Department of Biology, University of Konstanz, Konstanz, Germany.
Division of Cardiology, School of Medicine, University of California, San Diego, San Diego, CA.
J Gen Physiol. 2021 Jul 5;153(7). doi: 10.1085/jgp.202012766.
The N2A segment of titin is a main signaling hub in the sarcomeric I-band that recruits various signaling factors and processing enzymes. It has also been proposed to play a role in force production through its Ca2+-regulated association with actin. However, the molecular basis by which N2A performs these functions selectively within the repetitive and extensive titin chain remains poorly understood. Here, we analyze the structure of N2A components and their association with F-actin. Specifically, we characterized the structure of its Ig domains by elucidating the atomic structure of the I81-I83 tandem using x-ray crystallography and computing a homology model for I80. Structural data revealed these domains to present heterogeneous and divergent Ig folds, where I81 and I83 have unique loop structures. Notably, the I81-I83 tandem has a distinct rotational chain arrangement that confers it a unique multi-domain topography. However, we could not identify specific Ca2+-binding sites in these Ig domains, nor evidence of the association of titin N2A components with F-actin in transfected C2C12 myoblasts or C2C12-derived myotubes. In addition, F-actin cosedimentation assays failed to reveal binding to N2A. We conclude that N2A has a unique architecture that predictably supports its selective recruitment of binding partners in signaling, but that its mechanical role through interaction with F-actin awaits validation.
肌联蛋白的 N2A 片段是肌节 I 带中的主要信号枢纽，可招募各种信号因子和加工酶。它还通过其与肌动蛋白的 Ca2+调节性结合而被提议在力产生中发挥作用。然而，N2A 如何在重复和广泛的肌联蛋白链内选择性地执行这些功能的分子基础仍知之甚少。在这里，我们分析了 N2A 成分的结构及其与 F-肌动蛋白的关联。具体来说，我们通过阐明使用 X 射线晶体学阐明 I81-I83 串联的原子结构并为 I80 计算同源模型来表征其 Ig 结构域的结构。结构数据表明这些结构域呈现出异构和发散的 Ig 折叠，其中 I81 和 I83 具有独特的环结构。值得注意的是，I81-I83 串联具有独特的旋转链排列，赋予其独特的多结构域拓扑结构。然而，我们无法在这些 Ig 结构域中识别出特定的 Ca2+结合位点，也无法证明肌联蛋白 N2A 成分与转染的 C2C12 成肌细胞或 C2C12 衍生的肌管中的 F-肌动蛋白的关联。此外，F-肌动蛋白共沉淀测定未能揭示与 N2A 的结合。我们得出的结论是，N2A 具有独特的结构，可预测性地支持其在信号转导中选择性募集结合伴侣，但通过与 F-肌动蛋白的相互作用发挥机械作用仍有待验证。
