Binding model of human coactosin-like protein with filament actin revealed by mutagenesis
Binding model of human coactosin-like protein with filament actin revealed by mutagenesis
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DOI：
10.1016/j.bbapap.2006.06.017
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发表时间：
2006-11-01
影响因子：
3.2
通讯作者：
Shi, Yunyu
中科院分区：
文献类型：
--
作者：
Dai, Haiming;Huang, Wei;Shi, Yunyu
人辅肌动蛋白样蛋白（CLP）是一种小分子量（分子量约为17 kDa）的肌动蛋白结合蛋白。它可以结合肌动蛋白丝，但不能结合球状肌动蛋白，属于第四类含ADF-H结构域的蛋白质。人CLP还可以结合5LO，其在细胞白三烯合成中起重要作用。虽然hCLP的结构已经通过核磁共振和X射线实验确定，但hCLP如何与肌动蛋白丝结合仍然是一个有争议的问题。为了深入了解复合物的结构，我们使用多维NMR光谱研究了hCLP的三维结构和骨架动力学。以蛋白质溶液结构为指导，产生了一系列定点突变体，并通过高速共沉降法测定了它们的F-肌动蛋白结合活性。进一步利用计算对接方法，对hCLP-F-actin复合物的结构模型进行了初步构建。发现hCLP中的几个先前未测试的残基（包括T66、L 89、R91、K102、D116和E119）对于F-肌动蛋白结合活性是重要的。发现hCLP的β 4-β 5的延伸区域（残基66-75）非常灵活并且对于F-肌动蛋白结合非常重要。hCLP的C-末端残基不参与F-actin的结合，这与hCLP-60 B不同。基于我们的hCLP-F-肌动蛋白结合模型，解释了四类含ADF-H结构域的蛋白质对F-肌动蛋白的不同亲和力。(c)2006 Elsevier B. V.保留所有权利。
Human coactosin-like protein (CLP) is a small (MW similar to 17 kDa) evolutionarily conserved actin-binding protein. It can bind to actin filaments but not globular actin and belongs to the fourth class of ADF-H-domain-containing proteins. Human CLP can also bind to 5LO, which plays an important role in cellular leukotriene synthesis. Although the structure of hCLP has been determined by both NMR and X-ray experiments, how hCLP binds to the actin filament is still a controversial question. To obtain insights into the structure of the complex, we studied the three-dimensional structure and backbone dynamics of hCLP using multidimensional NMR spectroscopy. Guided by the solution structure of the protein, a series of site-directed mutants were generated and their F-actin-binding activities were measured by high-speed cosedimentation assays. Furthermore, the structure model of the hCLP-F-actin complex was proposed using computational docking with the docking results filtered by the mutation data. Several previously untested residues (including T66, L89, R91, K102, D116 and E119) in hCLP were found important for the F-actin-binding activity. The extended region of beta 4-beta 5 of hCLP (residue 66-75) was found very flexible and very important for F-actin binding. The C-terminal residues of hCLP were not involved in F-actin binding, which was different from UNC-60B. Based on our hCLP-F-actin-binding model, different affinities of the four classes of ADF-H domain containing proteins for F-actin were explained. (c) 2006 Elsevier B.V. All rights reserved.