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.
MRC Laboratory of Molecular Biology, Cambridge, U.K.
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Pope
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Maciver
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Weeds
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