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' co re domain of human topoisomerase II alpha (htopoII alpha) bind radiolabelle d etoposide specifically, even in the absence of DNA. The addition of ATP i mpairs etoposide binding to the holoenzyme and the N-terminal domain, but n ot to the core domain. To see if this interference resembles that between n ovobiocin and ATP in the bacterial GyrB subunit, we modeled the structure o f the N-terminal domain of htopoII alpha. and performed molecular docking a nalysis with etoposide. Mutagenesis of critical amino acids, predicted to s tabilize the drug within the N-terminal domain, reveals a less efficient bi nding of etoposide to the mutated proteins as monitored by direct drug bind ing assays, although the binding of ATP is not affected.