Structure of the topoisomerase II ATPase region and its mechanism of inhibition by the chemotherapeutic agent ICRF-187

Abstract

Type IIA topoisomerases both manage the topological state of chromosomal DNA and are the targets of a variety of clinical agents. Bisdioxopiperazines are anticancer agents that associate with ATP-bound eukaryotic topoisomerase II (topo II) and convert the enzyme into an inactive, salt-stable clamp around DNA. To better understand both topo II and bisdioxopiperazine function, we determined the structures of the adenosine 5'-[beta,gamma-imino]-triphosphate-bound yeast topo II ATPase region (ScT2-ATPase) alone and complexed with the bisdioxopiperazine ICRF-187. The drug-free form of the protein is similar in overall fold to the equivalent region of bacterial gyrase but unexpectedly displays significant conformational differences. The ternary drug-bound complex reveals that ICRF-187 acts by an unusual mechanism of inhibition in which the drug does not compete for the ATP-binding pocket, but bridges and stabilizes a transient dimer interface between two ATPase protomers. Our data explain why bisdioxopiperazines target ATP-bound topo II, provide a structural rationale for the effects of certain drug-resistance mutations, and point to regions of bisdioxopiperazines that might be modified to improve or alter drug specificity.

Fig. 1.

(a) Domain organization of eukaryotic type IIA topoisomerases. Key functional modules of Saccharomyces cerevisiae topo II are labeled as follows: ATPase, yellow; B′, red; A′, blue; and a nonconserved C-terminal region, black. (b) Bisdioxopiperazine inhibition and the topo II reaction cycle. The enzyme normally transports one DNA segment through another in an ATP-dependent manner. Bisdioxopiperazines are thought to lock the ATPase regions in a nucleotide-bound dimeric state around double-stranded DNA, preventing the enzyme from resetting for subsequent rounds of strand passage.

Fig. 2.

(a) Stereo diagram of the ScT2-ATPase dimer. The GHKL and transducer domains are colored gold and orange, respectively. A 22-aa β-hairpin unique to eukaryotic topo II is colored light blue. ICRF-187 is shown as blue spheres. All residues within 5 Å of the drug are colored green. ADPNP is shown as spheres and colored by atom. (b) Buried dimer surface area. This is a surface representation of a single protomer seen from the dimer interface, showing surfaces involved in dimer interactions. Distances between the surfaces of each protomer were calculated with grasp (29) and range from 0 Å (white) to >7 Å (red). The ICRF-187-binding pocket sits in the middle of the primary dimer interface.

Fig. 3.

ICRF-187-binding pocket and protein/drug interactions. (a) ICRF-187-binding pocket seen from the top of the dimer. An F_obs–F_calc simulated-anneal omit electron density map shown in green is contoured at 1.5σ around ICRF-187. ADPNP, ICRF-187, and residues within 5 Å of the drug are shown in stick representation. For one protomer the residue positions correlated with drug resistance are colored dark green and other neighboring amino acids have been colored yellow. Residue side chains from the second protomer are colored gray. (b) Schematic diagram of protein/drug interactions. ICRF-187 is blue. Residues contacting the drug from each of the two protomers are indicated by colored or black text. For the colored protomer, the green residues in a yellow box indicate that drug-resistance mutations have been isolated at these positions. Orange/yellow residues are within 5 Å of ICRF-187 but have not yet been shown to affect drug efficacy when mutated. Hydrogen bonds are indicated by dotted red lines, stacking interactions are indicated by horizontally dashed red lines, and van der Waals interactions are indicated by solid red lines with a flat end. The γ-phosphates of bound ADPNPs are indicated by yellow circles.

Fig. 5.

Details of bisdioxopiperazine compounds and ICRF-187-binding site. (a) Schematic of different bisdioxopiperazine compounds. (b) Stereo figure of drug-binding site. One protomer has been removed for clarity. ADPNP, ICRF-187, and residues within 5 Šof the drug are shown in stick representation and colored as in Fig. 3. Subtraction of the van der Waals volume of ICRF-187 (225 ų) from the volume of the drug-binding cavity (350 ų) reveals two unfilled cavities above and below the plane of the drug (light gray mesh cages) with volumes of 35 and 95 ų, respectively.

Fig. 4.

Surface representation of ScT2-ATPase and E. coli GyrB dimers. Each molecule has one protomer colored dark gray (GHKL) and light gray (transducer), and one protomer colored gold (GHKL) and orange (transducer). A 22-aa insert specific to eukaryotic topo II is colored light blue.