Molecular mechanism for inhibition of twinfilin by phosphoinositides
Molecular mechanism for inhibition of twinfilin by phosphoinositides
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DOI：
10.1074/jbc.ra117.000484
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发表时间：
2018-03-30
影响因子：
4.8
通讯作者：
Lappalainen, Pekka
中科院分区：
文献类型：
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作者：
Hakala, Markku;Kalimeri, Maria;Lappalainen, Pekka
膜磷酸肌醇通过调节几种关键的肌动蛋白结合蛋白的活性来控制肌动蛋白细胞骨架的组织和动力学。双联丝蛋白是一种进化上保守的蛋白质，它通过与肌动蛋白单体、丝状体以及异二聚体封端蛋白相互作用来促进细胞骨架动力学。双联丝蛋白也结合磷酸肌醇，这会抑制它与肌动蛋白的相互作用，但其潜在机制一直未知。在此，我们表明双联丝蛋白对磷酸肌醇的高亲和力结合位点位于C末端尾部区域，而双联丝蛋白的两个肌动蛋白解聚因子（ADF）/类辅肌动蛋白的ADF同源结构域仅以低亲和力结合磷酸肌醇。诱变和生化实验结合原子分子动力学模拟揭示，双联丝蛋白的C末端尾部通过多价静电相互作用与膜相互作用，且优先与磷脂酰肌醇3,5 - 二磷酸（PI(3,5)P₂）、磷脂酰肌醇4,5 - 二磷酸（PI(4,5)P₂）和磷脂酰肌醇3,4,5 - 三磷酸（PI(3,4,5)P₃）相互作用。这种初始相互作用使双联丝蛋白的肌动蛋白结合ADF同源结构域靠近膜，并随后促进它们与膜的结合，从而导致对肌动蛋白相互作用的抑制。为支持这一模型，一个缺失C末端尾部的双联丝蛋白突变体以一种对磷酸肌醇不敏感的方式抑制肌动蛋白丝的组装。我们的诱变数据还表明，磷酸肌醇和封端蛋白结合位点在双联丝蛋白的C末端尾部重叠，这表明磷酸肌醇结合还抑制了双联丝蛋白与异二聚体封端蛋白的相互作用。结果表明，双联丝蛋白保守的C末端尾部是一个多功能结合基序，它对于与异二聚体封端蛋白的相互作用以及将双联丝蛋白拴系在富含磷酸肌醇的膜上至关重要。
Membrane phosphoinositides control organization and dynamics of the actin cytoskeleton by regulating the activities of several key actin-binding proteins. Twinfilin is an evolutionarily conserved protein that contributes to cytoskeletal dynamics by interacting with actin monomers, filaments, and the heterodimeric capping protein. Twinfilin also binds phosphoinositides, which inhibit its interactions with actin, but the underlying mechanism has remained unknown. Here, we show that the high-affinity binding site of twinfilin for phosphoinositides is located at the C-terminal tail region, whereas the two actin-depolymerizing factor (ADF)/cofilin-like ADF homology domains of twinfilin bind phosphoinositides only with low affinity. Mutagenesis and biochemical experiments combined with atomistic molecular dynamics simulations reveal that the C-terminal tail of twinfilin interacts with membranes through a multivalent electrostatic interaction with a preference toward phosphatidylinositol 3,5-bisphosphate (PI(3,5)P-2), PI(4,5)P-2, and PI(3,4,5)P-3. This initial interaction places the actin-binding ADF homology domains of twinfilin in close proximity to the membrane and subsequently promotes their association with the membrane, thus leading to inhibition of the actin interactions. In support of this model, a twinfilin mutant lacking the C-terminal tail inhibits actin filament assembly in a phosphoinositide-insensitive manner. Our mutagenesis data also reveal that the phosphoinositide-and capping protein-binding sites overlap in the C-terminal tail of twinfilin, suggesting that phosphoinositide binding additionally inhibits the interactions of twinfilin with the heterodimeric capping protein. The results demonstrate that the conserved C-terminal tail of twinfilin is a multifunctional binding motif, which is crucial for interaction with the heterodimeric capping protein and for tethering twinfilin to phosphoinositide-rich membranes.