Villin is a gelsolin-like cytoskeleton regulator localized in the brush border at the apical end of epithelial cells. Villin regulates microvilli by bundling F-actin at low calcium levels and severing it at high calcium levels. The villin polypeptide consists of six gelsolin-like repeats (V1-V6) and the unique, actin binding C-terminal headpiece domain (HP). Villin modular fragment V6-HP requires calcium to stay monomeric and bundle F-actin. Our data show that isolated V6 is monomeric and does not bind F-actin at any level of calcium. We propose that the 40-residue unfolded V6-to-HP linker can be a key regulatory element in villin's functions such as its interactions with F-actin. Here we report a calcium-bound solution nuclear magnetic resonance (NMR) structure of V6, which has a gelsolin-like fold with the long α-helix in the extended conformation. Intrinsic tryptophan fluorescence quenching reveals two-Kd calcium binding in V6 (Kd1 of 22 μM and Kd2 of 2.8 mM). According to our NMR data, the conformation of V6 responds the most to micromolar calcium. We show that the long α-helix and the adjacent residues form the calcium-sensitive elements in V6. These observations are consistent with the calcium activation of F-actin severing by villin analogous to the gelsolin helix-straightening mechanism.

Villin is a modular protein that regulates F-actin bundles in the microvilli of absorptive epithelial cells in the intestine. At low (10-100 nM) calcium levels, Villin is an F-actin bundling agent supporting the specialized brush border membrane of the absorptive epithelium. At intermediate micromolar calcium levels, Villin nucleates and caps the barbed ends of F-actin and in high (> 100 μM) calcium Villin is an F-actin severing agent (Bretsher & Weber, 1980; Glenney et al., 1980, 1981; Mooseker et al. 1980). The amino acid sequence of Villin has seven modular domains. The first six Villin domains (D1-D6) form a “core” of ~50% sequence identity with Gelsolin; and contain a Ca 2+ -dependent actin-binding site associated with the D1-D3 fragment. The last domain, Villin’s unique C-terminal headpiece (HP), contains the other F-actin binding site, which is Ca 2+ -independent (Bretsher & Weber, 1980; Glenney et al., 1980, 1981; Mooseker et al. 1980). Recent investigation by Nuclear Magnetic Resonance (NMR) Spectroscopy and Negative-Stain Electron Microscopy (EM) of the backbone dynamics and actin-binding of Villin’s D6-HP, 208-residue, C-terminal modular fragment, revealed that: a) folded domains D6 and HP are interacting only via a largely unfolded 40-residue linker, and b) at millimolar calcium levels, the monomeric D6-HP fragment bundles F-actin and has two actin binding sites; one, which is previously known on HP, and the other is novel, cryptic and Ca 2+ -dependent, associated with domain D6 or the linker (Smirnov et al., 2007). We have investigated how the domain structure, domain-domain and linkerdomain interactions in D6-HP fragment of Villin define its actin regulation properties. v Toward this goal, we are: a) making the D6 and D6-HP NMR samples; b) determining the NMR resonance assignment of isolated D6; and c) elucidating the solution structure of D6 domain in isolation and within the D6-HP fragment. Our NMR data indicate that the D6 protein fragment in isolation likely adopts a Gelsolin-like fold and that HP and D6 structures in isolation resemble those in the context of the larger modular fragment D6-HP. The potential effect of the linker on the D6 and HP domains structure is exemplified by the noticeable chemical shift differences for residue 84 of D6 and residue 166 of HP ( 15 N-HSQC spectrum of D6-HP vs. D6 and HP in isolation). These two positions are ~23 residues away from either end of the linker and located on the surface of these domains. In the absence of calcium, Gelsolin adopts a compact, inactive conformation stabilized by the 12-residue C-terminal helix. This helix was suggested to keep together Gelsolin domains D2 and D6 as a “latch” closed in low calcium and released at higher calcium levels (Robinson et al., 1999). Our ensuing structural study of D6-HP will clarify whether the linker sequence in D6-HP corresponding to this C-terminal helix of Gelsolin forms a helix as well and thus may or not undergo a gelsolin-like, calciuminduced rearrangement. The solution structure of D6 will be determined by NMR and analyzed in combination with the complete solution structure of HP and known structural properties of D6-HP. Together with the calcium and F-actin binding properties of D6 in isolation (currently under study), these data will clarify the role of the C-terminal domains of Villin in its activity as a physiologically principal actin regulator of microvilli.