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Solution structures of the C-terminal headpiece subdomains of human villin and advillin, evaluation of headpiece F-actin-binding requirements.

ABSTRACT: Headpiece (HP) is a 76-residue F-actin-binding module at the C terminus of many cytoskeletal proteins. Its 35-residue C-terminal subdomain is one of the smallest known motifs capable of autonomously adopting a stable, folded structure in the absence of any disulfide bridges, metal ligands, or unnatural amino acids. We report the three-dimensional solution structures of the C-terminal headpiece subdomains of human villin (HVcHP) and human advillin (HAcHP), determined by two-dimensional 1H-NMR. They represent the second and third structures of such C-terminal headpiece subdomains to be elucidated so far. A comparison with the structure of the chicken villin C-terminal subdomain reveals a high structural conservation. Both C-terminal subdomains bind specifically to F-actin. Mutagenesis is used to demonstrate the involvement of Trp 64 in the F-actin-binding surface. The latter residue is part of a conserved structural feature, in which the surface-exposed indole ring is stacked on the proline and lysine side chain embedded in a PXWK sequence motif. On the basis of the structural and mutational data concerning Trp 64 reported here, the results of a cysteine-scanning mutagenesis study of full headpiece, and a phage display mutational study of the 69-74 fragment, we propose a modification of the model, elaborated by Vardar and coworkers, for the binding of headpiece to F-actin.

SUBMITTER: Vermeulen W

PROVIDER: S-EPMC2286768 | biostudies-literature | 2004 May

REPOSITORIES: biostudies-literature

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Solution structures of the C-terminal headpiece subdomains of human villin and advillin, evaluation of headpiece F-actin-binding requirements.

Vermeulen Wim W Vanhaesebrouck Peter P Van Troys Marleen M Verschueren Mieke M Fant Franky F Goethals Marc M Ampe Christophe C Martins José C JC Borremans Frans A M FA

Protein science : a publication of the Protein Society 20040501 5

Headpiece (HP) is a 76-residue F-actin-binding module at the C terminus of many cytoskeletal proteins. Its 35-residue C-terminal subdomain is one of the smallest known motifs capable of autonomously adopting a stable, folded structure in the absence of any disulfide bridges, metal ligands, or unnatural amino acids. We report the three-dimensional solution structures of the C-terminal headpiece subdomains of human villin (HVcHP) and human advillin (HAcHP), determined by two-dimensional 1H-NMR. Th ...[more]

PMID: 15096633

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Project description:The 36-residue helical subdomain of the villin headpiece, HP36, is one of the smallest cooperatively folded proteins, folding on the microsecond time scale. The domain is an extraordinarily popular model system for both experimental and computational studies of protein folding. The structure of HP36 has been determined using X-ray crystallography and NMR spectroscopy, with the resulting structures exhibiting differences in helix packing, van der Waals contacts, and hydrogen bonding. It is important to determine the solution structure of HP36 with as much accuracy as possible since this structure is widely used as a reference for simulations and experiments. We complement the existing data by using all-atom molecular dynamics simulations with explicit solvent to evaluate which of the experimental models is the better representation of HP36 in solution. After simulation for 50 ns initiated with the NMR structure, we observed that the protein spontaneously adopts structures with a backbone conformation, core packing, and C-capping motif on the third helix that are more consistent with the crystal structure. We also examined hydrogen bonding and side chain packing interactions between D44 and R55 and between F47 and R55, respectively, which were observed in the crystal structure but not in the NMR-based solution structure. Simulations showed large fluctuations in the distance between D44 and R55, while the distance between F47 and R55 remained stable, suggesting the formation of a cation-pi interaction between those residues. Experimental double mutant cycles confirmed that the F47-R55 pair has a larger energetic coupling than the D44-R55 interaction. Overall, these combined experimental and computational studies show that the X-ray crystal structure is the better reference structure for HP36 in solution at neutral pH. Our analysis also shows how detailed molecular dynamics simulations combined with experimental validation can help bridge the gap between NMR and crystallographic methods.

Dataset Information

Solution structures of the C-terminal headpiece subdomains of human villin and advillin, evaluation of headpiece F-actin-binding requirements.

Publications

Solution structures of the C-terminal headpiece subdomains of human villin and advillin, evaluation of headpiece F-actin-binding requirements.

Similar Datasets

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