Localization of the calcium-sensitive actin monomer binding site in gelsolin to segment 4 and identification of calcium binding sites.
Gelsolin is composed of six repeating segments of sequence (G1-6) and contains three distinct actin binding sites, two that bind to G-actin and one that binds to filaments. The calcium-dependent actin monomer binding site present in the carboxyl-terminal half of the protein (G4-6) plays a critical role both in the cooperative binding of actin by gelsolin and in its nucleating activity. Here we have localized this actin binding site to segment 4 (G4) by expressing the segments G4, G4-5, G5, and G5-6 in Escherichia coli and analyzing their actin binding properties. In addition we have measured their calcium binding. G4-5 and G5-6 each bind a single calcium ion, but there is no binding by G4 or G5. The affinity of binding by G5-6 is 10 times higher than that of G4-5, and calcium binding by G4-6 shows two sites of different affinity. Thus each actin binding site of gelsolin is restricted to a single segment (G1, G2, and G4), but the nonbinding segments G5 and G6 play an important role in the calcium regulation of actin binding and other activities of gelsolin.
Pope
B
B
MRC Laboratory of Molecular Biology, Cambridge, U.K.
Maciver
S
S
Weeds
A
A
D000199:Actins / Q000737:chemistry*; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000737:chemistry*; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D018260:Gelsolin / Q000737:chemistry*; D006801:Humans; D011485:Protein Binding; D012091:Repetitive Sequences, Nucleic Acid; D017421:Sequence Analysis
D000199:Actins; D018076:DNA, Complementary; D018260:Gelsolin; D002118:Calcium