Gelsolin-like Activation of Villin: Calcium Sensitivity of the Long Helix in Domain 6

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• 作者：Stanislav O. Fedechkin ; Jacob Brockerman ; Danielle A. Pfaff ; Lucian Burns ; Terry Webb ; Alexander Nelson ; Fengli Zhang ; Anton V. Sabantsev ; Alexey S. Melnikov ; C. James McKnight ; Serge L. Smirnov
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：November 12, 2013
• 年：2013
• 卷：52
• 期：45
• 页码：7890-7900
• 全文大小：456K
• 年卷期：v.52,no.45(November 12, 2013)
• ISSN：1520-4995

文摘

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鈥揤6) 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鈥檚 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 伪-helix in the extended conformation. Intrinsic tryptophan fluorescence quenching reveals two-K<sub>dsub> calcium binding in V6 (K<sub>d1sub> of 22 渭M and K<sub>d2sub> of 2.8 mM). According to our NMR data, the conformation of V6 responds the most to micromolar calcium. We show that the long 伪-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.