Kim Min-Kyu, Kim Ji-Hye, Kim Ji-Sun, Kang Sa-Ouk
Laboratory of Biophysics, School of Biological Sciences, and Institute of Microbiology, Seoul National University, Seoul 151-742, Republic of Korea.
Acta Crystallogr D Biol Crystallogr. 2015 Sep;71(Pt 9):1835-49. doi: 10.1107/S139900471501264X. Epub 2015 Aug 25.
The crystal structure of the 34 kDa F-actin-bundling protein ABP34 from Dictyostelium discoideum was solved by Ca(2+)/S-SAD phasing and refined at 1.89 Å resolution. ABP34 is a calcium-regulated actin-binding protein that cross-links actin filaments into bundles. Its in vitro F-actin-binding and F-actin-bundling activities were confirmed by a co-sedimentation assay and transmission electron microscopy. The co-localization of ABP34 with actin in cells was also verified. ABP34 adopts a two-domain structure with an EF-hand-containing N-domain and an actin-binding C-domain, but has no reported overall structural homologues. The EF-hand is occupied by a calcium ion with a pentagonal bipyramidal coordination as in the canonical EF-hand. The C-domain structure resembles a three-helical bundle and superposes well onto the rod-shaped helical structures of some cytoskeletal proteins. Residues 216-244 in the C-domain form part of the strongest actin-binding sites (193-254) and exhibit a conserved sequence with the actin-binding region of α-actinin and ABP120. Furthermore, the second helical region of the C-domain is kinked by a proline break, offering a convex surface towards the solvent area which is implicated in actin binding. The F-actin-binding model suggests that ABP34 binds to the side of the actin filament and residues 216-244 fit into a pocket between actin subdomains -1 and -2 through hydrophobic interactions. These studies provide insights into the calcium coordination in the EF-hand and F-actin-binding site in the C-domain of ABP34, which are associated through interdomain interactions.
通过Ca(2+)/S-SAD相位法解析了盘基网柄菌中34 kDa F-肌动蛋白成束蛋白ABP34的晶体结构，并在1.89 Å分辨率下进行了精修。ABP34是一种钙调节的肌动蛋白结合蛋白，可将肌动蛋白丝交联成束。通过共沉降分析和透射电子显微镜证实了其体外F-肌动蛋白结合和F-肌动蛋白成束活性。还验证了ABP34与细胞中肌动蛋白的共定位。ABP34采用双结构域结构，包含一个含EF手型的N结构域和一个肌动蛋白结合C结构域，但没有报道的整体结构同源物。EF手型被一个钙离子占据，其配位方式为五角双锥，与典型的EF手型相同。C结构域结构类似于三螺旋束，与一些细胞骨架蛋白的杆状螺旋结构很好地重叠。C结构域中的残基216-244构成了最强肌动蛋白结合位点（193-254）的一部分，并与α-辅肌动蛋白和ABP120的肌动蛋白结合区域具有保守序列。此外，C结构域的第二个螺旋区域因脯氨酸断裂而发生扭结，形成一个朝向溶剂区域的凸面，这与肌动蛋白结合有关。F-肌动蛋白结合模型表明，ABP34结合到肌动蛋白丝的侧面，残基216-244通过疏水相互作用嵌入肌动蛋白亚结构域-1和-2之间的一个口袋中。这些研究为ABP34的EF手型中的钙配位和C结构域中的F-肌动蛋白结合位点提供了见解，它们通过结构域间相互作用相关联。
