title: Calcium-sensitive activity and conformation of Caenorhabditis elegans gelsolin-like protein 1 are altered by mutations in the first gelsolin-like domain.
abstract: The gelsolin family of actin regulatory proteins is activated by Ca(2+) to sever and cap actin filaments. Gelsolin has six homologous gelsolin-like domains (G1-G6), and Ca(2+)-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 Ca(2+)-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 Ca(2+) 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 Ca(2+) sensitivity when compared with wild-type GSNL-1. The D83A/D84A mutation enhanced Ca(2+) 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 Ca(2+)-free and Ca(2+)-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.
authors.affiliation: Department of Pathology and Cell Biology, Emory University, Atlanta, Georgia 30322, USA.
authors.forename: Zhongmei
authors.initials: Z
authors.lastname: Liu
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Kanzawa
authors.forename: Shoichiro
authors.initials: S
authors.lastname: Ono
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D017173:Caenorhabditis elegans / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D052139:Intracellular Calcium-Sensing Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011817:Rabbits
chemical_list: D029742:Caenorhabditis elegans Proteins; C532447:GSNL-1 protein, C elegans; D052139:Intracellular Calcium-Sensing Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D002118:Calcium