Binding and Hydrolysis of a Single ATP Is Sufficient for N-Gate Closure and DNA Supercoiling by Gyrase

Abstract

Topoisomerases catalyze the relaxation, supercoiling, catenation, and decatenation of DNA. Gyrase is a bacterial topoisomerase that introduces negative supercoils into DNA in an ATP-dependent reaction. The enzyme consists of two GyrB subunits, containing the ATPase domains, and two GyrA subunits. Nucleotide binding to gyrase B GyrB causes closing of the N-gate in gyrase, which orients bound DNA for supercoiling. N-gate re-opening after ATP hydrolysis, at the end of the supercoiling reaction, resets the enzyme for subsequent catalytic cycles. Gyrase binds and hydrolyzes two ATP molecules per catalytic cycle. Here, we dissect the role of these two binding and hydrolysis events using gyrase with one ATP-binding- and hydrolysis-deficient subunit, or with one binding-competent, but hydrolysis-deficient ATPase domain. We show that binding of a single ATP molecule induces N-gate closure. Gyrase that can only bind and hydrolyze a single ATP undergoes opening and closing of the N-gate in synchrony with ATP hydrolysis, and promotes DNA supercoiling under catalytic conditions. In contrast, gyrase that can bind two ATP molecules, but hydrolyzes only one, only supercoils DNA under stoichiometric conditions. Here, ATP bound to the hydrolysis-deficient subunit keeps the N-gate closed after hydrolysis of the other ATP and prevents further turnovers. Gyrase with only one functional ATPase domain hydrolyzes ATP with a similar rate to wild-type, but its supercoiling efficiency is reduced. Binding and hydrolysis of the second ATP may thus ensure efficient coupling of the nucleotide cycle with the supercoiling reaction by stabilizing the closed N-gate and by acting as a timer for N-gate re-opening.

Keywords: ATP hydrolysis; conformational cycle; gyrase; single-molecule FRET; supercoiling mechanism.