Amino acids 39-456 of the large subunit and 210-262 of the small subunit constitute the minimal functionally interacting fragments of the unusual heterodimeric topoisomerase IB of Leishmania.
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 LdTOPIL(39-456) (lacking amino acids 1-39 and 457-635) and LdTOPIS(210-262) (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.
Bosedasgupta
Somdeb
S
Molecular Parasitology Laboratory, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata-700032, India.
Das
Benu Brata
BB
Sengupta
Souvik
S
Ganguly
Agneyo
A
Roy
Amit
A
Tripathi
Gayatri
G
Majumder
Hemanta K
HK
D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D001483:Base Sequence; D020134:Catalytic Domain; D004247:DNA / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D007891:Leishmania / Q000201:enzymology*; D008239:Lysine / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D021122:Protein Subunits; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D001224:Aspartic Acid; D004247:DNA; D004264:DNA Topoisomerases, Type I; D008239:Lysine