Elucidation of an essential function of the unique charged domain of Plasmodium topoisomerase III.

Topoisomerase III (TopoIII) along with RecQ helicases are required for the resolution of abnormal DNA structures that result from the stalling of replication forks. Sequence analyses have identified a putative TopoIII in the Plasmodium falciparum genome (PfTopoIII). PfTopoIII shows dual nuclear and mitochondrial localization. The expression and association of PfTopoIII with mtDNA is tightly linked to the asexual replication of the parasite. In this study, we observed that PfTopoIII physically interacts with PfBlm and PfWrn. Sequence alignment and domain analyses have revealed that it contains a unique positively charged region, spanning 85 amino acids, within domain II. A molecular dynamics simulation study revealed that this unstructured domain communicates with DNA and attains a thermodynamically stable state upon DNA binding. Here, we found that the association between PfTopoIII and the mitochondrial genome is negatively affected by the absence of the charged domain. Our study shows that PfTOPOIII can completely rescue the slow growth phenotype of the ΔtopoIII strain in Saccharomyces cerevisiae, but neither PfY421FtopoIII (catalytic-active site mutant) nor Pf(Δ259-337)topoIII (charged region deletion mutant) can functionally complement ScTOPOIII. Hydroxyurea (HU) led to stalling of the replication fork during the S phase, caused moderate toxicity to the growth of P. falciparum, and was associated with concomitant transcriptional upregulation of PfTOPOIII. In addition, ectopic expression of PfTOPOIII reversed HU-induced toxicity. Interestingly, the expression of Pf(Δ259-337)topoIII failed to reverse HU-mediated toxicity. Taken together, our results establish the importance of TopoIII during Plasmodium replication and emphasize the essential requirement of the charged domain in PfTopoIII function.

Molecular docking and molecular dynamics simulation identify a novel Radicicol derivative that predicts exclusive binding to Plasmodium falciparum Topoisomerase VIB.

Plasmodium falciparum harbors a unique type II topoisomerase, Topoisomerase VIB (PfTopoVIB), expressed specifically at the actively replicating stage of the parasite. An earlier study showed that Radicicol inhibits the decatenation activity of PfTopoVIB and thereby arrests the parasites at the schizont stage. Radicicol targets a unique ATP-binding fold called the Bergerat fold, which is also present in the N-terminal domain of the heat shock protein 90 (PfHsp90). Hence, Radicicol may manifest off-target activity within the parasite. We speculate that the affinity of Radicicol towards PfTopoVIB could be enhanced by modifying its structure so that it shows preferential binding towards PfTopoVIB but not to PfHsp90. Here, we have performed the docking and affinity studies of 97 derivatives (structural analogs) of Radicicol and have identified 3 analogs that show selective binding only to PfTopoVIB and no binding with PfHsp90 at all. Molecular dynamics simulation study was performed for 50 ns in triplicate with those 3 analogs and we find that one of them shows a stable association with Radicicol. This study identifies the structural molecule which could be instrumental in blocking the function of PfTopoVIB and hence can serve as an important inhibitor for malaria pathogenesis. Communicated by Ramaswamy H. Sarma.