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

Abstract

All proteins of the villin superfamily, which includes the actin-capping and -severing proteins such as gelsolin, scinderin, and severin, are calcium-regulated actinmodifying proteins. Like some of these proteins, villin has morphologically distinct effects on actin assembly depending on the free calcium concentrations. At physiological calcium (Ca 2؉ ) villin nucleates and bundles actin, whereas at higher concentrations it caps (>50 M) and severs (>200 M) 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 Ca 2؉ with a K d of 80.5 M, a stoichiometry of 5.97, and a Hill's coefficient of 1.2. Using the NMR structure of villin 14T and the gelsolin-actin/Ca 2؉ 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.

Helix Straightening as an Activation Mechanism in the Gelsolin Superfamily of Actin Regulatory Proteins

“…In addition, at low pH, gelsolin is able to sever actin in the complete absence of free Ca 2ϩ . Furthermore, villin has calcium requirements for activation quite different from those of gelsolin (8,9). The importance of Ca 2ϩ in activation of members of this protein superfamily appears to vary with both protein identity and environmental setting, despite the high degree of conservation in Ca 2ϩ -binding residues.…”

“…Each of the six domains contains a complete and evolutionarily conserved site, termed type 2, whereas G1 and G4 provide partial Ca 2ϩ coordination at interfaces with actin through sites termed type 1. Sequential mutagenesis of these sites in villin has identified six functional Ca 2ϩ -binding sites (8): two major sites, one each of type 1 and type 2, in V1, plus four type 2 sites in V2-V6. The type 1 site in V1 regulates F-actin-capping and F-actin-severing activities, whereas the lower affinity type 2 site in V1 only affects severing (9).…”

Interaction of Phospholipase C-γ1with Villin Regulates Epithelial Cell Migration

“…Urea Denaturation Assay-To determine the effects of specific mutations on the overall stability of the villin molecules, fluorescence-monitored urea denaturation was performed on each recombinant protein as described previously (25). Fluorescence measurements were taken at an excitation wavelength of 280 nm, and an emission scan was performed from 335 to 360 nm.…”

“…4A). In order to assess whether the single point mutants maintained the conformation of the wild-type villin protein, the unfolding profiles of wild-type and mutant villin proteins expressed in the absence of IAA (nonphosphorylated proteins) as a function of the urea concentration were recorded by measuring the intrinsic tryptophan fluorescence emission spectrum essentially as described before (25). The results indicate that single point mutants express similar unfolding transitions as wild-type villin, thus maintaining a comparable overall conformation as the wild-type recombinant villin protein (Fig.…”

“…Epithelial cells of the intestine and kidney express more than one protein of this family (villin, gelsolin, and adseverin) (22). We and others have reported previously that although these proteins share structural homology they are not functionally identical (18,(23)(24)(25)(26). The identification of the tyrosine phosphorylation sites and their molecular characterization in these proteins will facilitate our understanding of their functional diversity.…”