Functional dissection of the C-terminal domain of type II DNA topoisomerase from the kinetoplastid hemoflagellate Leishmania donovani.
The amino acid sequences of the C-terminal domain (CTD) of the type II DNA topoisomerases are divergent and species specific as compared with the highly conserved N-terminal and central domains. A set of C-terminal deletion mutants of Leishmania donovani topoisomerase II was constructed. Removal of more than 178 amino acids out of 1236 amino acid residues from the C-terminus inactivates the enzyme, whereas removal of 118 amino acids or less has no apparent effect on the ability of the parasite enzyme to complement a temperature-sensitive mutation of the Saccharomyces cerevisiae topoisomerase II gene. Deletion analysis revealed a potent nuclear localization signal (NLS) within the amino acid residues 998-1058. Immunomicroscopy results suggest that the removal of an NLS in the CTD is likely to contribute to the physiological dysfunction of these proteins. Modeling of the LdTOP2 based on the crystal structure of the yeast type II DNA topoisomerase showed that the parasite protein assumes a structure similar to its yeast counterpart harboring all the conserved residues in a structurally similar position. However, a marked difference in electrostatic potential was found in a span of 60 amino acid residues (998-1058), which also do not have any homology with topoisomerase II sequences. Such significant differences can be exploited by the structure-based design of selective inhibitors using the structure of the Leishmania enzyme as a template.
Sengupta
Tanushri
T
Department of Molecular Parasitology, Indian Institute of Chemical Biology, Kolkata 700032, India.
Mukherjee
Mandira
M
Mandal
Chhabinath
C
Das
Aditi
A
Majumder
Hemanta K
HK
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D005816:Genetic Complementation Test; D007893:Leishmania donovani / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D012333:RNA, Messenger; D004250:DNA Topoisomerases, Type II