The Isolated Sixth Gelsolin Repeat and Headpiece Domain of Villin Bundle F-Actin in the Presence of Calcium and Are Linked by a 40-Residue Unstructured Sequence

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• 作者：Serge L. Smirnov ; Nancy G. Isern ; Zhenghui G. Jiang ; David W. Hoyt ; C. James McKnight
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：June 26, 2007
• 年：2007
• 卷：46
• 期：25
• 页码：7488 - 7496
• 全文大小：230K
• 年卷期：v.46,no.25(June 26, 2007)
• ISSN：1520-4995

文摘

Villin is an F-actin regulating, modular protein with a gelsolin-like core and a distinct C-terminal"headpiece" domain. Localized in the microvilli of the absorptive epithelium, villin can bundle F-actinand, at higher calcium concentrations, is capable of a gelsolin-like F-actin severing. The headpiece domaincan, in isolation, bind F-actin and is crucial for F-actin bundling by villin. While the three-dimensionalstructure of the isolated headpiece is known, its conformation in the context of attachment to the villincore remains unexplored. Furthermore, the dynamics of the linkage of the headpiece to the core has notbeen determined. To address these issues, we employ a 208-residue modular fragment of villin, D6-HP,which consists of the sixth gelsolin-like domain of villin (D6) and the headpiece (HP). We demonstratethat this protein fragment requires calcium for structural stability and, surprisingly, is capable of Ca²⁺-dependent F-actin bundling, suggesting that D6 contains a cryptic F-actin binding site. NMR resonanceassignments and ¹⁵N relaxation measurements of D6-HP in 5 mM Ca²⁺ demonstrate that D6-HP consistsof two independent structural domains (D6 and HP) connected by an unfolded 40-residue linker sequence.The headpiece domain in D6-HP retains its structure and interacts with D6 only through the linker sequencewithout engaging in other interactions. Chemical shift values indicate essentially the same secondarystructure elements for D6 in D6-HP as in the highly homologous gelsolin domain 6. Thus, the headpiecedomain of villin is structurally and functionally independent of the core domain.