Cryo-EM structure of the complete E. coli DNA gyrase nucleoprotein complex

Abstract

DNA gyrase is an essential enzyme involved in the homeostatic control of DNA supercoiling and the target of successful antibacterial compounds. Despite extensive studies, a detailed architecture of the full-length DNA gyrase from the model organism E. coli is still missing. Herein, we report the complete structure of the E. coli DNA gyrase nucleoprotein complex trapped by the antibiotic gepotidacin, using phase-plate single-particle cryo-electron microscopy. Our data unveil the structural and spatial organization of the functional domains, their connections and the position of the conserved GyrA-box motif. The deconvolution of two states of the DNA-binding/cleavage domain provides a better understanding of the allosteric movements of the enzyme complex. The local atomic resolution in the DNA-bound area reaching up to 3.0 Å enables the identification of the antibiotic density. Altogether, this study paves the way for the cryo-EM determination of gyrase complexes with antibiotics and opens perspectives for targeting conformational intermediates.

Structural basis for the allosteric regulation of Human Topoisomerase 2α

“…However, the hTop2α DNA binding/cleavage domain is still able to oscillate between the closed and pre-opening states in presence of etoposide, despite the fact that the G-segment base pairs remain annealed (see supplemental analysis). This conformational oscillation has also been observed in different bacterial DNA gyrase complexes bound to antibiotics 30,31 (Figure 3, Supplementary Fig. 3 and 8).…”

“…The complete architecture of the hTop2α reveals the intertwined arrangement of the two subunits, a feature that was deduced from the yeast enzyme crystal structure and recently observed in the cryo-EM models of the prokaryotic Topo II 12,30 (Figure 2d and Figure 3d-f). The dimeric ATPase domain sits in a ~95°orthogonal orientation above the DNA-gate, similar to what was previously observed with the yeast Top2 (~90° orientation) 12 (Figure 3d-f).…”

“…6). Previous crystal structures of type 2 topoisomerases isolated domains or cryo-EM reconstructions of the bacterial homolog DNA gyrase reported distinct DNA/binding domain conformation but so far without connection with the position of the ATPase domain 15,30,38 . Our structures highlight how subtle movements in the DNA-gate propagate to the N-gate through networks of interactions mediated by conserved motifs, in particular in the transducer helices.…”

“…Interestingly the orientation of the linkers suggests the CTD domain could be positioned in an orthogonal direction relative to the plane of the Top2 catalytic domains. On the contrary, in prokaryotic enzymes, the linkers of the CTD domains are positioned in the same plane 24,30 . The CTD linker sequence of the Top2α displays a high content in positively charged residues, as well as several conserved residues at positions 1209 and 1212-1214 also found in the Top2β isoform and other eukaryotic species (Figure 5b).…”

The interplay between DNA topoisomerase 2α post-translational modifications and drug resistance

“…Strikingly, most of the SUMO sites in the catalytic domains were identified along the dimeric interface of the Top2a structure [ Figure 3]. Structure determination of Top2 has shown that the subunits form an intertwined dimer structure and that the buried surface could be accessible during the catalytic cycle [ Figure 1B] [66][67][68][69] .…”