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.

Figure 1.

DNA strand passage by type II topoisomerases. A generic scheme is shown whereby a transported DNA segment (maroon) is passed through a transiently broken gate-DNA (green) by coordinated action of N- (or ATPase), DNA- and C-gates. The diagram shows the putative open (1) and closed clamp states (2–4) and putative conformational transitions driven by ATP binding/hydrolysis and ADP release (see text). ATPase and TOPRIM domains are shown in red and yellow, respectively. The DNA breakage-reunion and C-terminal β-pinwheel domains are depicted in grey and silver, respectively.

Figure 2.

Streptococcus pneumoniae topo IV and its E and V cleavage sites on DNA. (A) Domain structures of 72 K ParE and 93K ParC subunits of S. pneumoniae topo IV. Figure shows the recombinant non-fused (top), fused (middle) and native (bottom) ParE and ParC subunits. ATP and TOPRIM denote N-terminal ATPase and C-terminal Mg²⁺ binding domains of ParE. CTD denotes the DNA binding C-terminal domain of ParC that orients DNA for transport and which adopts a β-pinwheel structure. WHD indicates the winged helix domain. Numbers denote amino acid residues in the 647 residue ParE and 823-residue ParC proteins. (B) DNA sequences of the 34mer V-site and 26mer E-site used in crystallization studies. The 4-bp overhanging sequence which is generated upon cleavage between −1 and +1 nucleotides (arrows) is shown in red.

Figure 3.

High-resolution ‘open clamp’ structure of S. pneumoniae topoisomerase IV bound to 34mer V-site DNA. The surface (left) and cartoon (right) representations of the dimeric complex of fused full-length ParE-ParC55 are shown in orthogonal views. ParC55 is in silver, ParE N-terminal gate domain (including the ATPase subdomain) is in red, ParE TOPRIM domain is in light pink, G-segment DNA is in green, the second DNA bound to α-helices near the C-gate is in blue.

Figure 4.

G-segment DNA in the high-resolution structure. Orthogonal views of the bound G-segment V-site DNA with the 2F_obs-F_calc electron density map at 1.5σ overlapped and the corresponding active site tyrosines-118 and arginines-117 displayed. The DNA form is indicated according to w3DNA (48).

Figure 5.

ParC subunit interactions with the second DNA duplex. Four topo IV dimers coordinate the B-form DNA duplex in the crystal with the corresponding ParC residues involved are shown in red, yellow, green and blue. Green and red spheres show the positions of water molecules. The DNA form is indicated according to w3DNA (48).

Figure 6.

Putative binding positions of the modeled C-terminal β-pinwheel domain of ParC relative to the solved biological dimer and the second DNA-binding site. ParC55 is in cyan, ParE N-terminal gate domain (including the ATPase subdomain) is in red, ParE TOPRIM domain is in light pink, G-segment DNA is in green, the second DNA bound to α-helices near the C-gate is in blue. The model of the C-terminal β-pinwheel domain of ParC is in silver with additional semi-transparent surface representation.

Figure 7.

The ATPase domains. Shown are cartoon representations of the ATPase domains of ParE or GyrB in S. pneumoniae (top left), E. coli [right top and bottom for GyrB (39) and ParE (49), respectively) and X. oryzae ParE (Heo, Y.-S. Crystal structure of ParE subunit. PDB code: 3LNU). Where applicable, the domains are in their dimerized form stabilized by ADPNP, a non-hydrolysable ATP analogue (displayed in Licorice mode). An ‘ATPase-active site covering’ α-helix present in S. pneumoniae ParE and its analogue in other structures is shown in red. ADPNP in B and D is shown in gold.

Figure 8.

Proposed model for the T-segment DNA capture and transport based on the high-resolution structure of the open N-gate topoisomerase complex with the V34 G-segment DNA. The individual distinct stages are numbered 1 to 14. The protein is shown in Cartoon representation, the DNA is shown in surface representation. The ParE ATPase and TOPRIM domains are shown in red/light red for one side and yellow/light yellow for the other one, with dotted lines indicating the flexible poorly ordered protein pivot corresponding to S. pneumoniae ParE residues 399–413. The G-segment is in green, the T-segment is in cyan and ParC55 is in silver. ATP/ADP + leaving phosphates are shown in van der Waals representation and coloured according to the atom type. Residues 303–316 of ParE comprising the K-loop (23) are shown in blue where applicable and the corresponding arginine/lysine Cα-s are shown in purple using van der Waals representation (stages 6 and 12, indicated by asterisks and arrows). Small arrows indicate general movements of the domains, whereas the larger arrows indicate progress through the topo II cycle. The open clamp state (stage 2 and 14) and its conversion to the closed clamp form (stage 5) are new findings from our study. There is uncertainty about exactly how the two ATP molecules are used and whether all type II topoisomerase systems follow the same hydrolytic mechanism. For simplicity and to focus on the pathway of DNA transport, we show both ATP molecules hydrolyzed at stage 12, though one molecule could be hydrolysed earlier (at stage 7 and 8) as postulated for yeast topo II (2,3).

Figure 9.

‘Open’ and ‘closed’ ATP gate conformations known from crystallographic structures. (A) ‘open’ ATP gate structure of pneumococcal topo IV (this work); (B) ‘closed’ ATP gate structure of yeast topo II (23). The structures are displayed in ‘surface’ mode in orthogonal views. Corresponding ParE domains are shown in red/yellow. G-segment is in green. ParC55 domains in (A) are in cyan. ParC55-equivalent domains in (B) are in white. (C) Diagram superimposing the hinge regions of open and closed clamps of type II topoisomerases. For the open clamp topo IV structure, linkage of ATPase and TOPRIM domains (shown in yellow) through their respective ParE S399 and G414 residues (black dotted line) occurs on the same side of the complex, whereas for the closed clamp of yeast topo II, the flexible hinge linking E408 and to S421 (red dotted line) passes over the bound DNA (shown in green) to connect ATPase and TOPRIM domains on opposite sides of the gate.

Figure 10.

Structural implications for rational drug design. (A) Previously published cleavage complex of topo IV from S. pneumoniae stabilized by PD 0305970 (19) with the bound drug molecules shown in blue. (B) Corresponding view of the high-resolution structure of topo IV from S. pneumoniae comprising the full-length ParE domains. ParC55 is in silver, G-segment DNA is in green, ParE is in red/yellow. The area available for rational drug design exploration and exploitation is indicated by the blue/white ellipse. The locations of the flexible pivot links between the ATPase and TOPRIM domains of ParE are in white/red circles.