The unusual, heterodimeric topoisomerase IB of Leishmania shows 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 C-terminal-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 and therefore co-purified fragments showed reduced DNA binding, cleavage and relaxation properties compared with the wild-type enzyme. The minimal fragments could complement their respective 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 co-operativity of this heterodimeric enzyme.

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