Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Proc Natl Acad Sci U S A. 2012 Dec 11;109(50):E3424-33. doi: 10.1073/pnas.1203035109. Epub 2012 Nov 16.
Formin proteins and their associated factors cooperate to assemble unbranched actin filaments in diverse cellular structures. The Saccharomyces cerevisiae formin Bni1 and its associated nucleation-promoting factor (NPF) Bud6 generate actin cables and mediate polarized cell growth. Bud6 binds to both the tail of the formin and G-actin, thereby recruiting monomeric actin to the formin to create a nucleation seed. Here, we structurally and functionally dissect the nucleation-promoting C-terminal region of Bud6 into a Bni1-binding "core" domain and a G-actin binding "flank" domain. The ∼2-Å resolution crystal structure of the Bud6 core domain reveals an elongated dimeric rod with a unique fold resembling a triple-helical coiled-coil. Binding and actin-assembly assays show that conserved residues on the surface of this domain mediate binding to Bni1 and are required for NPF activity. We find that the Bni1 dimer binds two Bud6 dimers and that the Bud6 flank binds a single G-actin molecule. These findings suggest a model in which a Bni1/Bud6 complex with a 2:4 subunit stoichiometry assembles a nucleation seed with Bud6 coordinating up to four actin subunits.
formin 蛋白及其相关因子合作组装不同细胞结构中的无分支肌动蛋白丝。酿酒酵母formin Bni1 及其相关的成核促进因子（NPF）Bud6 生成肌动蛋白电缆并介导极化细胞生长。Bud6 结合formin 的尾部和 G-肌动蛋白，从而将单体肌动蛋白募集到 formin 上以形成成核种子。在这里，我们从结构和功能上对 Bud6 的成核促进 C 端区域进行了细分，得到了一个与 Bni1 结合的“核心”结构域和一个与 G-肌动蛋白结合的“侧翼”结构域。Bud6 核心结构域的 ∼2-Å 分辨率晶体结构揭示了一个细长的二聚体棒，具有独特的折叠，类似于三螺旋卷曲螺旋。结合和肌动蛋白组装测定表明，该结构域表面上的保守残基介导与 Bni1 的结合，并且是 NPF 活性所必需的。我们发现 Bni1 二聚体结合两个 Bud6 二聚体，而 Bud6 侧翼结合一个单个 G-肌动蛋白分子。这些发现表明，具有 2:4 亚基比例的 Bni1/Bud6 复合物组装成具有 Bud6 协调多达四个肌动蛋白亚基的成核种子的模型。
