Tropomodulin contains two actin filament pointed end-capping domains
Tropomodulin contains two actin filament pointed end-capping domains
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DOI：
10.1074/jbc.m306895200
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发表时间：
2003-10-10
影响因子：
4.8
通讯作者：
Moyer, J
中科院分区：
文献类型：
--
作者：
Fowler, VM;Greenfield, NJ;Moyer, J
原调节蛋白1 （Tropomodulin 1, Tmod1）是一种类似于40 kda原肌球蛋白结合蛋白和肌动蛋白细丝尖端封盖蛋白的蛋白，在横纹肌中调节尖端动力学和控制细丝长度。在体外，Tmod1对原肌球蛋白-肌动蛋白丝的旋盖亲和力（K-d近似于50 pM）比对纯肌动蛋白丝的旋盖亲和力（K-d近似于0.1 muM）高几千倍。Tmod1的原肌球蛋白结合区定位于残基1和130之间的氨基末端部分，但肌动蛋白capping结构域的位置尚不清楚。我们现在已经确定了Tmod1上的两个不同的肌动蛋白封盖区域，通过测试一系列重组Tmod1片段，用pyrenee -actin聚合测定它们抑制凝胶-肌动蛋白种子的肌动蛋白延伸的能力。Tmod1的羧基末端部分（残基160-359）具有主要的肌动蛋白旋盖活性（K-d类似于0.4 muM），需要残基在323 -359之间才能完全发挥活性，而Tmod1的氨基末端部分（残基1-130）具有第二弱的肌动蛋白旋盖活性（K-d类似于1.8 muM）。有趣的是，160-359而不是1-130增强了肌动蛋白自发成核，这表明羧基末端结构域可以结合两个肌动蛋白亚基，而氨基末端结构域可能只结合一个肌动蛋白亚基。另一方面，在骨骼肌原肌球蛋白的存在下，Tmod1的氨基末端而不是羧基末端的肌动蛋白封顶活性增强了数千倍。我们得出结论，Tmod1的羧基末端旋盖结构域包含一个与tm无关的肌动蛋白末端旋盖活性，而氨基末端结构域包含一个tm调节的肌动蛋白末端旋盖活性。
Tropomodulin 1 (Tmod1) is a similar to40-kDa tropomyosin binding and actin filament pointed end-capping protein that regulates pointed end dynamics and controls thin filament length in striated muscle. In vitro, the capping affinity of Tmod1 for tropomyosin-actin filaments (K-d similar to 50 pM) is several thousand-fold greater than for capping of pure actin filaments (K-d similar to 0.1 muM). The tropomyosin-binding region of Tmod1 has been localized to the amino-terminal portion between residues 1 and 130, but the location of the actin-capping domain is not known. We have now identified two distinct actin-capping regions on Tmod1 by testing a series of recombinant Tmod1 fragments for their ability to inhibit actin elongation from gelsolin-actin seeds using pyrene-actin polymerization assays. The carboxyl-terminal portion of Tmod1 (residues 160-359) contains the principal actin-capping activity (K-d similar to 0.4 muM), requiring residues between 323 and 359 for full activity, whereas the amino-terminal portion of Tmod1 (residues 1-130) contains a second, weaker actin-capping activity (K-d similar to 1.8 muM). Interestingly, 160-359 but not 1-130 enhances spontaneous actin nucleation, suggesting that the carboxyl-terminal domain may bind to two actin subunits across the actin helix at the pointed end, whereas the amino-terminal domain may bind to only one actin subunit. On the other hand, the actin-capping activity of the amino-terminal but not the carboxyl-terminal portion of Tmod1 is enhanced several thousand-fold in the presence of skeletal muscle tropomyosin. We conclude that the carboxyl-terminal capping domain of Tmod1 contains a TM-independent actin pointed end-capping activity, whereas the amino-terminal domain contains a TM-regulated pointed end actin-capping activity.