Mok Y K, Lo K W, Zhang M
Department of Biochemistry, the Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, People's Republic of China.
J Biol Chem. 2001 Apr 27;276(17):14067-74. doi: 10.1074/jbc.M011358200. Epub 2001 Jan 8.
The minus-ended microtubule motor cytoplasmic dynein contains a number of low molecular weight light chains including the 14-kDa Tctex-1. The assembly of Tctex-1 in the dynein complex and its function are largely unknown. Using partially deuterated, (15)N,(13)C-labeled protein samples and transverse relaxation-optimized NMR spectroscopic techniques, the secondary structure and overall topology of Tctex-1 were determined based on the backbone nuclear Overhauser effect pattern and the chemical shift values of the protein. The data showed that Tctex-1 adopts a structure remarkably similar to that of the 8-kDa light chain of the motor complex (DLC8), although the two light chains share no amino acid sequence homology. We further demonstrated that Tctex-1 binds directly to the intermediate chain (DIC) of dynein. The Tctex-1 binding site on DIC was mapped to a 19-residue fragment immediately following the second alternative splicing site of DIC. Titration of Tctex-1 with a peptide derived from DIC, which contains a consensus sequence R/KR/KXXR/K found in various Tctex-1 target proteins, indicated that Tctex-1 binds to its targets in a manner similar to that of DLC8. The experimental results presented in this study suggest that Tctex-1 is likely to be a specific cargo adaptor for the dynein motor complex.
负端微管马达胞质动力蛋白包含许多低分子量轻链，其中包括14 kDa的Tctex-1。Tctex-1在动力蛋白复合物中的组装及其功能在很大程度上尚不清楚。利用部分氘代的、（15）N、（13）C标记的蛋白质样品和横向弛豫优化的核磁共振光谱技术，基于蛋白质的主链核Overhauser效应模式和化学位移值确定了Tctex-1的二级结构和整体拓扑结构。数据显示，Tctex-1采用的结构与马达复合物（DLC8）的8 kDa轻链的结构非常相似，尽管这两条轻链没有氨基酸序列同源性。我们进一步证明，Tctex-1直接与动力蛋白的中间链（DIC）结合。DIC上的Tctex-1结合位点被定位到紧跟DIC第二个可变剪接位点后的一个19个残基的片段。用源自DIC的肽对Tctex-1进行滴定，该肽包含在各种Tctex-1靶蛋白中发现的共有序列R/KR/KXXR/K，表明Tctex-1以类似于DLC8的方式与其靶标结合。本研究中呈现的实验结果表明，Tctex-1可能是动力蛋白马达复合物的一种特异性货物衔接蛋白。
