An Unconventional Form of Actin in Protozoan Hemoflagellate, Leishmania

Leishmania actin was cloned, overexpressed in baculovirus-insect cell system, and purified to homogeneity. The purified protein polymerized optimally in the presence of Mg2+ and ATP, but differed from conventional actins in its following properties: (i) it did not polymerize in the presence of Mg2+...

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Bibliographic Details
Published in:The Journal of biological chemistry 2008-08, Vol.283 (33), p.22760-22773
Main Authors: Kapoor, Prabodh, Sahasrabuddhe, Amogh A., Kumar, Ashutosh, Mitra, Kalyan, Siddiqi, Mohammad Imran, Gupta, Chhitar M.
Format: Article
Language:English
Subjects:
Actins - chemistry
Actins - metabolism
Actins - ultrastructure
Adenosine Triphosphate - metabolism
Animals
Base Sequence
Computer Simulation
Hydrogen-Ion Concentration
Kinetics
Leishmania donovani - chemistry
Leishmania donovani - physiology
Magnesium - metabolism
Molecular Sequence Data
Polymerase Chain Reaction
Protozoan Proteins - chemistry
Protozoan Proteins - metabolism
Protozoan Proteins - ultrastructure
Spodoptera - parasitology
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Summary:Leishmania actin was cloned, overexpressed in baculovirus-insect cell system, and purified to homogeneity. The purified protein polymerized optimally in the presence of Mg2+ and ATP, but differed from conventional actins in its following properties: (i) it did not polymerize in the presence of Mg2+ alone, (ii) it polymerized in a restricted range of pH 7.0-8.5, (iii) its critical concentration for polymerization was found to be 3-4-fold lower than of muscle actin, (iv) it predominantly formed bundles rather than single filaments at pH 8.0, (v) it displayed considerably higher ATPase activity during polymerization, (vi) it did not inhibit DNase-I activity, and (vii) it did not bind the F-actin-binding toxin phalloidin or the actin polymerization disrupting agent Latrunculin B. Computational and molecular modeling studies revealed that the observed unconventional behavior of Leishmania actin is related to the diverged amino acid stretches in its sequence, which may lead to changes in the overall charge distribution on its solvent-exposed surface, ATP binding cleft, Mg2+ binding sites, and the hydrophobic loop that is involved in monomer-monomer interactions. Phylogenetically, it is related to ciliate actins, but to the best of our knowledge, no other actin with such unconventional properties has been reported to date. It is therefore suggested that actin in Leishmania may serve as a novel target for design of new antileishmanial drugs.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M800213200