The structure of DNA-bound human topoisomerase II alpha: conformational mechanisms for coordinating inter-subunit interactions with DNA cleavage

Abstract

Type II topoisomerases are required for the management of DNA superhelicity and chromosome segregation, and serve as frontline targets for a variety of small-molecule therapeutics. To better understand how these enzymes act in both contexts, we determined the 2.9-Å-resolution structure of the DNA cleavage core of human topoisomerase IIα (TOP2A) bound to a doubly nicked, 30-bp duplex oligonucleotide. In accord with prior biochemical and structural studies, TOP2A significantly bends its DNA substrate using a bipartite, nucleolytic center formed at an N-terminal dimerization interface of the cleavage core. However, the protein also adopts a global conformation in which the second of its two inter-protomer contact points, one at the C-terminus, has separated. This finding, together with comparative structural analyses, reveals that the principal site of DNA engagement undergoes highly quantized conformational transitions between distinct binding, cleavage, and drug-inhibited states that correlate with the control of subunit-subunit interactions. Additional consideration of our TOP2A model in light of an etoposide-inhibited complex of human topoisomerase IIβ (TOP2B) suggests possible modification points for developing paralog-specific inhibitors to overcome the tendency of topoisomerase II-targeting chemotherapeutics to generate secondary malignancies.

Fig. 1

Crystallization constructs. (a) Primary structure of human TOP2A. The region crystallized (residues 431–1193) is shown in color. Specific domains (TOPRIM, WHD, tower) and dimerization gates are labeled with important residues and features highlighted: catalytic tyrosine and associated arginine, RY; intercalating isoleucine, I; metal-binding triad, DxD and E. (b) Schematic of the doubly nicked, 30-bp DNA substrate. Nicks and intercalation sites are highlighted.

Fig. 2

Structure of the human TOP2A DNA binding and cleavage core bound to DNA. (a) Refined 2F_o − F_c map around the nucleolytic center (1.4 σ contour). The active site is formed around DNA (yellow) at the interface between the TOPRIM domain (cyan) and WHD (magenta) of opposing protomers. (b) Refined 2F_o − F_c electron density map (2 σ contour) clearly shows the bound DNA substrate. (c) The human TOP2A·DNA complex as viewed from the front (left) and above (right). Domains are colored as in Fig. 1a, with DNA in dark orange. The G-segment is bound in the DNA-gate, and the C-gate dimerization interface is dissociated.

Fig. 3

Comparison of TOP2A and etoposide-bound TOP2B. (a) The global conformation of TOP2A and TOP2B differs. Superposition of the two human isoforms shows that the TOPRIM domain in the etoposide-bound TOP2B cleavage complex (PDB ID 3QX3) is rotated relative to the WHD and tower domain in the TOP2A structure (left panel, only one protomer shown). The dimeric TOP2B cleavage complex has a closed C-gate compared to TOP2A (right panel). (b) Structure and ring numbering of etoposide. (c) Only two amino acids differ in the etoposide-binding pocket of TOP2A and TOP2B. The WHD and TOPRIM of TOP2A (cyan sticks and colored surface) are shown individually superposed on the TOP2B (salmon sticks) drug-binding site. Two residues that differ—Met762 in TOP2A versus Gln778 in TOP2B, and Ser800 versus Ala816—are labeled, as are three amino acids that are preserved (Arg487/503, Asp463/479, and Met766/782). Etoposide is shown as gray/red sticks; the additional thienyl group of the related anticancer drug teniposide is modeled in magenta to show its relation to the Ser800/Ala816 region.

Fig. 4

Comparison of TOP2A and S cerevisiae topoisomerase II. (a) The quaternary conformation of the human TOP2A·DNA complex most closely resembles the yeast Top2–DNA complex with a doubly nicked substrate. Architectural differences between the two structures are most pronounced in the Greek key motif and in the C-gate (boxed regions). (b) Differences in the TOPRIM insert region. A tyrosine (Tyr684) in the 670–686 insert of TOP2A forms hydrogen bonds with Glu597 in the Greek key element of the TOPRIM domain. Interactions seen in TOP2A, but not yeast topoisomerase II, are shown as sticks/broken lines and labeled. (c) Differences in the C-gate. A β-hairpin in the yeast topoisomerase II C-gate is replaced by an α-helix (A′α15′) in TOP2A.

