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Protein Science (2004), 13:1276-1287. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Solution structures of the C-terminal headpiece subdomains of human villin and advillin, evaluation of headpiece F-actin-binding requirements

Wim Vermeulen1, Peter Vanhaesebrouck1, Marleen Van Troys2, Mieke Verschueren1, Franky Fant1, Marc Goethals2, Christophe Ampe2, José C. Martins1 and Frans A.M. Borremans1

1 NMR and Structure Analysis Unit, Department of Organic Chemistry, Faculty of Sciences, Ghent University, 9000 Ghent, Belgium
2 Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University and Department of Medical Protein Science, Flanders Interuniversity Institute for Biotechnology (VIB09), 9000 Ghent, Belgium

(RECEIVED November 19, 2003; FINAL REVISION February 12, 2004; ACCEPTED February 14, 2004)



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.

Keywords: headpiece subdomain; villin; advillin; NMR solution structure; actin-binding protein

Abbreviations: CD, circular dichroism • cHP, C-terminal headpiece subdomain • CVcHP, chicken villin C-terminal headpiece subdomain • CVHP67, chicken villin headpiece without N-terminal linker • DQF-COSY, double quantum filtered correlation spectroscopy • DSS-d6, 2,2-dimethyl-2-sila 3,3,4,4,5,5-hexadeutero-pentane sulphonic acid • DTT, dithiothreitol • E.COSY, exclusive correlation spectroscopy • HP, headpiece • HAcHP, human advillin C-terminal headpiece subdomain • HVcHP, human villin C-terminal headpiece subdomain • NOE, nuclear Overhauser effect • NOESY, nuclear Overhauser effect spectroscopy • rEM, restrained energy minimization • rMD, restrained molecular dynamics • r.m.s.d., root-mean-square deviation • TOCSY, total correlation spectroscopy • TPPI, time proportional phase incrementation • TrisHCL, [tris-(hydroxymethyl)aminomethane]hydrochloride

Reprint requests to: José C. Martins, NMR and Structure Analysis Unit, Department of Organic Chemistry, Faculty of Sciences, Ghent University, Krijgslaan 281 S4, 9000 Ghent, Belgium; e-mail: jose.martins{at}UGent.be; fax: 32-9-264-49-72; or Christophe Ampe, Department of Biochemistry, Faculty of Medicine and Health Sciences, Flanders Interuniversity Institute for Biotechnology (VIB09), Albert Baertsoenkaai 3, 900 Ghent, Belgium; e-mail: christophe.ampe{at}UGent.be; fax: 32-9-264-94-88.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03518104.

Supplemental material: see www.proteinscience.org


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