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J Biol Chem. 2004 Oct 22;279(43):45036-46. Epub 2004 Jul 21.

Functional dissection and molecular characterization of calcium-sensitive actin-capping and actin-depolymerizing sites in villin.

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1
Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA.

Abstract

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 microm) and severs (>200 microM) actin filaments. Although Ca(2+)-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 Kd of 80.5 microM, 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 Ca(2+)-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 Ca(2+)-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.

PMID:
15272027
DOI:
10.1074/jbc.M405424200
[Indexed for MEDLINE]
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