Microtubule-binding sites of the CH domain of EB1 and its autoinhibition revealed by NMR
Microtubule-binding sites of the CH domain of EB1 and its autoinhibition revealed by NMR
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DOI：
10.1016/j.bbapap.2012.10.013
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发表时间：
2013-02-01
影响因子：
3.2
通讯作者：
Mishima, Masaki
中科院分区：
文献类型：
--
作者：
Kanaba, Teppei;Maesaki, Ryoko;Mishima, Masaki
末端结合蛋白 1 (EB1) 是研究最充分的正末端跟踪蛋白之一。众所周知，EB1 特异性结合微管 (MT) 的正端并促进 MT 生长。 EB1 活性被认为是通过分子内相互作用而自动抑制的。最近的冷冻电镜分析表明，Mal3p（粟酒裂殖酵母 EB1 同源物）的 CH 结构域与 GMPCPP-MT 结合（Sandblad, L. Cell 127 (2006) 1415-24），并与 GTP gamma S-MT 强烈结合，建议比 GMPCPP-MT 更好地模拟 MT 加末端（Maurer S.P. 等人 Cell 149 (2012)） 371-82）。在这里，我们报告了使用转移交叉饱和方法通过 NMR 显示的 EB1 CH 结构域的 MT 结合位点。在本研究中，我们使用 GMPCPP-MT，发现 MT 结合位点与冷冻电镜 (Maurer S.P. et al. Cell 149 (2012) 371-82) 所建议的 GTP gamma S-MT 结合位点非常相似。值得注意的是，CH 结构域的螺旋 α 6 的 N 末端并未与 GMPCPP-MT 接触，这与冷冻电镜研究相反，冷冻电镜研究表明它靠近 GTP γ S-MT 中 β-微管蛋白的推定开关区域（Maurer S.P. 等人，Cell 149 (2012) 371-82）。此外，我们发现EB1的分子内相互作用位点与MT结合位点重叠，表明MT结合位点被与C端结构域的相互作用所掩盖。我们提出了一种自抑制的结构观点及其通过与腺瘤性息肉病大肠杆菌蛋白等结合伙伴竞争结合的释放机制。 (c) 2012 Elsevier B.V. 保留所有权利。
End-binding protein 1 (EB1) is one of the best studied plus-end tracking proteins. It is known that EB1 specifically binds the plus ends of microtubules (MTs) and promotes MT growth. EB1 activity is thought to be autoinhibited by an intramolecular interaction. Recent cryo-EM analyses showed that the CH domain of Mal3p (Schizosaccharomyces pombe EB1 homolog) binds to GMPCPP-MT (Sandblad, L. Cell 127 (2006) 1415-24), and strongly binds GTP gamma S-MT which is proposed to mimic MT plus ends better than GMPCPP-MT (Maurer S.P. et al. Cell 149 (2012) 371-82). Here, we report on the MT binding sites of the CH domain of EB1 as revealed by NMR using the transferred cross-saturation method. In this study, we used GMPCPP-MT and found that the MT binding sites are very similar to the binding site for GTP gamma S-MT as suggested by cryo-EM (Maurer S.P. et al. Cell 149 (2012) 371-82). Notably, the N-terminal tip of helix alpha 6 of the CH domain did not make contact with GMPCPP-MT, in contrast to the cryo-EM study which showed that it is closely located to a putative switch region of beta-tubulin in GTP gamma S-MT (Maurer S.P. et al. Cell 149 (2012) 371-82). Further, we found that the intramolecular interaction site of EB1 overlaps the MT binding sites, indicating that the MT binding sites are masked by interaction with the C-terminal domain. We propose a structural view of autoinhibition and its release mechanism through competition binding with binding partners such as adenomatous polyposis coli protein. (c) 2012 Elsevier B.V. All rights reserved.