Structural basis of topoisomerase targeting by delafloxacin

Abstract

Delafloxacin is a potent anionic fluoroquinolone approved for the treatment of respiratory infections that acts by trapping the DNA cleavage complexes of bacterial topoisomerase IV and gyrase. Its N-1-pyridinyl-, C-7-azetidinyl- and C-8-chlorine substituents confer enhanced antibiotic activity against bacteria resistant to other fluoroquinolones, but its mode of action is unclear. Here we present the X-ray crystal structures of a delafloxacin-DNA cleavage complex obtained by co-crystallization with Streptococcus pneumoniae topo IV using a graphene nucleant and solved at 2.0 and 2.4 Å resolution. The two Mg²⁺-chelated delafloxacin molecules intercalated at the DNA cleavage site are bound in an unusual conformation involving interacting out-of-plane N-1-aromatic- and C-8-chlorine- substituents. The unprecedented resolution allows comprehensive imaging of water-metal ion links integrating enzyme and DNA through drug-bound and active-site Mg²⁺ ions plus the discovery of enzyme-bound K⁺ ions. Our studies on delafloxacin action suggest that intrinsic target affinity contributes to its activity against quinolone-resistant bacteria.

Fig. 1. Chemical structures of fluoroquinolones.

Structures of the clinically important fluoroquinolones delafloxacin, levofloxacin, moxifloxacin and ciprofloxacin are shown alongside clinafloxacin and trovafloxacin, two potent investigational agents.

Fig. 2. Mechanism and structure of type IIA topoisomerases.

a Proposed catalytic cycle of a type II topoisomerase illustrated for topo IV. The transported T-segment DNA (see stage 1) is captured by closure of the N-gate formed by ATP-induced dimerisation of the ParE ATPase domains (red) which allows its presentation to the cleavage core of the enzyme (2), passage through the G-segment DNA bound at the DNA cleavage gate (3) and subsequent release through the protein C-gate (4). Hydrolysis of bound ATP (pink dots) resets the enzyme for another cycle. The breakage-reunion (ParC55) and C-terminal domain (CTD) of ParC are in blue and silver; the TOPRIM domain of ParE (ParE30) is in yellow. G-gate DNA and transported (T) segment DNA are shown in green and purple, respectively. Bound ATP is shown by pink circles (Reproduced with permission under a Creative Commons licence from Fig. 1 of Laponogov, I et al., Nucleic Acids Res, 41, 9911-9923 (2013). Complexes 1, 2 and 3 (either with or lacking the ATPase domains and CTDs as in the core complex) can be captured as a cleavage complex by fluoroquinolones. b Domain organisation for the fused ParE-ParC cleavage core of S. pneumoniae topo IV (top) shown with that of the corresponding full-length ParE and ParC subunits (bottom). The open triangle shows the location of a potassium ion binding motif. c 18-mer E-site (E18) and V-site (V18) DNA duplexes are strongly cleaved (arrows) by S. pneumoniae topo IV in the presence of quinolones generating a 4 bp overhang (underlined).

Fig. 3. Delafloxacin stabilises the cleavage complex of pneumocococal topo IV and gyrase.

a. Delafloxacin-mediated DNA cleavage is more efficient for S. pneumoniae topo IV than gyrase. Supercoiled pBR322 DNA (0.4 µg) was incubated in cleavage buffer (40 mM Tris-HCl (pH 7.5), 6 mM MgCl₂, 10 mM dithiothreitol, 200 mM potassium glutamate, 50 µg/ml bovine serum albumin) with either topo IV (reconstituted from 0.45 µg ParC and 1 µg ParE) or gyrase (0.45 µg GyrA/1 µg GyrB) in the absence or presence of delafloxacin (Dela) at the indicated concentrations. After incubation for 60 min at 37 ^oC, samples were treated with sodium dodecyl sulphate and proteinase K. Plasmid DNA products were separated by gel electrophoresis in 1% agarose run in TBE buffer. Subsequent gel staining with ethidium bromide allowed DNA visualisation and photography under UV light. Lanes C, supercoiled pBR322 DNA. S, L and N denote supercoiled, linear and nicked DNA products. b The ParE30-ParC55 fusion protein is active in DNA cleavage and targeted more efficiently by delafloxacin than levofloxacin. The protein (0.4 µg) was incubated in cleavage buffer with supercoiled pBR322 DNA (0.4 µg) in the absence or presence of delafloxacin or levofloxacin at the concentrations indicated on the figure. Induction of cleavage and analysis of DNA cleavage products was carried out as described for Fig. 3a. N, R, L and S denote nicked, relaxed, linear and supercoiled DNA, respectively. Cleavage experiments were conducted once primarily to aid structural studies (rather than provide inhibition parameters to decimal place precision). Experiments were carefully done and show that as for other quinolones, delafloxacin captures a cleavage complex markedly more efficiently (10-20-fold) with topo IV than gyrase. Studies in Fig. 3b show delafloxacin is comparably active against the topo IV fusion and holoenzyme complexes and for levofloxacin recapitulate results we previously published using the same conditions and levels of drug, fusion protein and pBR322 DNA.

