Structure of an ‘open’ clamp type II topoisomerase-DNA complex provides a mechanism for DNA capture and transport

Abstract

Type II topoisomerases regulate DNA supercoiling and chromosome segregation. They act as ATP-operated clamps that capture a DNA duplex and pass it through a transient DNA break in a second DNA segment via the sequential opening and closure of ATPase-, G-DNA- and C-gates. Here, we present the first ‘open clamp’ structures of a 3-gate topoisomerase II-DNA complex, the seminal complex engaged in DNA recognition and capture. A high-resolution structure was solved for a (full-length ParE-ParC55)2 dimer of Streptococcus pneumoniae topoisomerase IV bound to two DNA molecules: a closed DNA gate in a B-A-B form double-helical conformation and a second B-form duplex associated with closed C-gate helices at a novel site neighbouring the catalytically important β-pinwheel DNA-binding domain. The protein N gate is present in an ‘arms-wide-open’ state with the undimerized N-terminal ParE ATPase domains connected to TOPRIM domains via a flexible joint and folded back allowing ready access both for gate and transported DNA segments and cleavage-stabilizing antibacterial drugs. The structure shows the molecular conformations of all three gates at 3.7 Å, the highest resolution achieved for the full complex to date, and illuminates the mechanism of DNA capture and transport by a type II topoisomerase.

Insights into the Mechanism of Inhibition of Novel Bacterial Topoisomerase Inhibitors from Characterization of Resistant Mutants of Staphylococcus aureus

“…The biochemical data and the resistance substitutions with NBTI compounds in previous studies (20,22), together with the co-crystal structure, have helped to differentiate both the binding mode and the mechanism of inhibition of this class of inhibitors from those of the fluoroquinolones (16,39,40). While the general mechanistic differences have been well established, the specific pathways of conformational changes that result in this allosteric inhibition remain ill defined.…”

“…While the general mechanistic differences have been well established, the specific pathways of conformational changes that result in this allosteric inhibition remain ill defined. Recently, structural advances in the field of bacterial (13,20,34,39,40) topoisomerases have significantly improved the understanding of this complex machinery, where association and dissociation of various subunits result in DNA cleavage/religation and DNA strand passage. However, with a limited number of inhibitors having unique inhibition mechanisms, resistance-based validation of these hypotheses is sparse.…”

Direct control of type IIA topoisomerase activity by a chromosomally encoded regulatory protein

“…7B). CoaE is the last enzyme in the CoA biosynthetic pathway (Mishra et al 2001;Leonardi et al 2005) and acts by adding phosphate to the 39 ribose moiety of dephospho-CoA (Leonardi et al 2005); the operonic Figure 1A. Gyrase introduces negative supercoils into DNA in the presence of ATP.…”

“…2; Yoshida et al 1991;Piton et al 2010). The QRDR loop is unstructured in almost all type IIA topoisomerases in the absence of DNA (Fig 2C;Fu et al 2009;Piton et al 2010;Schoeffler et al 2010) but becomes ordered and visible when DNA is present (Fig 2D;Dong and Berger 2007;Bax et al 2010;Laponogov et al 2010Laponogov et al , 2013Schmidt et al 2010;Wohlkonig et al 2010;Wendorff et al 2012). Ordering of the QRDR loop through DNA binding masks the hydrophobic pocket occupied by Trp40 of YacG, whereas the pocket is exposed when the loop is unstructured.…”

“…1A), an explanation for this effect was not immediately evident from the structure. To determine whether YacG might have some additional effect on the organization or architecture of the gyrase holoenzyme, we modeled our structure onto previously crystallized, near-full-length crystallographic structures of type IIA topoisomerases from Saccharomyces cerevisiae (topo II; PDB ID 4FGH) and Streptococcus pneumoniae (topo IV; PDB ID 4I3H) (Laponogov et al 2013), which exhibit closed and open conformations of the ATPase domains, respectively. Inspection of the resultant models suggested that YacG binding to the GyrB TOPRIM fold might allow the protein to interact with the ATPase domains when they are closed, thereby interfering with reopening to impede ATP turnover.…”