Solution structures of the C-terminal headpiece subdomains of human villin and advillin, evaluation of headpiece F-actin-binding requirements.
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.
Vermeulen
Wim
W
NMR and Structure Analysis Unit, Department of Organic Chemistry, Faculty of Sciences, Ghent University, 9000 Ghent, Belgium.
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
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D016900:Neurofilament Proteins / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry
C114228:AVIL protein, human; D000199:Actins; D008840:Microfilament Proteins; D016900:Neurofilament Proteins; D010446:Peptide Fragments; C026615:villin; C115223:villin headpiece subdomain peptide; D014364:Tryptophan