Department of Molecular Physiology and Biophysics, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
J Biol Chem. 2010 Dec 24;285(52):40654-65. doi: 10.1074/jbc.M110.166041. Epub 2010 Oct 22.
Catch force maintenance in invertebrate smooth muscles is probably mediated by a force-bearing tether other than myosin cross-bridges between thick and thin filaments. The phosphorylation state of the mini-titin twitchin controls catch. The C-terminal phosphorylation site (D2) of twitchin with its flanking Ig domains forms a phosphorylation-sensitive complex with actin and myosin, suggesting that twitchin is the tether (Funabara, D., Osawa, R., Ueda, M., Kanoh, S., Hartshorne, D. J., and Watabe, S. (2009) J. Biol. Chem. 284, 18015-18020). Here we show that a region near the N terminus of twitchin also interacts with thick and thin filaments from Mytilus anterior byssus retractor muscles. Both a recombinant protein, including the D1 and DX phosphorylation sites with flanking 7th and 8th Ig domains, and a protein containing just the linker region bind to thin filaments with about a 1:1 mol ratio to actin and K(d) values of 1 and 15 μM, respectively. Both proteins show a decrease in binding when phosphorylated. The unphosphorylated proteins increase force in partially activated permeabilized muscles, suggesting that they are sufficient to tether thick and thin filaments. There are two sites of thin filament interaction in this region because both a 52-residue peptide surrounding the DX site and a 47-residue peptide surrounding the D1 site show phosphorylation-dependent binding to thin filaments. The peptides relax catch force, confirming the region's central role in the mechanism of catch. The multiple sites of thin filament interaction in the N terminus of twitchin in addition to those in the C terminus provide an especially secure and redundant mechanical link between thick and thin filaments in catch.
在无脊椎动物平滑肌中，力的维持可能是通过一种力承载的系绳来实现的，而不是肌球蛋白横桥在粗丝和细丝之间。迷你肌球蛋白抽搐的磷酸化状态控制着捕获。抽搐的 C 端磷酸化位点（D2）及其侧翼免疫球蛋白结构域与肌动蛋白和肌球蛋白形成一个磷酸化敏感的复合物，这表明抽搐是系绳（Funabara，D.，Osawa，R.，Ueda，M.，Kanoh，S.，Hartshorne，D. J.，和 Watabe，S.（2009）J. Biol. Chem. 284，18015-18020）。在这里，我们展示了抽搐的 N 端附近的一个区域也与贻贝前闭壳肌的粗丝和细丝相互作用。包括 D1 和 DX 磷酸化位点及其侧翼第 7 和第 8 个免疫球蛋白结构域的重组蛋白，以及仅包含连接区的蛋白，与肌动蛋白以约 1:1 的摩尔比结合，Kd 值分别为 1 和 15 μM。两种蛋白在磷酸化后结合能力均下降。未磷酸化的蛋白增加部分激活的通透肌肉的力，这表明它们足以将粗丝和细丝系紧。该区域有两个细丝结合点，因为 DX 位点周围的 52 个残基肽和 D1 位点周围的 47 个残基肽都显示出与细丝的磷酸化依赖性结合。这些肽放松了捕获力，证实了该区域在捕获机制中的核心作用。抽搐的 N 端除了 C 端的多个细丝结合点外，还为粗丝和细丝之间提供了一个特别安全和冗余的机械连接，以保持捕获状态。
