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.