Photon Factory Activity Report 2014 #32 (2015) B

BL-5A/2012G519

Crystal structure of the bacterial A site in complex with β-fluoro neomycin analog
Hiroki Kanazawa1, Juan Pablo Maianti2, Stephen Hanessian2 and Jiro Kondo1,*1
Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University,
7-1 Kioi-cho, Chiyoda-ku, Tokyo 102-8554, Japan, 2Department of Chemistry, Université deMontréal, CP 6128 Succ. Centre-Ville, Montréal Québec, H3C3J7, Canada

1 Introduction
Aminoglycosides (AGs) comprise a diverse group ofpotent broad-spectrum natural and semi-synthetic
bacterial antibiotics used in clinical practice worldwide.
Kidney toxicity has known as side effect of AGs andcorrelates with the number of protonated and nonprotonated amino groups on the AG sugar scaffolds, yetthese features are also a requirement for potent
antibacterial action. Before the present study, we havedesigned and synthesized some fluorinated novel AGs.
Compound JPM5177 (Fig. 1), one of these AGs, hassmaller kidney toxicity through decreasing the pKa ofamino groups caused by fluorination. And surprisingly,
the bactericidal effects of JPM5177 against several
bacteria are remarkably increased. In the present study,
we have performed X-ray analysis of the bacterial rRNA
molecular switch in complex with the JPM5177 to reveal
a role of the fluorine in the AG binding [1].

switch in the “on” state through three hydrogen bonds.
These interactions are common among AG series and arerequired when they bind to the A-site molecular switch.
The hydroxy-aminobutyric acyl group (HABA) observedin the RNA/JPM5177 and RNA/amikacin complexes [2]are shown in Fig. 3. The unmodified HABA of amikacinmakes three hydrogen bonds with RNA. On the otherhand, the HABA with a fluorine atom of JPM5177 makes
two additional hydrogen bonds. Although the original
purpose is controlling the pKa of amino groups by thefluorination, we found that the fluorine itself contributes
to the direct interactions with the RNA molecule.

Fig. 2: Binding (a) and detailed interactions (b) ofJPM5177 with the bacterial A site.

Fig. 1: Chemical structure of JPM5177
2 Experiment
The RNA oligomer designed to fold as a double helix
containing two asymmetrical internal loops of thebacterial A site was chemically synthesized.
Crystallizations of the RNA/JPM5177 complex wereperformed by the hanging-drop vapor diffusion method.
X-ray data were collected at 100K with synchrotron
radiation at the structural biology beamline BL-5A in thePhoton Factory. Initial phase of the complex crystal wasderived with the molecular replacement program AutoMR
from the Phenix suite. The atomic parameters of thecrystal structure was refined using the program CNS.
3 Results and Discussion
The bacterial A-site molecular switch forms an A-form
duplex and takes the “on” state with two bulged-out
adenines (Fig. 2a). JPM5177 binds to the A-site switch.
Ring A of JPM5177 stacks over G1491 and forms a
pseudo-pair with A1408. Ring B fixes the molecularFig. 3: Interactions between the HABA groups of (a)JPM5177 and (b) amikacin [2] and the bacterial A-site
molecular switch.
References[1] J. P. Maianti et al., ACS Chem. Biol. 9, 2067-2073(2014)[2] J. Kondo et al., Biochimie. 88, 1027-1031 (2006)* j.kondo@sophia.ac.jp