Research paper Interactions of amikacin with the RNA model of the ribosomal A-site: Computational, spectroscopic and calorimetric studies Marta Dudek a, b, c , Julia Romanowska d, e, 1 , Tomasz Witu1a a , Joanna Trylska a, * a Centre of New Technologies, University of Warsaw, Al. _ Zwirki i Wigury 93, 02-089 Warsaw, Poland b Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawi nskiego 5a, 02-106 Warsaw, Poland c First Faculty of Medicine, Department of Hematology, Oncology and Internal Diseases, Medical University of Warsaw, Al. _ Zwirki i Wigury 61, 02-091 Warsaw, Poland d Department of Biophysics, Faculty of Physics, University of Warsaw, Ho _ za 69, 00-681 Warsaw, Poland e Interdisciplinary Centre for Mathematical and Computational Modelling, University of Warsaw, Pawi nskiego 5a, 02-106 Warsaw, Poland article info Article history: Received 23 October 2013 Accepted 20 March 2014 Available online xxx Keywords: Ribosomal RNA A-site Amikacin Molecular dynamics simulations Melting temperatures Isothermal titration calorimetry abstract Amikacin is a 2-deoxystreptamine aminoglycoside antibiotic possessing a unique L-HABA (L-(-)-g-amino- a-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 thermody- namics 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 calo- rimetries, 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 ami- noglycosides, paromomycin and ribostamycin. Circular dichroism spectra suggest that the observed in- crease 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 oligonu- cleotide 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. Ó 2014 Elsevier Masson SAS. All rights reserved. 1. Introduction The most known role of RNA is to provide intermediates for translation in the form of messenger RNA. However, a plethora of noncoding, but nevertheless functional, RNAs has been described over the recent years [1]. Understanding their dy- namics at the atomistic level is a challenge that can be faced only by combining experimental and computational studies [2]. Due to diverse roles, RNA is a promising target for inhibitors that control RNA expression or modulate its function. In particular, many antibiotics target ribosomal RNA (rRNA) and inhibit protein synthesis in bacteria [3]. One class of such anti- biotics are aminoglycosides, which have been administered clinically since 1943, and despite adverse effects are still the antibiotics of choice used in hospitals [4]. The examples of dis- eases treatable with aminoglycosides include tuberculosis, conjunctivitis and systemic infections [5]. Many aminoglycosides, which include the 2-deoxystreptamine (2-DOS) core, bind to the rRNA of the small subunit of the bacte- rial ribosome (namely 16S rRNA). Their binding site overlaps with * Corresponding author. Tel.: þ48 22 5540843; fax: þ48 22 5540801. E-mail address: joanna@cent.uw.edu.pl (J. Trylska). 1 Current address: Heidelberg Institute for Theoretical Studies gGmbH, Schloss- Wolfsbrunnenweg 35, 69118 Heidelberg, Germany. Contents lists available at ScienceDirect Biochimie journal homepage: www.elsevier.com/locate/biochi http://dx.doi.org/10.1016/j.biochi.2014.03.009 0300-9084/Ó 2014 Elsevier Masson SAS. All rights reserved. Biochimie xxx (2014) 1e15 Please cite this article in press as: M. Dudek, et al., Interactions of amikacin with the RNA model of the ribosomal A-site: Computational, spectroscopic and calorimetric studies, Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.03.009