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J. Biol. Chem., Vol. 279, Issue 43, 45036-45046, October 22, 2004

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Functional Dissection and Molecular Characterization of Calcium-sensitive Actin-capping and Actin-depolymerizing Sites in Villin^*

Narendra Kumar, Alok Tomar, Abby L. Parrill, and Seema Khurana¶

From the Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163 and the Department of Chemistry, University of Memphis, Memphis, Tennessee 38152

All proteins of the villin superfamily, which includes the actin-capping and -severing proteins such as gelsolin, scinderin, and severin, are calcium-regulated actin-modifying proteins. Like some of these proteins, villin has morphologically distinct effects on actin assembly depending on the free calcium concentrations. At physiological calcium (Ca²⁺) villin nucleates and bundles actin, whereas at higher concentrations it caps (>50 µM) and severs (>200 µM) actin filaments. Although Ca²⁺-binding sites have been described in villin, the functional characterization of these sites has not been done previously. In the present study we functionally dissect the calcium-dependent actin-capping and -depolymerizing sites in villin. Our analysis reveals that villin binds Ca²⁺ with a K_d of 80.5 µM, a stoichiometry of 5.97, and a Hill's coefficient of 1.2. Using the NMR structure of villin 14T and the gelsolin-actin/Ca²⁺ crystal structure, six putative sites that result in Ca²⁺-induced conformational changes were identified in human villin and confirmed by mutational analysis. Molecular dynamics studies support the mutational analysis and provide a model for structural difference in the A93G mutant that prevents the calcium-induced conformational changes in the S1 domain of villin. Furthermore, we determined that villin expresses at least two types of Ca²⁺-sensitive sites that determine separate functional properties; site 1 (Glu-25, Asp-44, and Glu-74) regulates actin-capping, whereas sites 1 and 2 (Asp-86, Ala-93, and Asp-61), together with the intra-domain calcium-sensitive sites in villin, regulate actin depolymerization by villin. This is the first study that employs sequential mutagenesis to biochemically and functionally characterize the calcium-sensitive sites in villin. Such mutational analysis and functional characterization of the actin-capping and -depolymerizing sites are unknown for other proteins of the villin family.

Received for publication, May 14, 2004 , and in revised form, July 19, 2004.

^* This work was supported by NIDDK, National Institutes of Health Grants DK-65006 and DK-54755 (to S. K.). 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 material in the form of a list of primers used to confirm Ca²⁺-binding site mutations and graphs depicting tryptophan fluorescence measurements (Figs. S1 and S2).

^¶ To whom correspondence should be addressed: University of Tennessee, Health Science Center, 894 Union Ave., Nash 402, Memphis, TN 38163. Tel.: 901-448-3410; Fax: 901-448-3505; E-mail: skhurana{at}utmem.edu.

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