Identification of microtubule binding sites in the Ncd tail domain
Identification of microtubule binding sites in the Ncd tail domain
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DOI：
10.1021/bi981850i
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发表时间：
1999-02-09
期刊：
影响因子：
2.9
通讯作者：
Walker, RA
中科院分区：
文献类型：
--
作者：
Karabay, A;Walker, RA
非红细胞分离蛋白（Nonclaretdisjunctional，Ncd）是果蝇卵母细胞和早期胚胎减数分裂和早期有丝分裂过程中纺锤体组装和维持所必需的一种负末端定向的C-末端马达蛋白。Ncd在N-末端尾部结构域中具有ATP非依赖性MT结合位点，在C-末端马达结构域中具有ATP依赖性MT结合位点。Ncd通过-se结合位点的作用交联MT的能力对于Ncd在体内的功能可能是重要的。为了鉴定Ncd与ATP非依赖性MT相互作用的区域，在大肠杆菌中表达了12个编码Ncd尾区不同区域的cDNA。大肠杆菌作为C-末端融合硫氧还蛋白（Trx）。通过离子交换（S-Sepharose）和/或Talon金属亲和层析纯化Ncd尾融合蛋白（TrxNT）。在微管（MT）共沉降和捆绑测定中分析纯化的TrxNT和NT蛋白以鉴定哪些尾蛋白能够结合和捆绑MT。基于这些实验的结果，显示MT结合活性的所有TrxNT和NT蛋白也捆绑MT，并且在尾区中存在两个ATP非依赖性MT相互作用位点：一个在氨基酸83-100内，其显示构象非依赖性、高亲和力MT结合活性;另一个在氨基酸115-187内，其显示构象依赖性、较低亲和力NIT结合活性。这两个MT相互作用位点有可能在天然蛋白质中联合收割机形成单个MT结合位点，使Ncd尾能够在体内结合货物MT。
Nonclaret disjunctional (Ncd) is a minus end-directed, C-terminal motor protein that is required for spindle: assembly and maintenance during meiosis and early mitosis in Drosophila oocytes and early embryos. Ncd has an ATP-independent MT binding site in the N-terminal tail domain, and an ATP-dependent MT binding site in the C-terminal motor domain. The ability of Ncd to cross-link MTs through the action of the-se binding sites may be important for Ncd function in vivo. To identify the region(s) responsible for ATP-independenr MT interactions of Ncd, 12 cDNAs coding various regions of Ncd tail domain were expressed in E. coli as C-terminal fusions to thioredoxin (Trx). Ncd tail fusion proteins (TrxNT) were purified by ion exchange (S-Sepharose) and/or Talon metal affinity chromatography, Purified TrxNT and NT proteins were analyzed in microtubule (MT) cosedimentation and bundling assays to identify which tail proteins were able to bind and bundle MTs. Based on the results of these experiments, all TrxNT and NT proteins that showed MT binding activity also bundled MTs, and there are two ATP-independent MT interaction sites in the tail region: one within amino acids 83-100 that exhibits conformation-independent, high-affinity MT binding activity; and another within amino acids 115-187 hat exhibits conformation-dependent, lower affinity NIT binding activity. It is possible that both of these MT interacting sites combine in the native protein to form a single MT binding site that allows the Ncd tail to bind cargo MTs in vivo.