Cryo-EM structure of the completeE. coliDNA 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, the detailed architecture of DNA Gyrase from the model genetic organism E. coli, is still missing, impeding structure-function analysis of E. coli-specific catalytic regulation and limiting the study of conformational intermediates of this highly flexible macromolecule. Herein, we determined the complete molecular structure of the E. coli DNA Gyrase bound to a 180 bp DNA and the antibiotic Gepotidacin, using phase-plate single-particle cryo-electron microscopy. Our data unveil with unprecedented details the structural and spatial organization of the functional domains, their connections and the position of the conserved GyrA-box motif. The deconvolution of closed and pre-opening states of the DNA-binding domain provides a better understanding of the allosteric movements of the enzyme complex. In this region, the local atomic resolution reaching up to 3.0 Å enables the identification of the antibiotic density in the DNA complex. Altogether, this study paves the way for the cryo-EM determination of gyrase complexes with antibiotics and opens perspectives for targeting conformational intermediates.

Rapid, DNA-induced subunit exchange by DNA gyrase

“…Therefore, for the model to stand, it must be assumed that, in addition to the (+1) supercoils stabilized by the writhe of the loop, another +1 must be stabilized in the twist of the double helix within this short loop. We find this to be unlikely considering recent cryo-EM structures of gyrase with DNA (Vanden Broeck, Lotz et al 2019). However, a CTD-wrapped state with +1.7 supercoils has been detected in the absence of ATP (Basu, Schoeffler et al 2012).…”

“…We did not add a linker to our fusion and therefore the position of GyrB with respect to GyrA and DNA is expected to be more constrained than with free GyrB. It has been reported that the GyrB dimer is leaning to one side with respect to the dyad axis in the gyrase-DNA complex (Vanden Broeck, Lotz et al 2019). Therefore, in this complex, the position of each GyrB subunit with regard to GyrA is different and this flexibility of GyrB in the gyrase complex might be important for its activity.…”