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J. Biol. Chem., Vol. 283, Issue 33, 22760-22773, August 15, 2008

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An Unconventional Form of Actin in Protozoan Hemoflagellate, Leishmania^*

Prabodh Kapoor¹, Amogh A. Sahasrabuddhe, Ashutosh Kumar¹, Kalyan Mitra, Mohammad Imran Siddiqi, and Chhitar M. Gupta²

From the Division of Molecular and Structural Biology and Electron Microscopy Unit, Central Drug Research Institute, Lucknow-226001, India

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.

Received for publication, January 9, 2008 , and in revised form, May 12, 2008.

^* This work was supported in part by a research grant from CDRI and the Department of Biotechnology, Government of India, New Delhi (India) under the Distinguished Biotechnologist Award Scheme (to C. M. G.). This is communication 7446 from CDRI. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ Recipients of research fellowships from the Council of Scientific and Industrial Research, Rafi Marg, New Delhi (India).

² To whom correspondence should be addressed: Chattar Manzil Palace, M G Rd., Central Drug Research Institute, Lucknow-226001, India. Fax: 91-522-2623405; E-mail: drcmg{at}sify.com.

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