Molecular mechanism of topoisomerase poisoning by the peptide antibiotic albicidin

Abstract

The peptide antibiotic albicidin is a DNA topoisomerase inhibitor with low-nanomolar bactericidal activity towards fluoroquinolone-resistant Gram-negative pathogens. However, its mode of action is poorly understood. We determined a 2.6 Å resolution cryoelectron microscopy structure of a ternary complex between Escherichia coli topoisomerase DNA gyrase, a 217 bp double-stranded DNA fragment and albicidin. Albicidin employs a dual binding mechanism where one end of the molecule obstructs the crucial gyrase dimer interface, while the other intercalates between the fragments of cleaved DNA substrate. Thus, albicidin efficiently locks DNA gyrase, preventing it from religating DNA and completing its catalytic cycle. Two additional structures of this trapped state were determined using synthetic albicidin analogues that demonstrate improved solubility, and activity against a range of gyrase variants and E. coli topoisomerase IV. The extraordinary promiscuity of the DNA-intercalating region of albicidins and their excellent performance against fluoroquinolone-resistant bacteria holds great promise for the development of last-resort antibiotics.

Keywords: Antibiotics; Enzyme mechanisms; Mechanism of action; Structure-based drug design; Target validation.

Fig. 1. Structure of Gyr–Mu217–albicidin.

a, Scheme of GyrA and GyrB domains. The DNA cleavage–reunion complex is shown, along with the catalytic residue (Tyr122) and residues involved in quinolone resistance (GyrB Lys447 and Asp426 and GyrA Ser83 and Asp87). A consistent colour code is used throughout the manuscript: beige, GyrA; coral, GyrB. b, Chemical structure of albicidin. c, An overview of the Gyr–Mu217–albicidin cryo-EM map depicted as an overlay of two different contour level maps. Low-resolution contour (white) illustrates the position of GyrA CTDs and GyrB ATPase domains. High-resolution core part, including albicidin (in the zoomed-in image), is coloured according to the scheme in a: coral, GyrB; beige, GyrA; teal, DNA; yellow, albicidin. d, Cartoon representation of the overall model.

Fig. 2. Albicidin-binding pocket.

a, Enlarged view of the albicidin-binding site in the Gyr–Mu217–albicidin structure. Gyrase is represented as a cartoon, and albicidin as a stick representation. Van der Waals radii for albicidin atoms are shown as transparent yellow spheres. Two opposing GyrA helices α3 and α3′ at the dimer interface (DNA gate) form one part of the binding pocket, while DNA bases form another part. Distances (Å) between the modelled metal ion water shell and GyrB Glu744′ to Cya3 of albicidin are indicated. b, Schematic of albicidin binding in the context of the Mu cleavage site. Two potential binding pockets next to the scission sites ‘TG’ and ‘AA’ are labelled by the red arrows. Albicidin position is depicted by the sticks model (yellow), with the grey image indicating the potential alternative orientation not observed in the Gyr–Mu217–albicidin data. Two metal-binding sites, A and B, are indicated as half-circles. c, A LigPlot two-dimensional diagram of the albicidin-binding site. Hydrogen bonds and lengths (<4 Å) are indicated with dashed lines and the non-bonding and hydrophobic interactions (<4 Å) are labelled by the red and green spiked arcs, respectively. W, water coordinated to the metal (presumed Mg²⁺) ion.

Fig. 3. Binding of albicidin derivatives.

a, Chemical structure of parent albicidin, Albi-1, Albi-2 and Albi-3. Modifications in the N-terminal, central or C-terminal region of the molecule are highlighted in violet, orange or lime, respectively. b, An overlay of Albi-1 (brick, blue or grey sticks) bound in three positions (TG, AA and XT) found in the cryo-EM density. c, Comparison of albicidin (gold), Albi-1 (blue) and Albi-2 (pink) binding in the main (TG) binding pocket. Arabic numerals indicate the peptide residues numbers (Extended Data Fig. 7). To create the figure, GyrA subunits were aligned to the main Gyr–Mu217–albicidin model in ChimeraX and bound ligands shown in stick representation. Interacting residues of GyrA and GyrB are labelled, as is the distance (Å) between the triazole and the water shell of the Mg²⁺ ion.

Fig. 4. Effects of GyrA and GyrB mutations on susceptibility to albicidins.

a, CC₅₀ values for albicidin, Albi-1, Albi-2 and Albi-3 determined for WT E. coli gyrase and selected mutants. The mutations that notably increased resistance to the compound are shown in red. b–d, Mutated residues of GyrA (beige) and GyrB (coral), and albicidin (b, gold), Albi-1 (c, blue) and Albi-2 (d, pink) in stick representation. Main interactions are shown with the corresponding distances in Å. e, Plots used for CC₅₀ determination. Data plotted are means of triplicate measurements; error bars represent s.d. ND, not determined. Source data

Fig. 5. Mechanistic model of gyrase inhibition by albicidin.

a, Initial state: an apo-gyrase complex. b, G-segment DNA fragment is engaged and bound. c, T-segment DNA fragment is captured by the ATPase domains of GyrB and the DNA is cleaved. d, The T-segment is transported through the enzyme, stimulated by the ATP binding (blue hexagons), causing dimerization of the ATPase domains. e, The T-segment is transported to the bottom chamber of the enzyme and the DNA has to be religated to release the T-segment. Albicidin intercalates in DNA in outer-rotated conformation (XT state). f, Albicidin rotates and occupies the binding pocket between two GyrA monomers. g, Albicidin effectively jams the enzyme movement, blocking the escape into any productive state. Inset: an enlarged scheme of albicidin–DNA–gyrase interaction in the locked state. h, Scheme of albicidin molecule, consisting of two rigid fragments (N- and C-terminal), connected by a flexible hinge residue, While in our structure the compound has L shape (‘L’), it adopts a more open wide-angle V shape (‘V’) in complex with the AlbA resistance protein (PDB: 6ET8).

