Amino acids 39–456 of the Large Subunit and 210–262 of the Small Subunit Constitute the Minimal Functionally Interacting Fragments of the Unusualudheterodimeric topoisomerase IB of Leishmania

摘要

The unusual, heterodimeric topoisomerase IB of Leishmaniaudshows functional activity upon reconstitution of the DNA-binding large subunit (LdTOPIL; or L) and the catalytic small subunit (LdTOPIS; or S). In the present study, we generated N- and Cterminal- truncated deletion constructs of either subunit and identified proteins LdTOPIL39−456 (lacking amino acids 1–39 and 457–635) and LdTOPIS210−262 (lacking amino acids 1–210) as the minimal interacting fragments. The interacting region of LdTOPIL lies between residues 40–99 and 435–456, while for LdTOPIS it lies between residues 210–215 and 245–262. The heterodimerization between the two fragments is weak andudtherefore co-purified fragments showed reduced DNA binding,udcleavage and relaxation properties compared with the wild type enzyme. The minimal fragments could complement theirudrespective wild-type subunits inside parasites when the respective subunits were down-regulated by transfection with conditional antisense constructs. Site-directed mutagenesis studies identify Lys455 of LdTOPIL and Asp261 of LdTOPIS as two residues involved in subunit interaction. Taken together, the present study provides crucial insights into the mechanistic details for understanding the unusual structure and inter-subunit cooperativity of this heterodimeric enzyme.