Interactions of amikacin with the RNA model of the ribosomal A-site: computational, spectroscopic and calorimetric studies.
Amikacin is a 2-deoxystreptamine aminoglycoside antibiotic possessing a unique l-HABA (l-(-)-γ-amino-α-hydroxybutyric acid) group and applied in the treatment of hospital-acquired infections. Amikacin influences bacterial translation by binding to the decoding region of the small ribosomal subunit that overlaps with the binding site of aminoacylated-tRNA (A-site). Here, we have characterized thermodynamics of interactions of amikacin with a 27-mer RNA oligonucleotide mimicking the aminoglycoside binding site in the bacterial ribosome. We applied isothermal titration and differential scanning calorimetries, circular dichroism and thermal denaturation experiments, as well as computer simulations. Thermal denaturation studies have shown that amikacin affects only slightly the melting temperatures of the A-site mimicking RNA model suggesting a moderate stabilization of RNA by amikacin. Isothermal titration calorimetry gives the equilibrium dissociation constants for the binding reaction between amikacin and the A-site oligonucleotide in the micromolar range with a favorable enthalpic contribution. However, for amikacin we observe a positive entropic contribution to binding, contrary to other aminoglycosides, paromomycin and ribostamycin. Circular dichroism spectra suggest that the observed increase in entropy is not caused by structural changes of RNA because amikacin binding does not destabilize the helicity of the RNA model. To investigate the origins of this positive entropy change we performed all-atom molecular dynamics simulations in explicit solvent for the 27-mer RNA oligonucleotide mimicking one A-site and the crystal structure of an RNA duplex containing two A-sites. We observed that the diversity of the conformational states of the l-HABA group sampled in the simulations of the complex was larger than for the free amikacin in explicit water. Therefore, the larger flexibility of the l-HABA group in the bound form may contribute to an increase of entropy upon binding.
Centre of New Technologies, University of Warsaw, Al. Żwirki i Wigury 93, 02-089 Warsaw, Poland; Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland; First Faculty of Medicine, Department of Hematology, Oncology and Internal Diseases, Medical University of Warsaw, Al. Żwirki i Wigury 61, 02-091 Warsaw, Poland.
Marta
M
Dudek
Department of Biophysics, Faculty of Physics, University of Warsaw, Hoża 69, 00-681 Warsaw, Poland; Interdisciplinary Centre for Mathematical and Computational Modelling, University of Warsaw, Pawińskiego 5a, 02-106 Warsaw, Poland.
Julia
J
Romanowska
Centre of New Technologies, University of Warsaw, Al. Żwirki i Wigury 93, 02-089 Warsaw, Poland.
Tomasz
T
Wituła
Centre of New Technologies, University of Warsaw, Al. Żwirki i Wigury 93, 02-089 Warsaw, Poland. Electronic address: joanna@cent.uw.edu.pl.
Joanna
J
Trylska
D000583:Amikacin / Q000737:chemistry* / Q000378:metabolism / Q000627:therapeutic use; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003198:Computer Simulation; D003428:Cross Infection / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D009690:Nucleic Acid Conformation / Q000187:drug effects; D009841:Oligonucleotides / Q000737:chemistry; D012313:RNA / Q000378:metabolism*; D054679:Ribosome Subunits, Small / Q000737:chemistry / Q000187:drug effects; D013057:Spectrum Analysis; D013816:Thermodynamics
D009841:Oligonucleotides; D012313:RNA; D000583:Amikacin
Amikacin; Isothermal titration calorimetry; Melting temperatures; Molecular dynamics simulations; Ribosomal RNA A-site