Isolation and properties of two actin-binding domains in gelsolin.
Gelsolin is a Ca2+-sensitive 90-kDa protein which regulates actin filament length. A molecular variant of gelsolin is present in plasma as a 93-kDa protein. Functional studies have shown that gelsolin contains two actin-binding sites which are distinct in that after Ca2+-mediated binding, removal of free Ca2+ releases actin from one site but not from the other. We have partially cleaved human plasma gelsolin with alpha-chymotrypsin and identified two distinct actin-binding domains. Peptides CT17 and CT15, which contain one of the actin-binding domains, bind to actin independently of Ca2+; peptides CT54 and CT47, which contain the other domain, bind to actin reversibly in response to changes in Ca2+ concentration. These peptides sequester actin monomers inhibiting polymerization. Unlike intact gelsolin, neither group of peptides nucleates actin assembly or forms stable filament end caps. CT17 and CT15 can however sever actin filaments. Amino acid sequence analyses place CT17 at the NH2 terminus of gelsolin and CT47 at the carboxyl-terminal two-thirds of gelsolin. Circular dichroism measurements show that Ca2+ induces an increase in the alpha-helical content of CT47. These studies provide a structural basis for understanding the interaction of gelsolin with actin and allow comparison with other Ca2+-dependent actin filament severing proteins.
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000032:analysis*; D002846:Chromatography, Affinity; D002918:Chymotrypsin / Q000378:metabolism; D002942:Circular Dichroism; D003488:Cyanogen Bromide / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D005470:Fluorometry; D018260:Gelsolin; D006801:Humans; D008840:Microfilament Proteins / Q000032:analysis*; D011108:Polymers / Q000378:metabolism; D011487:Protein Conformation
D000199:Actins; D002135:Calcium-Binding Proteins; D018260:Gelsolin; D008840:Microfilament Proteins; D011108:Polymers; D004533:Egtazic Acid; D002918:Chymotrypsin; D003488:Cyanogen Bromide; D002118:Calcium