Photon Factory Activity Report 2014 #32 (2015) B

BL-17A/2012G519

Crystal structure of the bacterial A site in complex withaxial 4’-fluoro neomycin analog
Hiroki Kanazawa1, 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.
Recent years, one of the problems in clinical practice isresistances against AGs. One of the mechanisms is
modification of AG by AG-modifying enzymes such as
O-phosphotransferases (APHs), which phosphorylates theoxygen of AG and inactivates AG. The phosphorylation
depends on the nucleophilicity of the oxygen atoms.
Therefore, the phosphorylation can be prevented byreducing the nucleophilicity by introducing the fluorine
nearby. Before the present study, we have designed andsynthesized some fluorinated novel AGs. Compound
OS174 (Fig. 1), one of these AGs, is an axially 4’fluorinated aminoglycoside. A minimum inhibitory
concentration of OS174 against P. aeruginosa APH(3’)
indicates that OS174 is not inactivated. And surprisingly,
the bactericidal effects of OS174 against several bacteria
are remarkably increased. In the present study, we have
performed X-ray analysis of the bacterial A-site
molecular switch in complex with the OS174 to reveal arole of the fluorine in the AG binding [1].

3 Results and Discussion
OS174 specifically binds to the A-site switch andforces the two adenine residues to bulge out (Fig. 2a).
Interactions between the AGs and the A-site are highly
conserved in the Bact/OS174 complex. The axial 4’fluorine (F4’) of OS174 is accommodated by a fluorinearyl interaction with G1491 (Fig. 3a). The F4’ points
toward and is in close contact with C2, N3 and C4 in
G1491. The distances from F4’ to these atoms are quite
short compared to the sums of the van der Waals radii,
and are similar to the distances from the H4’ to the C2,N3 and C4 observed for the natural AG compounds[2](Fig. 3b), suggesting that there could be non-negligible
forces between these atoms. Since the F atom is a neutralelectron-rich atom, it can make interactions with electronpoor atoms. Nucleic acid bases have electron-depleted clouds. Therefore, it is reasonable that the axial F4’ inOS174 can form a strong lone pair- interaction withG1491.

Fig. 2: Binding (a) and detailed interactions (b) of OS174
with the bacterial A site.
Fig. 1: Chemical structure of OS174
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/OS174 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-17A 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.

Fig. 3: Stacking interactions between ring A of (a) OS174and (b) neomycin B [2] and G1491.
References[1] S. Hanessian et al., Chem. Sci. 5, 4621-4632 (2014)[2] B. Francois et al., Nuceic Acids Res. 33, 5677-5690(2005)* j.kondo@sophia.ac.jp