Mapping the cofilin binding site on yeast G-actin by chemical cross-linking
Mapping the cofilin binding site on yeast G-actin by chemical cross-linking
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DOI：
10.1016/j.jmb.2007.12.073
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发表时间：
2008-03-21
影响因子：
5.6
通讯作者：
Reisler, Emil
中科院分区：
文献类型：
--
作者：
Grintsevich, Elena E.;Benchaar, Sabrina A.;Reisler, Emil
Cofilin 是一种主要的细胞骨架蛋白，可与单体肌动蛋白 (G-肌动蛋白) 和聚合肌动蛋白 (F-肌动蛋白) 结合，并参与微丝动力学。尽管 G-肌动蛋白-肌动蛋白复合物的原子结构不存在，但已使用分子动力学模拟、结构同源性考虑和同步加速器辐射分解足迹数据建立了该复合物的模型。肌动蛋白亚结构域 1 和 3 之间的疏水性裂缝以及肌动蛋白亚结构域 I 和 2 之间的裂缝已被提议作为可能的高亲和力丝切蛋白结合位点。在本研究中，利用定点诱变、荧光标记和化学交联，对肌动蛋白丝切蛋白与 G-肌动蛋白上的子结构域 1/子结构域 3 区域的结合进行了探测，酵母肌动蛋白突变体在肌动蛋白疏水性裂隙中含有单个反应性半胱氨酸，而肌动蛋白丝切蛋白突变体在预测与 G-肌动蛋白结合的区域中携带反应性半胱氨酸。交联复合物的质谱分析表明，突变型 G-肌动蛋白亚结构域 I 中的半胱氨酸 345 与丝切蛋白上的天然半胱氨酸 62 交联。在 α 3 螺旋（残基 95）中携带半胱氨酸取代的肌动蛋白丝切蛋白突变体与肌动蛋白子结构域 3 中的残基 144 形成交联。这些交联施加的距离限制为肌动蛋白亚结构域 I 和 3 之间的肌动蛋白丝切蛋白结合提供了实验证据，并适合相应的基于对接的复合物结构。还记录了重组酵母丝切蛋白的 N 末端区域与肌动蛋白残基 346 和 374 与二硫代-双-马来酰亚胺乙烷（12.4 埃）并通过二硫键形成的交联。这组交联数据证实了丝切蛋白 N 末端片段在与 G 肌动蛋白相互作用中的重要作用。 (C) 2008 Elsevier Ltd. 保留所有权利。
Cofilin is a major cytoskeletal protein that binds to both monomeric actin (G-actin) and polymeric actin (F-actin) and is involved in microfilament dynamics. Although an atomic structure of the G-actin-cofilin complex does not exist, models of the complex have been built using molecular dynamics simulations, structural homology considerations, and synchrotron radiolytic footprinting data. The hydrophobic cleft between actin subdomains 1 and 3 and, alternatively, the cleft between actin subdomains I and 2 have been proposed as possible high-affinity cofilin binding sites. In this study, the proposed binding of cofilin to the subdomain 1/subdomain 3 region on G-actin has been probed using site-directed mutagenesis, fluorescence labeling, and chemical cross-linking, with yeast actin mutants containing single reactive cysteines in the actin hydrophobic cleft and with cofilin mutants carrying reactive cysteines in the regions predicted to bind to G-actin. Mass spectrometry analysis of the cross-linked complex revealed that cysteine 345 in subdomain I of mutant G-actin was cross-linked to native cysteine 62 on cofilin. A cofilin mutant that carried a cysteine substitution in the alpha 3-helix (residue 95) formed a cross-link with residue 144 in actin subdomain 3. Distance constraints imposed by these cross-links provide experimental evidence for cofilin binding between actin subdomains I and 3 and fit a corresponding docking-based structure of the complex. The cross-linking of the N-terminal region of recombinant yeast cofilin to actin residues 346 and 374 with dithio-bis-maleimidoethane (12.4 angstrom) and via disulfide bond formation was also documented. This set of cross-linking data confirms the important role of the N-terminal segment of cofilin in interactions with G-actin. (C) 2008 Elsevier Ltd. All rights reserved.