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

Shoichiro Ono,Zhongmei Liu,Nobuyuki Kanzawa

doi:10.1074/jbc.m111.237404

Shoichiro Ono, Zhongmei Liu + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m111.237404

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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.

Highlights

The gelsolin family of actin-severing/capping proteins is calcium-regulated
We demonstrated that Asp-83 and Asp-84 of C. elegans gelsolin-like protein-1 (GSNL-1) are dispensable for actin filament-severing activity but important for Ca2ϩ-dependent activation and conformational changes of GSNL-1
Mutations at Asp-83 and Asp-84 caused remarkable changes in the conformation as determined by susceptibility to chymotrypsin digestion, suggesting specific roles for Asp-83 and Asp-84 in maintaining Ca2ϩ-free and Ca2ϩ-bound conformations, respectively. Because these aspartic acid residues are conserved in vertebrate gelsolin, we expect that they have similar regulatory functions for Ca2ϩ-dependent properties of vertebrate gelsolin

Summary

Background

Results: Two acidic residues of Caenorhabditis elegans gelsolin-like protein 1 (GSNL-1) were important for calcium regulation. 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 have previously reported that Caenorhabditis elegans gelsolin-like protein-1 (GSNL-1), which has only four G domains (G1–G4), severs actin filaments and caps barbed ends in a similar manner to gelsolin [14]. Calcium Regulation of GSNL-1 actin filaments in a Ca2ϩ-dependent manner [15], whereas the equivalent fragments of vertebrate gelsolin exhibit Ca2ϩ-independent actin-severing activity [17, 18]. We propose that this sequence is an important determinant for maintaining active and inactive conformations of the gelsolin family

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DISCUSSION