Functional elements within the dynein microtubule-binding domain
Functional elements within the dynein microtubule-binding domain
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DOI：
10.1091/mbc.11.2.523
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发表时间：
2000-02-01
影响因子：
3.3
通讯作者：
Tikhonenko, I
中科院分区：
文献类型：
--
作者：
Koonce, MP;Tikhonenko, I
动力蛋白通过一个ATP敏感的连接与微管相互作用，该连接映射到重链的第四个P环基序之后的结构复杂区域。事实上，关于ATP水解过程中如何实现和调节结合亲和力，我们一无所知。我们已经进行了详细的解剖微管接触网站，使用片段表达，丙氨酸取代，和肽竞争。我们的工作确定了三个簇的氨基酸与微管的物理接触的重要性，其中两个属于一个区域内共享序列同源性与MAP 1B，第三个在一个区域的下游。这些区域中的任何一个内的氨基酸取代可以消除或减弱微管结合（KK 3379，80，E3385，K3387，K3397，KK 3410，11，W3414，RKK 3418 -20，F3426，R3464，S3466和K3467），表明它们的活性是高度协调的。一种主动取代微管中MAP 1B的肽干扰了动力蛋白的结合，支持了以前类似相互作用位点的证据。我们还确定了四个氨基酸的取代影响释放的电机从微管（E3413，R3444，E3460和C3469）。这些表明，核苷酸敏感的亲和力可能是局部控制的网站的接触。我们的工作是第一次详细描述动力蛋白-微管蛋白相互作用，并为理解亲和力如何实现和调节提供了框架。
Dynein interacts with microtubules through an ATP-sensitive linkage mapped to a structurally complex region of the heavy chain following the fourth P-loop motif. Virtually nothing is known regarding how binding affinity is achieved and modulated during ATP hydrolysis. We have performed a detailed dissection of the microtubule contact site, using fragment expression, alanine substitution, and peptide competition. Our work identifies three clusters of amino acids important for the physical contact with microtubules; two of these fall within a region sharing sequence homology with MAP1B, the third in a region just downstream. Amino acid substitutions within any one of these regions can eliminate or weaken microtubule binding (KK3379,80, E3385, K3387, K3397, KK3410,11, W3414, RKK3418-20, F3426, R3464, S3466, and K3467), suggesting that their activities are highly coordinated. A peptide that actively displaces MAP1B from microtubules perturbs dynein binding, supporting previous evidence for similar sites of interaction. We have also identified four amino acids whose substitutions affect release of the motor from the microtubule (E3413, R3444, E3460, and C3469). These suggest that nucleotide-sensitive affinity may be locally controlled at the site of contact. Our work is the first detailed description of dynein-tubulin interactions and provides a framework for understanding how affinity is achieved and modulated.