Park Kyoung Ryoung, An Jun Yop, Kang Jung Youn, Lee Jung-Gyu, Lee Youngjin, Mun Sang A, Jun Chang-Duk, Song Woo Keun, Eom Soo Hyun
School of Life Science, Gwangju Institute of Science and Technology (GIST), 123 Cheomdangwagi-ro, Buk-gu, Gwangju 61005, Republic of Korea; Steitz Center for Structural Biology, Gwangju Institute of Science and Technology (GIST), 123 Cheomdangwagi-ro, Buk-gu, Gwangju 61005, Republic of Korea.
School of Life Science, Gwangju Institute of Science and Technology (GIST), 123 Cheomdangwagi-ro, Buk-gu, Gwangju 61005, Republic of Korea.
Biochem Biophys Res Commun. 2017 Jan 29;483(1):442-448. doi: 10.1016/j.bbrc.2016.12.124. Epub 2016 Dec 21.
EF-hand domain-containing protein D2/Swiprosin-1 (EFhd2) is an actin-binding protein mainly expressed in the central nervous and the immune systems of mammals. Intracellular events linked to EFhd2, such as membrane protrusion formation, cell adhesion, and BCR signaling, are triggered by the association of EFhd2 and F-actin. We previously reported that Ca enhances the F-actin-bundling ability of EFhd2 through maintaining a rigid parallel EFhd2-homodimer structure. It was also reported that the F-actin-bundling ability of EFhd2 is regulated by a phosphorylation-dependent mechanism. EGF-induced phosphorylation at Ser183 of EFhd2 has been shown to inhibit F-actin-bundling, leading to irregular actin dynamics at the leading edges of cells. However, the underlying mechanism of this inhibition has remained elusive. Here, we report the crystal structure of a phospho-mimicking mutant (S183E) of the EFhd2 core domain, where the actin-binding sites are located. Although the overall structure of the phospho-mimicking mutant is similar to the one of the unphosphorylated form, we observed a conformational transition from ordered to disordered structure in the linker region at the C-terminus of the mutant. Based on our structural and biochemical analyses, we suggest that phosphorylation at Ser183 of EFhd2 causes changes in the local conformational dynamics and the surface charge distribution of the actin-binding site, resulting in a re-coordination of the actin-binding sites in the dimer structure and a reduction of F-actin-bundling activity without affecting the F-actin-binding capacity.
含EF手型结构域蛋白D2/丝氨酸蛋白酶抑制蛋白-1（EFhd2）是一种肌动蛋白结合蛋白，主要在哺乳动物的中枢神经系统和免疫系统中表达。与EFhd2相关的细胞内事件，如膜突起形成、细胞黏附及BCR信号传导，由EFhd2与F-肌动蛋白的结合触发。我们之前报道过，钙离子通过维持刚性平行的EFhd2同源二聚体结构增强EFhd2的F-肌动蛋白束集能力。也有报道称，EFhd2的F-肌动蛋白束集能力受磷酸化依赖性机制调控。已证实，表皮生长因子（EGF）诱导的EFhd2第183位丝氨酸磷酸化会抑制F-肌动蛋白束集，导致细胞前缘肌动蛋白动力学不规则。然而，这种抑制的潜在机制仍不清楚。在此，我们报道了EFhd2核心结构域磷酸化模拟突变体（S183E）的晶体结构，肌动蛋白结合位点位于该结构域。虽然磷酸化模拟突变体的整体结构与未磷酸化形式相似，但我们观察到突变体C末端连接区从有序结构向无序结构的构象转变。基于我们的结构和生化分析，我们认为EFhd2第183位丝氨酸磷酸化导致肌动蛋白结合位点局部构象动力学和表面电荷分布发生变化，从而导致二聚体结构中肌动蛋白结合位点重新配位，降低F-肌动蛋白束集活性，而不影响F-肌动蛋白结合能力。
