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.

中文翻译：

---

凝溶胶蛋白超家族蛋白fragmin中新的域间Ca 2+结合位点。

凝溶胶蛋白超家族蛋白，由多个结构域（通常为六个）组成，切断肌动蛋白丝，并以Ca ²⁺依赖性方式覆盖带刺的末端。在该家族中已经鉴定出两种类型的进化保守的Ca ²⁺结合位点。1型（凝溶胶蛋白和肌动蛋白之间）和2型（凝溶胶蛋白域内）。Fragmin是粘液霉菌Physarum polycephalum的成员，由三个域（F1-F3）组成，与哺乳动物凝溶胶蛋白（G1-G3）N端的一半高度相似。尽管它们具有相似性，但这两种蛋白在Ca ²⁺依赖性上仍表现出显着差异。F1-F3绝对需要Ca ²⁺切断灯丝，而G1-G3则不行。在这项研究中，我们使用生化和结构方法研究了香精素对Ca ²⁺的强烈依赖性。我们的共同沉淀试验表明，Ca ²⁺显着增强了F2-F3与肌动蛋白的结合。我们确定了在Ca ²⁺存在下F2-F3的晶体结构。F2-F3总共结合三个钙离子。虽然其中两个位于F2或F3的2型位点，但其余一个位于F2长螺旋和F3短螺旋之间。域间Ca ²⁺配位似乎以紧密堆积的构型稳定了F2-F3。值得注意的是，在存在Ca的情况下，F3长螺旋的弯曲构象与直G3长螺旋不同。²⁺。我们的结果为凝溶胶蛋白超家族蛋白中存在非常规的Ca ²⁺结合位点提供了第一个结构证据。