Structural basis of chiral wrap and T-segment capture by Escherichia coli DNA gyrase

What is it about?

Gyrase is an important enzyme that bacteria need to maintain, pack and copy their DNA and has the unique ability to twist DNA, negatively supercoiling it using energy from ATP hydrolysis. Because of this, it is a main target for antibiotics like fluoroquinolones, which kill bacteria by stopping gyrase working at a point at which it has broken the bacterial DNA but not yet resealed it –something which is lethal for the cell. A model proposed 40 years ago suggested gyrase works by stabilising and inverting a DNA loop, but this was never directly observed. We now have detailed cryoEM images of E. coli gyrase attached to a 217 base pair DNA strand, and another version with the antibiotic moxifloxacin. These images show the DNA loop in a figure-eight shape, ready to pass one DNA segment through a temporary break in another segment. The loop is held steady by a part of the GyrA subunit that looks like a flat disc, working akin to a mini nucleosome to keep the DNA loop in place. Our data revealed that the enzyme's ATPase parts move significantly to push a segment of DNA through the break in the loop during the process. By comparing the enzyme's structures with and without the drug, we see gyrase in a ready-to-react state with one metal ion in its active site. We also suggest that gyrase uses a "ratchet and pawl" method to turn ATP energy into mechanical force.

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Why is it important?

Our model helps us understand how the parts of gyrase work together. It's especially exciting to use techniques like smFRET48, which measures tiny distances between molecules, or EPR, which studies materials with unpaired electrons, to see these changes directly. These experiments could also look at the important role of the C-tail (the end of an amino acid chain in a protein) in moving between different conformational stages of gyrase. This tail is crucial for the structural changes and interactions that happen during the enzyme's activity. Previous research on making and testing mixed complexes with inactive parts on one side has given us useful information. The levels of DNA supercoiling differ even among closely related organisms, and even more so in heat-loving bacteria and archaea. We hope this framework will spark more discussions and set the stage for future studies on how this and other molecular machines work.

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