Extended Data Fig. 1. Length dependency of albicidin-mediated DNA cleavage.

Position of cleaved DNA indicated by arrows and red dots. ATP and ADPNP were added as indicated, (----) stands for no nucleotide added. 20 nM gyrase, 1 µM albicidin and 25 nM DNA was used in each reaction.

Extended Data Fig. 2. Cryo-EM data processing for Gyr-Mu217-albicidin.

a. A representative micrograph with example gyrase particles indicated. b. A selection of 2D classes, box size in angstroms indicated. c. FSC curve for the highest-resolution reconstruction as outputted by cryoSPARC. d. Euler angle distribution as output by cryoSPARC. e. Processing scheme (see Methods for description). f. Local resolution maps contoured at two different levels (5σ; 15σ) to illustrate resolution distribution from >2.5 Å next to the DNA and compound to <4 Å at the ends of the TOPRIM insert. g. Map-to-model curve.

Extended Data Fig. 3. Coulomb potential density maps for albicidin and derivatives Albi-1 and Albi-2.

Residue numbers are indicated. All maps are contoured at 11 σ level.

Extended Data Fig. 4. Comparison of albicidin, FQ and gepotidacin binding sites.

Gyr-Mu217-albicidin structure is shown as cartoon representation with albicidin shown as yellow sticks. GyrA is shown in beige, DNA in teal and GyrB in coral. Catalytic Tyr122 and parts of GyrB are not shown for clarity. Albicidin residues, GyrA/GyrA’ interface (α3/α3’) and DNA bases next to the break (+1/-1) are indicated. To compare binding sites of different compounds, E. coli gyrase-gepotidacin cryo-EM structure (PDB:6RKS) and moxifloxacin (MFX) - Staphylococcus aureus crystal structure (PDB:5CDQ) were superimposed onto Gyr-Mu217-albicidin and aligned to the GyrA’ protomer using match maker in ChimeraX. Two MFX molecules (MFX, MFX’) are shown as orange stick representations. A single bound gepotidacin molecule is shown as magenta sticks, with DNA-binding left-hand-side (LHS) and hydrophobic pocket binding right-hand-side (RHS) labelled. Note the overlap between the gepotidacin RHS and the methoxy group of pMBA5. MFX is bound via a so called ‘water–metal ion bridge’ to Ser83 and Asp87. The distance between the Asp87 and MFX in one protomer versus another illustrates large-scale movement of the albicidin-bound gyrase in comparison to the moxifloxacin-bound structure.

Extended Data Fig. 5. Photocrosslinking of diazirine-labelled albicidin (photo-Albi) to the DNA.

a. Structures of N-terminal photo-Albi and control compound. b. DNA cleavage assay with Mu217 fragment and indicated concentrations of compounds. The labelled DNA band appearing upon irradiation of a photo-compound when bound to the complex is marked by a yellow star. c. Ciprofloxacin competition experiment. Pre-incubation with excess CFX prevents albicidin binding and crosslinking. Details of the experiment are available in Methods section.

Extended Data Fig. 6. Metal binding site in Gyr-Mu217-albicidin.

The water shell around the postulated Mg²⁺ ion was modelled and refined with ideal geometrical restraints (see Methods). Map density contoured at 9σ level. Distances in Å to the closest atoms are indicated.

Extended Data Fig. 7. Structures of all albicidin derivatives used in this study.

Individual residues are marked as in Fig. 1.

Extended Data Fig. 8. Inhibition of supercoiling by Albi-1/2/3.

a. Chemical structures of Albi-1, Albi-2 and Albi-3. b. Example supercoiling inhibition assay gels: Albi-1, decreasing concentrations (320, 160, 80, 40, 20, 10, 5, 2.5 nM); Albi-2 (320, 160, 80, 40, 20 nM); Albi-3 (960, 800, 640, 480, 320, 160, 80, 40 nM). First lane - relaxed pBR322; second lane - DNA gyrase. 40 nM albicidin was used as positive control (third lane). Different concentrations were tested and representative gels are shown.

Extended Data Fig. 9. Cryo-EM data processing for Gyr-Mu217-Albi-1.

a. Processing scheme (see Methods for description). b. FSC curves as output by cryoSPARC. c. Euler angle distributions as output by cryoSPARC. d. Map-to-model plots.

Extended Data Fig. 10. Cryo-EM data processing for Gyr-Mu217-Albi-2.

a. Processing scheme (see Methods for description). b. FSC curve as output by cryoSPARC. c. Euler angle distribution as output by cryoSPARC. d. Map-to-model plot.