Calcium-sensitive Activity and Conformation of Caenorhabditis elegans Gelsolin-like Protein 1 Are Altered by Mutations in the First Gelsolin-like Domain

摘要

The gelsolin family of actin regulatory proteins is activated by Ca²⁺ to sever and cap actin filaments. Gelsolin has six homologous gelsolin-like domains (G1–G6), and Ca²⁺-dependent conformational changes regulate its accessibility to actin. Caenorhabditis elegans gelsolin-like protein-1 (GSNL-1) has only four gelsolin-like domains (G1–G4) and still exhibits Ca²⁺-dependent actin filament-severing and -capping activities. We found that acidic residues (Asp-83 and Asp-84) in G1 of GSNL-1 are important for its Ca²⁺ activation. These residues are conserved in GSNL-1 and gelsolin and previously implicated in actin-severing activity of the gelsolin family. We found that alanine mutations at Asp-83 and Asp-84 (D83A/D84A mutation) did not disrupt actin-severing or -capping activity. Instead, the mutants exhibited altered Ca²⁺ sensitivity when compared with wild-type GSNL-1. The D83A/D84A mutation enhanced Ca²⁺ sensitivity for actin severing and capping and its susceptibility to proteolytic digestion, suggesting a conformational change. Single mutations caused minimal changes in its activity, whereas Asp-83 and Asp-84 were required to stabilize Ca²⁺-free and Ca²⁺-bound conformations, respectively. On the other hand, the D83A/D84A mutation suppressed sensitivity of GSNL-1 to phosphatidylinositol 4,5-bisphosphate inhibition. The structure of an inactive form of gelsolin shows that the equivalent acidic residues are in close contact with G3, which may maintain an inactive conformation of the gelsolin family.