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

AbstractHeadpiece (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.