Analysis of etoposide binding to subdomains of human DNA topoisomerase II alpha in the absence of DNA.
Epipodophyllotoxins are effective anti-tumor drugs that inhibit eukaryotic DNA topoisomerase II by trapping the enzyme in a covalent complex with DNA. We show that both the recombinant N-terminal ATPase domain and the B'A' core domain of human topoisomerase IIalpha (htopoIIalpha) bind radiolabeled etoposide specifically, even in the absence of DNA. The addition of ATP impairs etoposide binding to the holoenzyme and the N-terminal domain, but not to the core domain. To see if this interference resembles that between novobiocin and ATP in the bacterial GyrB subunit, we modeled the structure of the N-terminal domain of htopoIIalpha and performed molecular docking analysis with etoposide. Mutagenesis of critical amino acids, predicted to stabilize the drug within the N-terminal domain, reveals a less efficient binding of etoposide to the mutated proteins as monitored by direct drug binding assays, although the binding of ATP is not affected.
Leroy
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D
Swiss Institute for Experimental Cancer Research (ISREC), CH-1066 Epalinges/Lausanne, Switzerland.
Kajava
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AV
Frei
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C
Gasser
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SM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000951:Antigens, Neoplasm; D000972:Antineoplastic Agents, Phytogenic / Q000378:metabolism*; D001667:Binding, Competitive / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D005047:Etoposide / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D059005:Topoisomerase II Inhibitors
D000951:Antigens, Neoplasm; D000972:Antineoplastic Agents, Phytogenic; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D059005:Topoisomerase II Inhibitors; D005047:Etoposide; D000255:Adenosine Triphosphate; D004247:DNA; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II