Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Lucknow 226031, India.
Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Lucknow 226031, India; Academy of Scientific and Innovative Research, New Delhi, 110025, India.
Biochim Biophys Acta Proteins Proteom. 2018 Aug;1866(8):885-898. doi: 10.1016/j.bbapap.2018.04.010. Epub 2018 Apr 30.
Twinstar is an ADF/cofilin family protein, which is expressed by the tsr gene in Drosophila melanogaster. Twinstar is one of the main regulators of actin cytoskeleton remodelling and is essential for vital cellular processes like cytokinesis and endocytosis.
We have characterized the structure and dynamics of Twinstar by solution NMR spectroscopy, the interaction of Twinstar with rabbit muscle actin by ITC, and biochemical activities of Twinstar through different biochemical assays using fluorescence spectroscopy and ultra-centrifugation.
The solution structure of Twinstar shows characteristic ADF-H fold with well-formed G/F-site and F-site for interaction with actin. The structure possesses an extended F-loop, which is rigid at the base, but flexible towards its apical region. Twinstar shares similar dynamics for the G/F-site with C. elegans homologs, UNC-60A and UNC-60B. However, the dynamics of its F-loop are different from its C. elegans homologs. Twinstar shows strong affinity for ADP-G-Actin and ATP-G-Actin with Ks of ~7.6 nM and ~0.4 μM, respectively. It shows mild F-actin depolymerizing activity and stable interaction with F-actin with a K of ~5.0 μM. It inhibits the rate of the nucleotide exchange in a dose dependent manner.
On the basis of structure, dynamics, and biochemical activity, Twinstar can be taken to execute its biochemical role by facilitating directional growth and maintenance of length of actin filaments.
This study characterizes the structure, backbone dynamics, and biochemical activities of Twinstar of Drosophila, which provides an insight into the regulation of actin dynamics in the member of phylum insecta.
Twinstar 是 ADF/cofilin 家族蛋白，在黑腹果蝇中由 tsr 基因表达。Twinstar 是肌动蛋白细胞骨架重排的主要调节剂之一，对于细胞分裂和胞吞等重要细胞过程是必不可少的。
我们通过溶液 NMR 光谱学、ITC 测定 Twinstar 与兔肌动蛋白的相互作用、以及通过荧光光谱学和超速离心等不同生化测定方法测定 Twinstar 的生化活性，对 Twinstar 的结构和动力学进行了表征。
Twinstar 的溶液结构显示出特征性的 ADF-H 折叠，具有良好形成的 G/F 位和 F 位，用于与肌动蛋白相互作用。该结构具有延伸的 F 环，其基部刚性，但顶端区域灵活。Twinstar 与线虫同源物 UNC-60A 和 UNC-60B 共享 G/F 位相似的动力学。然而，其 F 环的动力学与其线虫同源物不同。Twinstar 对 ADP-G-肌动蛋白和 ATP-G-肌动蛋白表现出强烈的亲和力，Ks 值分别约为 7.6 nM 和 0.4 μM。它表现出轻度的 F-肌动蛋白解聚活性和与 F-肌动蛋白的稳定相互作用，K 值约为 5.0 μM。它以剂量依赖的方式抑制核苷酸交换的速率。
基于结构、动力学和生化活性，Twinstar 可以通过促进肌动蛋白丝的定向生长和长度维持来执行其生化功能。
本研究对果蝇 Twinstar 的结构、骨架动力学和生化活性进行了表征，为了解节肢动物门成员中肌动蛋白动力学的调节提供了线索。
