A Thermostable 35-Residue Subdomain within Villin Headpiece

Abstract

The actin-bundling protein villin contains, at its extreme C terminus, a compact f-actin binding domain called "headpiece". This 76-amino acid domain from chicken is highly thermostable. Here, we show that the stable folded structure in headpiece is localized to a subdomain formed by the C-terminal 35 residues. The subdomain, denoted HP-35, is monomeric and retains high thermostability, with a Tm of 70( +/- 1) degree C at PH 7.0. There are no cysteine residues in HP-35 and its folding is not dependent on the binding of metals or other ligands. HP-35 is not a molten globule, but instead, has properties expected for a fully folded protein with a unique structure. In particular, the slowly exchanging amide protons in HP-35 have protection factors that are slightly larger than those predicted if exchange occurred only from globally unfolded molecules. NMR studies indicate that the headpiece subdomain contains three short alpha-helices, and that these same helices are present in the corresponding regions of intact headpiece. HP-35 is the smallest monomeric polypeptide characterized consisting of only naturally occurring amino acids that autonomously folds into a unique and thermostable structure without disulfide bonds or ligand binding.