Mycobacterium tuberculosis DNA gyrase ATPase domain structures suggest a dissociative mechanism that explains how ATP hydrolysis is coupled to domain motion

Abstract

DNA gyrase, a type II topoisomerase, regulates DNA topology by creating a double-stranded break in one DNA duplex and transporting another DNA duplex [T-DNA (transported DNA)] through this break. The ATPase domains dimerize, in the presence of ATP, to trap the T-DNA segment. Hydrolysis of only one of the two ATPs, and release of the resulting Pi, is rate-limiting in DNA strand passage. A long unresolved puzzle is how the non-hydrolysable ATP analogue AMP-PNP (adenosine 5'-[β,γ-imido]triphosphate) can catalyse one round of DNA strand passage without Pi release. In the present paper we discuss two crystal structures of the Mycobacterium tuberculosis DNA gyrase ATPase domain: one complexed with AMP-PCP (adenosine 5'-[β,γ-methylene]triphosphate) was unexpectedly monomeric, the other, an AMP-PNP complex, crystallized as a dimer. In the AMP-PNP structure, the unprotonated nitrogen (P-N=P imino) accepts hydrogen bonds from a well-ordered 'ATP lid', which is known to be required for dimerization. The equivalent CH2 group, in AMP-PCP, cannot accept hydrogen bonds, leaving the 'ATP lid' region disordered. Further analysis suggested that AMP-PNP can be converted from the imino (P-N=P) form into the imido form (P-NH-P) during the catalytic cycle. A main-chain NH is proposed to move to either protonate AMP-P-N=P to AMP-P-NH-P, or to protonate ATP to initiate ATP hydrolysis. This suggests a novel dissociative mechanism for ATP hydrolysis that could be applicable not only to GHKL phosphotransferases, but also to unrelated ATPases and GTPases such as Ras. On the basis of the domain orientation in our AMP-PCP structure we propose a mechanochemical scheme to explain how ATP hydrolysis is coupled to domain motion.

Structure of the N-Terminal Gyrase B Fragment in Complex with ADP⋅Pi Reveals Rigid-Body Motion Induced by ATP Hydrolysis

“…(PDB code: 1EI1) [13] contains a mutation (Y5S) in the N-terminal arm. Furthermore, inter-species comparison between GyrB from E. coli (PDB code: 1EI1) [13] and Mycobacterium tuberculosis (PDB code: 3ZKB) [25] reveals a very well conserved ligand-protein interaction network (Fig. S1).…”

Alpha-carboxy nucleoside phosphonates as universal nucleoside triphosphate mimics

“…The design of α-CNPs may also represent a technological tool for facilitating biochemical and structural studies of (d)NTPbinding proteins in the presence of α-CNPs as nonhydrolysable (d)NTP mimics. In fact, the nonhydrolysable ATP mimic adenosine 5′-(β,γ-methylene)triphosphate has been demonstrated to be instrumental for an improved crystallography of proteins that use ATP as one of their ligands and to obtain mechanistic insights in the role of ATP in enzyme interaction and catalysis (34,35). Therefore, α-CNPs should be further investigated for their interaction with (d)ATP-, (d)CTP-, or (d)TTP-binding proteins from a crystallographic and mechanistic viewpoint.…”

Getting the chemistry right: protonation, tautomers and the importance of H atoms in biological chemistry

“…In solution the N atom between the -phosphate andphosphate is normally protonated and the compound is known as adenosine-5 0 -(,-imido)triphosphate (AMPP-NH-P). In some crystal structures of AMPPNP with proteins this bridging N atom accepts hydrogen bonds and is in the unprotonated imino form: adenosine-5 0 -(,-imino)triphosphate (AMPP-N P) (Dauter & Dauter, 2011;Agrawal et al, 2013). Comparison of the fitting of (a) AMPP-NH-P or (b) AMPP-N P into a structure.…”