Department of Chemistry, Western Washington University , 516 High Street, Bellingham, Washington 98225-9150, United States.
Biochemistry. 2013 Nov 12;52(45):7890-900. doi: 10.1021/bi400699s. Epub 2013 Oct 29.
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.
微丝束蛋白是一种肌动蛋白结合蛋白，定位于上皮细胞的顶端刷状缘。微丝束蛋白通过在低钙水平下束集 F-肌动蛋白和在高钙水平下切断 F-肌动蛋白来调节微绒毛。微丝束蛋白由六个类似肌球蛋白的重复序列（V1-V6）和独特的、肌动蛋白结合的 C 端头部结构域（HP）组成。微丝束蛋白的模块化片段 V6-HP 需要钙才能保持单体状态并束集 F-肌动蛋白。我们的数据表明，分离的 V6 是单体，并且在任何钙水平下都不结合 F-肌动蛋白。我们提出，40 个残基的未折叠的 V6 到 HP 接头可以是微丝束蛋白功能的关键调节元件，例如其与 F-肌动蛋白的相互作用。在这里，我们报道了 V6 的一个结合钙的溶液核磁共振（NMR）结构，它具有一个类似肌球蛋白的折叠，其中长 α-螺旋处于伸展构象。固有色氨酸荧光猝灭揭示了 V6 中存在两个 Kd 值的钙结合（Kd1 为 22 μM，Kd2 为 2.8 mM）。根据我们的 NMR 数据，V6 的构象对微摩尔钙的响应最敏感。我们表明，长 α-螺旋和相邻的残基形成了 V6 中钙敏感的元件。这些观察结果与微丝束蛋白通过类似于肌球蛋白的螺旋拉直机制激活 F-肌动蛋白切割的钙激活一致。
