Cysteine scanning mutagenesis at 40 of 76 positions in villin headpiece maps the F-actin binding site and structural features of the domain.
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).
Doering
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DS
Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge 02142, USA. doering@aquapharm.com
Matsudaira
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D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002645:Chickens; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D000199:Actins; D002352:Carrier Proteins; D008840:Microfilament Proteins; D011994:Recombinant Proteins; C026615:villin; D003545:Cysteine