Novel inter-domain Ca2+-binding site in the gelsolin superfamily protein fragmin.

Gelsolin superfamily proteins, consisting of multiple domains (usually six), sever actin filaments and cap the barbed ends in a Ca2+-dependent manner. Two types of evolutionally conserved Ca2+-binding sites have been identified in this family; type-1 (between gelsolin and actin) and type-2 (within the gelsolin domain). Fragmin, a member in the slime mold Physarum polycephalum, consists of three domains (F1–F3) that are highly similar to the N-terminal half of mammalian gelsolin (G1–G3). Despite their similarities, the two proteins exhibit a significant difference in the Ca2+ dependency; F1–F3 absolutely requires Ca2+ for the filament severing whereas G1–G3 does not. In this study, we examined the strong dependency of fragmin on Ca2+ using biochemical and structural approaches. Our co-sedimentation assay demonstrated that Ca2+ significantly enhanced the binding of F2–F3 to actin. We determined the crystal structure of F2–F3 in the presence of Ca2+. F2–F3 binds a total of three calcium ions; while two are located in type-2 sites within F2 or F3, the remaining one resides between the F2 long helix and the F3 short helix. The inter-domain Ca2+-coordination appears to stabilize F2-F3 in a closely packed configuration. Notably, the F3 long helix exhibits a bent conformation which is different from the straight G3 long helix in the presence of Ca2+. Our results provide the first structural evidence for the existence of an unconventional Ca2+-binding site in the gelsolin superfamily proteins.