From the Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037.
Center for Systems and Computational Biology, The Wistar Institute, Philadelphia, Pennsylvania 19104.
J Biol Chem. 2010 Oct 22;285(43):33265-33280. doi: 10.1074/jbc.M110.144873. Epub 2010 Jul 21.
Many actin-binding proteins have been shown to possess multiple activities to regulate filament dynamics. Tropomodulins (Tmod1-4) are a conserved family of actin filament pointed end-capping proteins. Our previous work has demonstrated that Tmod3 binds to monomeric actin in addition to capping pointed ends. Here, we show a novel actin-nucleating activity in mammalian Tmods. Comparison of Tmod isoforms revealed that Tmod1-3 but not Tmod4 nucleate actin filament assembly. All Tmods bind to monomeric actin, and Tmod3 forms a 1:1 complex with actin. By truncation and mutagenesis studies, we demonstrated that the second α-helix in the N-terminal domain of Tmod3 is essential for actin monomer binding. Chemical cross-linking and LC-MS/MS further indicated that residues in this second α-helix interact with actin subdomain 2, whereas Tmod3 N-terminal domain peptides distal to this α-helix interact with actin subdomain 1. Mutagenesis of Leu-73 to Asp, which disrupts the second α-helix of Tmod3, decreases both its actin monomer-binding and -nucleating activities. On the other hand, point mutations of residues in the C-terminal leucine-rich repeat domain of Tmod3 (Lys-317 in the fifth leucine-rich repeat β-sheet and Lys-344 or Arg-345/Arg-346 in the C-terminal α6-helix) significantly reduced pointed end-capping and nucleation without altering actin monomer binding. Taken together, our data indicate that Tmod3 binds actin monomers over an extended interface and that nucleating activity depends on actin monomer binding and pointed end-capping activities, contributed by N- and C-terminal domains of Tmod3, respectively. Tmod3 nucleation of actin assembly may regulate the cytoskeleton in dynamic cellular contexts.
许多肌动蛋白结合蛋白已被证明具有多种活性，以调节纤维丝动态。原肌球蛋白（Tmod1-4）是肌动蛋白纤维丝尖端帽状蛋白的一个保守家族。我们之前的工作表明，Tmod3 除了帽状纤维丝的尖端外，还与单体肌动蛋白结合。在这里，我们展示了哺乳动物 Tmod 中的一种新的肌动蛋白成核活性。Tmod 同工型的比较表明，Tmod1-3 而不是 Tmod4 成核肌动蛋白丝组装。所有 Tmod 都与单体肌动蛋白结合，Tmod3 与肌动蛋白形成 1:1 的复合物。通过截短和突变研究，我们证明了 Tmod3 N 端结构域中的第二个α-螺旋对于肌动蛋白单体结合是必不可少的。化学交联和 LC-MS/MS 进一步表明，该第二个α-螺旋中的残基与肌动蛋白亚基 2 相互作用，而 Tmod3 N 端结构域中远离该α-螺旋的肽与肌动蛋白亚基 1 相互作用。将 Tmod3 中的 Leu-73 突变为 Asp，破坏了 Tmod3 的第二个α-螺旋，降低了其肌动蛋白单体结合和成核活性。另一方面，Tmod3 C 端富含亮氨酸重复结构域中的残基（第五个富含亮氨酸重复β-折叠中的 Lys-317 和 C 端α6-螺旋中的 Lys-344 或 Arg-345/Arg-346）的点突变显著降低了尖端帽状和成核作用，而不改变肌动蛋白单体结合。总之，我们的数据表明，Tmod3 在扩展的界面上结合肌动蛋白单体，并且成核活性取决于肌动蛋白单体结合和 Tmod3 的 N 端和 C 端结构域分别贡献的尖端帽状活性。Tmod3 成核肌动蛋白组装可能调节动态细胞环境中的细胞骨架。
