Hydrogen-deuterium exchange points to inter-domain interactions in reverse gyrase that modulate conformational changes in positive DNA supercoiling.

Reverse gyrase is a DNA topoisomerase that catalyzes positive DNA supercoiling in an ATP-dependent reaction. The enzyme consists of a helicase and a topoisomerase domain. The isolated helicase domain is a DNA-stimulated ATPase, and the isolated topoisomerase domain can relax supercoiled DNA. In reverse gyrase, these two domains need to cooperate to achieve positive supercoiling of DNA, presumably through conformational changes. Here, we probed conformational changes of Thermotoga maritima reverse gyrase on DNA and nucleotide binding using hydrogen-deuterium exchange mass spectrometry. Although 5'-adenylyl-β,γ-imidotriphosphate binding does not induce major changes in HDX, binding of DNA leads to increased exposure of a region at the helicase-topoisomerase interface, encompassing R507 and E701 whose side chains engage in electrostatic interactions. Removing the charges in an R507A/E701A variant leads to a moderate increase in supercoiling activity, suggesting a slightly inhibitory role of these interactions. A R507C/E701C variant, crosslinked to fix the two cysteine side chains in a disulfide bond, on the other hand, is inactive. Collectively, these data show that rearrangements at the helicase and topoisomerase interface are important for the functional cooperation of the domains and for the enzymatic activity of reverse gyrase and provide a first structural model of the reverse gyrase/DNA complex.