Abstract
Type II topoisomerases (Top2s) alter DNA topology via the formation of an enzyme–DNA adduct termed cleavage complex, which harbors a transient double-strand break in one DNA to allow the passage of another. Agents targeting human Top2s are clinically active anticancer drugs whose trapping of Top2-mediated DNA breakage effectively induces genome fragmentation and cell death. To understand the structural basis of this drug action, we previously determined the structure of human Top2 β-isoform forming a cleavage complex with the drug etoposide and DNA, and described the insertion of drug into DNA cleavage site and drug-induced decoupling of catalytic groups. By developing a post-crystallization drug replacement procedure that simplifies structural characterization of drug-stabilized cleavage complexes, we have extended the analysis toward other structurally distinct drugs, m-AMSA and mitoxantrone. Besides the expected drug intercalation, a switch in ribose puckering in the 3′-nucleotide of the cleavage site was robustly observed in the new structures, representing a new mechanism for trapping the Top2 cleavage complex. Analysis of drug-binding modes and the conformational landscapes of the drug-binding pockets provide rationalization of the drugs’ structural-activity relationships and explain why Top2 mutants exhibit differential effects toward each drug. Drug design guidelines were proposed to facilitate the development of isoform-specific Top2-targeting anticancer agents.
Highlights
Type II topoisomerases (Top2s) are a group of essential enzymes that are capable of passing two duplex DNA segments through each other; such an activity allows these enzymes to manipulate DNA topology and resolve geometric DNA entanglements that originate from cellular DNA transactions [1,2]
Our group has previously reported the method by which high-quality crystals of etoposide-stabilized Top2 cleavage complex (Top2cc) can be produced for X-ray diffraction analysis [10]
Other anticancer drugs were introduced by placing the pre-soaked crystals in a solution that contained the different drug to produce new drug-stabilized Top2cc crystals. Such post-crystallization treatments have allowed the structures of ternary Top2cc stabilized by m-AMSA or mitoxantrone to be successfully determined at 2.7 and 2.55 Aresolution, respectively; the binding of new drug molecules, the formation of a pair of 50-phosphotyrosyl covalent linkages and the presence of a double-strand DNA break are unambiguously defined in the resulting electron density maps (Figure 2A and B; Supplementary Figure S1)
Summary
Type II topoisomerases (Top2s) are a group of essential enzymes that are capable of passing two duplex DNA segments through each other; such an activity allows these enzymes to manipulate DNA topology and resolve geometric DNA entanglements that originate from cellular DNA transactions [1,2]. Each cycle of Top2-catalyzed DNA topological transformation constitutes the following steps in sequential order: binding one DNA duplex to the enzyme’s DNA-binding and cleavage core (Top2core); producing a double-strand break on the bound DNA via the formation of a covalent enzyme-DNA adduct termed a Top cleavage complex (Top2cc); capturing and transporting a second DNA duplex through the transiently formed break; and resealing the cleaved DNA backbones followed by enzyme resetting [for recent reviews and development on the catalytic mechanism of Top, see [2,3,4,5]]. The DNA cleavage activity of Top is accomplished by a transesterification reaction between a pair of dyadrelated tyrosine residues and two phosphodiester bonds 4-bp apart on opposite DNA strands. Many clinically active anticancer and antimicrobial drugs exploit this latent jeopardy of Top and exert their cell-killing effects by trapping and accumulating the usually short-lived Top2cc, leading
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