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
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-actin and, at higher calcium concentration, is capable of a gelsolin-like F-actin severing. The headpiece domain can, in isolation, bind F-actin and is crucial for F-actin bundling by villin. While the three-dimensional structure of the isolated headpiece is known, its conformation in the context of attachment to the villin core remains unexplored. Furthermore, the dynamics of the linkage of headpiece to the core has not been 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 demonstrate that 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 resonance assignments and 15N-relaxation measurements of D6-HP in 5 mM Ca2+ demonstrate that D6-HP consists of 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 domain only through the linker sequence without engaging in other interactions. Chemical shift values indicate essentially the same secondary structure elements for the D6 domain in D6-HP as in the highly homologous gelsolin domain 6. Thus, the headpiece domain of villin is structurally and functionally independent from the core domain.
- Research Organization:
- Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 910265
- Report Number(s):
- PNNL-SA-55624; 17990; KP1303000
- Journal Information:
- Biochemistry, 46(25):7488-7496, Journal Name: Biochemistry, 46(25):7488-7496 Journal Issue: 25 Vol. 46
- Country of Publication:
- United States
- Language:
- English
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