Demonstration of three distinct calcium-binding sites in villin, a modulator of actin assembly

Villin, a Ca2+-modulated F-actin-binding protein (95,000 daltons) present in microvillus core filament bundles, has been shown to contain multiple Ca2+-binding sites. 45Ca Hummel-Dreyer chromatography reveals the presence of two rapidly exchanging Ca2+-binding sites with an apparent dissociation constant, Kd, equal to 4.6 X 10(-6) M. Use of the two proteolytically separable domains of the molecule revealed that one site is located on the 90,000-dalton core (apparent Kd = 3.5 X 10(-6) M) while the second site is provided by the 8,800-dalton headpiece fragment (apparent Kd = 7.4 X 10(-6) M). In addition villin displays a further very slowly exchanging or nonexchangeable high affinity Ca2+-binding site, which is situated in the core domain. Secondary structural predictions and a comparison of the amino acid sequence of headpiece with other known Ca2+-binding proteins indicates one region suggestive of a Ca2+-binding site, although headpiece seems not to exhibit a classical "EF-hand" Ca2+-binding structure.