Fowler Velia M, Greenfield Norma J, Moyer Jeannette
Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
J Biol Chem. 2003 Oct 10;278(41):40000-9. doi: 10.1074/jbc.M306895200. Epub 2003 Jul 14.
Tropomodulin 1 (Tmod1) is a approximately 40-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 (Kd approximately 50 pm) is several thousand-fold greater than for capping of pure actin filaments (Kd approximately 0.1 microM). 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 (Kd approximately 0.4 microM), 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 (Kd approximately 1.8 microM). 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.
原肌球蛋白调节蛋白1（Tmod1）是一种分子量约为40 kDa的原肌球蛋白结合蛋白，可封闭肌动蛋白丝的尖端，调节其尖端动力学，并控制横纹肌中细肌丝的长度。在体外，Tmod1对原肌球蛋白 - 肌动蛋白丝的封闭亲和力（解离常数Kd约为50 pm）比对纯肌动蛋白丝的封闭亲和力（Kd约为0.1 μM）高数千倍。Tmod1的原肌球蛋白结合区域定位于第1至130位残基之间的氨基末端部分，但肌动蛋白封闭结构域的位置尚不清楚。我们现在通过使用芘 - 肌动蛋白聚合测定法测试一系列重组Tmod1片段抑制凝溶胶蛋白 - 肌动蛋白种子引发的肌动蛋白伸长的能力，确定了Tmod1上两个不同的肌动蛋白封闭区域。Tmod1的羧基末端部分（第160 - 359位残基）具有主要的肌动蛋白封闭活性（Kd约为0.4 μM），其完整活性需要第323至359位残基，而Tmod1的氨基末端部分（第1至130位残基）具有第二种较弱的肌动蛋白封闭活性（Kd约为1.8 μM）。有趣的是，第160 - 359位残基区域而非第1至130位残基区域增强了肌动蛋白的自发成核作用，这表明羧基末端结构域可能在尖端跨肌动蛋白螺旋与两个肌动蛋白亚基结合，而氨基末端结构域可能仅与一个肌动蛋白亚基结合。另一方面，在骨骼肌原肌球蛋白存在的情况下，Tmod1氨基末端部分而非羧基末端部分的肌动蛋白封闭活性增强了数千倍。我们得出结论，Tmod1的羧基末端封闭结构域包含一种不依赖原肌球蛋白的肌动蛋白尖端封闭活性，而氨基末端结构域包含一种受原肌球蛋白调节的尖端肌动蛋白封闭活性。
