Regulatory role of the second gelsolin‐like domain of Caenorhabditis elegans gelsolin‐like protein 1 (GSNL‐1) in its calcium‐dependent conformation and actin‐regulatory activities

Caenorhabditis elegans gelsolin‐like protein‐1 (GSNL‐1) is an unconventional member of the gelsolin family of actin‐regulatory proteins. Unlike typical gelsolin‐related proteins with three or six G domains, GSNL‐1 has four gelsolin‐like (G) domains (G1–G4) and exhibits calcium‐dependent actin filament severing and capping activities. The first G domain (G1) of GSNL‐1 is necessary for its actin‐regulatory activities. However, how other domains in GSNL‐1 participate in regulation of its functions is not understood. Here, we report biochemical evidence that the second G domain (G2) of GSNL‐1 has a regulatory role in its calcium‐dependent conformation and actin‐regulatory activities. Comparison of the sequences of gelsolin‐related proteins from various species indicates that sequences of G2 are highly conserved. Among the conserved residues in G2, we focused on D162 of GSNL‐1, since equivalent residues in gelsolin and severin are part of the calcium‐binding sites and is a pathogenic mutation site in human gelsolin causing familial amyloidosis, Finish‐type. The D162N mutation does not alter the inactive and fully calcium‐activated states of GSNL‐1 for actin filament severing (at 20 nM GSNL‐1) and capping activities (at 50 nM GSNL‐1). However, under these conditions, the mutant shows reduced calcium sensitivity for activation. By contrast, the D162N mutation strongly enhances susceptibility of GSNL‐1 to chymotrypsin digestion only at high calcium concentrations but not at low calcium concentrations. The mutation also reduces affinity of GSNL‐1 with actin monomers. These results suggest that G2 of GSNL‐1 functions as a regulatory domain for its calcium‐dependent actin‐regulatory activities by mediating conformational changes of the GSNL‐1 molecule. © 2013 Wiley Periodicals, Inc