RESUMEN
Villin headpiece, the 76 amino acid, C-terminal domain of villin, is one of the two F-actin binding sites in villin necessary for F-actin bundling activity. Expression and study of recombinant headpiece revealed the domain to be remarkably thermostable (Tm = 74 degrees C) for a non-disulfide-bonded domain. Forty independent point mutations to cysteine of headpiece have been purified and tested for their actin binding activity, cysteine reactivity, and thermal stability. These assays identify two segments of headpiece, near amino acids 38 and 70 of headpiece, in which mutations to cysteine significantly disrupt cosedimentation of headpiece with F-actin. Assay of the thermal stability of these mutants and assay of the reactivity of the introduced cysteine show that these amino acids are mutations at the protein surface that do not perturb the overall structure of the domain. The actin binding mutants are replacements to cysteine of Lys38, Glu39, Lys65, Lys70, Lys71, Leu75, and Phe76 of headpiece. We propose that these discontinuous segments of charged amino acids define the F-actin binding contacts of the headpiece domain. The assay of mutants for effects on the thermal stability of helical structure as well as the assay of reactivity of the introduced sulfhydryl group identify candidate positions that are involved in the stabilizing core and internal structure of the domain. The cysteine scanning mutagenesis also identifies an amino-terminal subdomain (Val1-Leu35) and a predominantly helical carboxy-terminal subdomain (Pro36-Phe76).
Asunto(s)
Actinas/metabolismo , Proteínas Portadoras/química , Cisteína/química , Proteínas de Microfilamentos/química , Conformación Proteica , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Pollos , Dicroismo Circular , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Desnaturalización Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , TemperaturaRESUMEN
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.