Fig. 5

Substrate-dependent movements in the type IIA topoisomerase DNA-gate are quantized. This figure depicts the superposition of 22 type IIA topoisomerase structures, aligned using the WHD of one protomer to reveal the relative displacements of its partner WHD. The juxtapositions fall into six groups. Cartoons depict just the three-helix bundles of the two WHDs; surface representations of TOP2A are provided for orientation. (a) Overview of the six juxtapositions: (1) no DNA bound, C-gate open [A. baumannii topo IV (2XKJ)]; (2) no DNA bound, C-gate closed [Escherichia coli gyrase (3NUH), also E. coli gyrase (1AB4), M. tuberculosis gyrase (3ILW)]; (3) DNA noncovalently bound, C-gate open [human TOP2A (4FM9), also S. cerevisiae topoisomerase II (2RGR)]; (4) DNA cleavage complex, C-gate closed [S. cerevisiae topoisomerase II (3L4K)]; (5) DNA cleavage complex, C-gate closed, initial crystallization with drug bound [A. baumannii topo IV bound to moxifloxacin (2XKK), also S. pneumoniae topo IV cleavage complex bound to moxifloxacin (3FOF), clinafloxacin (3FOE), levofloxacin (3K9F), a dione inhibitor (3LTN), the dione complex back soaked with EDTA (3KSA), and the EDTA-soaked complex resealed by a MgCl₂ soak (3KSB). The S. aureus gyrase noncovalent complex bound to GSK299943 (2XCR, 2XCS) and cleavage complex bound to ciprofloxacin (2XCT) further map to this group]; (6) DNA cleavage complex, C-gate closed, etoposide bound [human TOP2B (3QX3)]. The red broken line indicates the demarcation in relative WHD rotation angles between open and closed C-gate states. The black broken line indicates the demarcation of lateral WHD displacement between DNA-bound and free forms. (b) Binding of DNA causes the WHDs in opposing protomers to slide past each other by one helical turn along the α3 helix (A′α3 helix in eukaryotic topoisomerase IIs). This sliding can be quantized into juxtapositions taken by the apoenzyme [conformations 1 and 2 in (a)] and juxtapositions taken by DNA-bound enzymes [conformations 3–5 in (a)]. A more extreme lateral shift is seen in the etoposide-bound TOP2B complex where the DNA-gate interface has been disrupted [conformation 6 in (a)]. (c) DNA cleavage and C-gate dimerization are correlated to a rocking of the WHDs against each other. This rotation is quantized into juxtapositions with an open C-gate and noncovalently bound DNA [conformations 1 and 3 in (a)], a closed C-gate and covalently bound DNA [conformations 2 and 4 in (a)], and a closed C-gate, covalently bound DNA, and drug binding [conformations 5 and 6 in (a)].

Fig. 6

Comparison of metal occupancy between type IIA topoisomerase structures. (a) The structure of S. cerevisiae topoisomerase II trapped in a cleavage intermediate by a phosphorothiolate suicide substrate has two metals, A and B, bound in the active site (PDB 3L4K shown). (b) In structures solved with nicked substrates, only metal B is present, as metal A binding requires coordination by the missing scissile phosphate (PDB 4FM9 shown). (c) Structures bound to a type IIA topoisomerase poison bind only metal B, due to a shift of the phosphotyrosine out of the metal A binding site (PDB 2XKK shown). (d) In structures bound to a non-intercalative drug (e.g., GSK29994), or where poison has been removed following co-crystallization, only metal A is present. In these structures, residues in the DxD motif do not form appropriate geometry for metal B coordination, due possibly to the retention of an altered DNA-gate state (cf. Fig. 5) (PDB 2XCS shown).

Fig. 7

Model relating DNA-gate status to C-gate dimerization and metal occupancy in the context of the type IIA topoisomerase reaction. Topoisomerase IIA dimers exist in an equilibrium between different combinations of associated or dissociated proteinaceous interfaces. Where double arrows are shown, the relative thickness of the arrows denotes the likely weighted distribution of the two states. Prior to DNA binding, DNA-gate closure can occur with either a closed (1) or an open (2) C-gate. G-segment binding promotes the TOPRIM domain, WHD, and tower domain of both protomers to clamp around the DNA (3); this event may promote C-gate opening (4). ATP binding and capture of a second duplex, the T-segment, promote cleavage of the G-segment (5), favoring C-gate closure. The T-segment exits through an open C-gate following its passage through the G-segment and resealing of the cleaved DNA (6). Binding of topoisomerase II poisons and inhibitors such as GSK299943 perturb the DNA-gate interface to a more extended rotation that is impaired for religation and may help maintain the C-gate in a closed conformation (7). Relative WHD displacements as observed between different ligand-bound and/or free states are shown as cartoon cylinders and colored as per Fig. 5. The relative occupancies of the two metal-binding sites are also shown, with question marks denoting states that have yet to be imaged. The lone question mark associated with site A, state (4), reflects the fact that the DNAs found in these models lack a scissile phosphate and, hence, may not capable of binding a metal ion accordingly.