An actin footprint on villin. Single site substitutions in a cluster of basic residues inhibit the actin severing but not capping activity of villin.
Villin is a multidomain protein that severs, caps, and bundles actin filaments. We employed a chemical modification/cleavage strategy to identify residues whose chemical reactivities are reduced when villin is complexed with actin. We found that actin protects 3 methionine residues, Met125, Met379, and Met711 from oxidation by N-chlorosuccinimide. Because Met125 lies within the actin-severing domain of villin (44T), we probed this region for actin binding sites using a series of overlapping peptides each with an additional cysteine residue at their C terminus. Each peptide, as a disulfide-bonded dimer, was examined for actin cross-linking activity by electron microscopy and light scattering. Our results with M3R suggest this region contains an F-actin binding site and are consistent with proteolysis and deletion mutagenesis studies of gelsolin. Single substitution of the basic residues modulated actin severing but not capping activity of 44T. Circular dichroism and protease digestions did not detect alterations in secondary structure or conformational changes in the mutants, although some are cleaved more rapidly, thereby suggesting a change in the packing of the domains. Our results highlight that basic residues comprise part of the F-actin binding site that is involved in the actin severing activity of villin.
de Arruda
M V
MV
Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge 02142.
Bazari
H
H
Wallek
M
M
Matsudaira
P
P
D000199:Actins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002645:Chickens; D002942:Circular Dichroism; D003488:Cyanogen Bromide; D004563:Electrochemistry; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D008715:Methionine / Q000378:metabolism; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D013388:Succinimides / Q000494:pharmacology
D000199:Actins; D002352:Carrier Proteins; D008840:Microfilament Proteins; D013388:Succinimides; C026615:villin; C015752:N-chlorosuccinimide; D008715:Methionine; D003488:Cyanogen Bromide