Structural basis for tubulin recognition by cytoplasmic linker protein 170 and its autoinhibition
Structural basis for tubulin recognition by cytoplasmic linker protein 170 and its autoinhibition
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DOI：
10.1073/pnas.0703876104
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发表时间：
2007-06-19
影响因子：
11.1
通讯作者：
Hakoshima, Toshio
中科院分区：
文献类型：
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作者：
Mishima, Masaki;Maesaki, Ryoko;Hakoshima, Toshio
细胞质连接蛋白170 （CLIP-170）是调节微管动力学的正端跟踪蛋白的原型，但CLIP-170如何识别微管正端并有助于聚合拯救尚不清楚。对CLIP-170的两个串联细胞骨架相关蛋白富甘氨酸（CAP-Gly）结构域CAP-Gly-1和CAP-Gly-2的晶体学、核磁共振和突变研究显示，CAP-Gly结构域都具有带正电的微管蛋白结合基本凹槽，而CAP-Gly-2结构域具有更基本的凹槽，并直接结合α -微管蛋白c端酸尾末端的eexey /F基序。值得注意的是，带有较少正电荷表面的p150(glue) CAP-Gly结构域仅与α -微管蛋白酸性尾部弱相互作用。突变研究表明，这种酸性六元基序是CAP-Gly结合的最小区域。CLIP-170的c端锌关节结构域与碱性凹槽结合，抑制与酸性尾部的结合。这些结果为提出的CLIP-170与微管+端微管蛋白共聚提供了结构基础。CLIP-170与EB1的酸性尾部以及α -微管蛋白的酸性尾部强烈结合，表明位于正端的EB1有助于CLIP-170募集到正端。我们认为，CLIP-170刺激微管聚合和/或成核是通过中和小管的负电荷和CLIP-170的CAP-Gly结构域的高正电荷来实现的。一旦CLIP-170结合微管，释放的锌关节结构域可能通过与p150（glue）和LIS1相互作用将动力蛋白招募到正端。因此，我们的结构为微管+端特定动力蛋白的加载提供了结构基础。
Cytoplasmic linker protein 170 (CLIP-170) is a prototype of the plus end-tracking proteins that regulate microtubule dynamics, but it is obscure how CLIP-170 recognizes the microtubule plus end and contributes to polymerization rescue. Crystallographic, NMR, and mutation studies of two tandem cytoskeleton-associated protein glycine-rich (CAP-Gly) domains of CLIP-170, CAP-Gly-1 and CAP-Gly-2, revealed positively charged basic grooves of both CAP-Gly domains for tubulin binding, whereas the CAP-Gly-2 domain possesses a more basic groove and directly binds the EExEEY/F motif of the C-terminal acidic-tail ends of alpha-tubulin. Notably, the p150(Glued) CAP-Gly domain that is furnished with a less positively charged surface only weakly interacts with the alpha-tubulin acidic tail. Mutation studies showed that this acidic sextette motif is the minimum region for CAP-Gly binding. The C-terminal zinc knuckle domains of CLIP-170 bind the basic groove to inhibit the binding to the acidic tails. These results provide a structural basis for the proposed CLIP-170 copolymerization with tubulin on the microtubule plus end. CLIP-170 strongly binds the acidic tails of EB1 as well as those of alpha-tubulins, indicating that EB1 localized at the plus end contributes to CLIP-170 recruitment to the plus end. We suggest that CLIP-170 stimulates microtubule polymerization and/or nucleation by neutralizing the negative charges of tubulins with the highly positive charges of the CLIP-170 CAP-Gly domains. Once CLIP-170 binds microtubule, the released zinc knuckle domain may serve to recruit dynein to the plus end by interacting with p150(Glued) and LIS1. Thus, our structures provide the structural basis for the specific dynein loading on the microtubule plus end.