XMAP215 polymerase activity is built by combining multiple tubulin-binding TOG domains and a basic lattice-binding region
XMAP215 polymerase activity is built by combining multiple tubulin-binding TOG domains and a basic lattice-binding region
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DOI：
10.1073/pnas.1016498108
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发表时间：
2011-02-15
影响因子：
11.1
通讯作者：
Howard, Jonathon
中科院分区：
文献类型：
--
作者：
Widlund, Per O.;Stear, Jeffrey H.;Howard, Jonathon
XMAP 215/Dis 1家族蛋白正向调节微管生长。在其N末端的重复序列，称为TOG结构域，对于该功能是重要的。虽然TOG结构域直接结合微管蛋白二聚体，但尚不清楚这种相互作用如何转化为聚合酶活性。了解TOG结构域的功能作用是进一步复杂的事实，这些结构域存在于不同物种的蛋白质的数量不同。在这里，我们利用最近的晶体结构的第三TOG域秀丽隐杆线虫，Zyg 9，和突变的关键残基在每个TOG域的XMAP 215预测是重要的微管蛋白异二聚体的相互作用。我们确定了个别TOG域微管生长的贡献。我们表明，TOG域是绝对需要结合自由微管蛋白和域差异有助于XMAP 215的整体亲和力自由微管蛋白。突变体对游离微管蛋白的总体亲和力与聚合酶活性很好地相关。此外，我们证明了一个额外的碱性区域对于靶向微管晶格是重要的，并且对于XMAP 215在生理浓度下发挥作用是至关重要的。利用这些信息，我们设计了一种“盆景”蛋白，具有两个TOG结构域和一个碱性区域，几乎具有完全的聚合酶活性。
XMAP215/Dis1 family proteins positively regulate microtubule growth. Repeats at their N termini, called TOG domains, are important for this function. While TOG domains directly bind tubulin dimers, it is unclear how this interaction translates to polymerase activity. Understanding the functional roles of TOG domains is further complicated by the fact that the number of these domains present in the proteins of different species varies. Here, we take advantage of a recent crystal structure of the third TOG domain from Caenorhabditis elegans, Zyg9, and mutate key residues in each TOG domain of XMAP215 that are predicted to be important for interaction with the tubulin heterodimer. We determined the contributions of the individual TOG domains to microtubule growth. We show that the TOG domains are absolutely required to bind free tubulin and that the domains differentially contribute to XMAP215's overall affinity for free tubulin. The mutants' overall affinity for free tubulin correlates well with polymerase activity. Furthermore, we demonstrate that an additional basic region is important for targeting to the microtubule lattice and is critical for XMAP215 to function at physiological concentrations. Using this information, we have engineered a "bonsai" protein, with two TOG domains and a basic region, that has almost full polymerase activity.