 and 37 degrees C reveal that aminoglycoside-RNA complexation is associated with a negative heat capacity change (Delta C(p)), the magnitude of which becomes greater with increasing pH. (v) The observed RNA binding affinities of the aminoglycosides decrease with increasing pH and/or Na(+) concentration. In addition, the thermodynamic forces underlying these RNA binding affinities also change as a function of pH. Specifically, with increasing pH, the enthalpic contribution to the observed RNA binding affinity increases, while the corresponding entropic contribution to binding decreases. (vi) The affinities of the aminoglycosides for the host RNA follow the hierarchy neomycin > paromomycin > ribostamycin. The enhanced affinity of neomycin relative to either paromomycin or ribostamycin is primarily, if not entirely, enthalpic in origin. (vii) The salt dependencies of the RNA binding affinities of neomycin and paromomycin are consistent with at least three drug NH(3)(+) groups participating in electrostatic interactions with the host RNA. In the aggregate, our results reveal the impact of specific alterations in aminoglycoside structure on the thermodynamics of binding to an A-site model RNA oligonucleotide. Such systematic comparative studies are critical first steps toward establishing the thermodynamic database required for enhancing our understanding of the molecular forces that dictate and control aminoglycoside recognition of RNA.
authors.affiliation: Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854-5635, USA.
authors.forename: Malvika
authors.initials: M
authors.lastname: Kaul
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Pilch
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D009355:Neomycin / Q000737:chemistry* / Q000378:metabolism; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000378:metabolism; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D010303:Paromomycin / Q000737:chemistry / Q000378:metabolism; D011522:Protons; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D012336:RNA, Ribosomal, 16S / Q000737:chemistry* / Q000378:metabolism; D012271:Ribostamycin / Q000737:chemistry / Q000378:metabolism; D012492:Salts / Q000737:chemistry; D012964:Sodium / Q000737:chemistry; D055672:Static Electricity; D013816:Thermodynamics
chemical_list: D000900:Anti-Bacterial Agents; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; D011522:Protons; D000644:Quaternary Ammonium Compounds; D012336:RNA, Ribosomal, 16S; D012492:Salts; D012271:Ribostamycin; D010303:Paromomycin; D012964:Sodium; D009355:Neomycin