Fig. 4. Orthogonal views in surface representation of the 2.0 Å X-ray crystal structure of the delafloxacin-core cleavage complex of S.pneumoniae topo IV with 18-mer V gate DNA (PDB ID: 8QMB).

The ParE TOPRIM domains are in yellow, ParC domains in blue, DNA is in red (cartoon representation) and the two drug molecules are in pink (solid sphere representation). The two surface potassium ions bound one to each ParC tower are in dark purple (solid sphere representation) highlighted by dark circles (top) and arrows (bottom left).

Fig. 5. Unusual conformation of delafloxacin in the 2.0 Å complex of S.pneumoniae topo IV with V18 DNA (PDB ID: 8QMB) featuring out-of-plane N-1 aromatic and C-8 chlorine substituents.

a, b Opposing views of the delafloxacin molecule with its chelated magnesium ion and associated waters with the electron density from the (Fo–Fc) map denoted by a mesh contoured at 1.5σ (limited to 2.3 Å range). The chelated magnesium ion is shown in purple and bound water shown in red. a, b show views perpendicular and parallel to the bicyclic quinolone ring, respectively. c Representation in full van der Waal radius of the delafloxacin molecule from the 2.0 Å topo IV X-ray crystal structure, indicating that the large electronegative Cl atom (shown in green) interacts directly to facilitate/stabilise tilting of the N1 heteroaromatic ring (dark grey).

Fig. 6. Details of the topo IV-DNA binding site for delafloxacin, one of two sites present in the 2.0 A X-ray crystal structure with the V18 gate-DNA (PDB ID: 8QMB).

The figure shows opposing views of the drug binding site. The ParC backbone is in blue, the ParE backbone is in yellow, the DNA backbone is in light blue, the drug molecule is shown in black, and magnesium ions are shown in purple. Active site tyrosine (Y118) and arginine (R117) sidechains from ParC are in orange, DNA bases and sugars are in silver. The active site magnesium-coordinating residues are in purple, and the ParC S79 and D83 residues whose mutation is associated with drug resistance are shown in red.

Fig. 7. A magnesium ion coordinates drug binding to topo IV and DNA via multiple water-metal ion links.

a, b Schematic representation of the water coordination spheres of delafloxacin chelated Mg²⁺ derived from 2.0 Å V-18 (a) and (better resolved) in 2.4 Å E18 (b) X-ray crystal structures of DNA cleavage complexes with topo IV (PDB ID: 8QMB). Important distances and likely hydrogen bonds are shown by dashed lines; only water molecules that could be individually imaged are shown (in red). c Imaging of the water molecule that forms a water-metal ion bridge between the delafloxacin and the ParC D83 sidechain (2mFo-DFc map contoured at 1.5σ) (PDB ID: 8C41).

Fig. 8. Top-down view of the DNA cleft occupied by delafloxacin in the 2.0 Å V−18 X-ray crystal structure (PDB ID: 8QMB).

The ParC backbone is in blue, the ParE backbone is in yellow, DNA backbone is in light blue, the intercalated drug molecule is in red, magnesium ions are in purple, and the active site tyrosine (Y118) and arginine residues (R117) are in orange, DNA bases and sugars are in silver, active site magnesium-coordinating residues are in purple. The positions of ParC S79 and D83 residues responsible for quinolone resistance upon mutation are indicated by arrows.

Fig. 9. Electron density and schematic diagram showing the full coordination sphere and multiple water mediated linkages of the active site magnesium in the 2.0 Å delafloxacin-core complex X-ray crystal structure (PDB ID:8QMB), with the 2Fo–Fc electron density map contoured at the 1.5σ level.

ParE backbone is in yellow, DNA backbone is in light blue, magnesium ions are in purple, ParE residue side chains are in light purple, DNA bases and sugars are in silver, coordinated water molecules in red. Metal coordination is shown by dashed red lines; likely hydrogen bonds between the coordinated water molecules and the residues are shown as dashed dark blue lines. Bottom figure: Mg2+ coordination and links directly or via water molecules to protein or DNA are shown by black dashed lines.

Fig. 10. Coordination sphere and binding motif of the newly found surface potassium ions located one to each ParC tower domain.

The figure is from the 2.0 Å V18 delafloxacin core complex X-ray crystal structure (PDB ID: 8QMB) with the 2Fo–Fc electron density map contoured at the 1.5σ level. The ParC backbone is in blue, ParC sidechains are in orange, the potassium ion is in pink and coordinated water molecules are in red. Metal coordination is shown by dashed red lines, and likely hydrogen bonds between the coordinated water molecules and protein residues are denoted by dashed dark blue lines.