Structure and Function of Heterodimeric Kinesin-2 Motor Head Domains

 描述（由申请人提供）：本申请提出通过3-D冷冻电子显微镜（cryo-EM）对小鼠KIF 3AB和KIF 3AC的异二聚体驱动蛋白-2马达结构域进行结构和功能研究，以确定其机械化学、分子内通讯以及与微管晶格的结构关系。与许多其他驱动蛋白不同的是，关于驱动蛋白-2的生物化学和结构数据仍然很少。在这里，我们想调查为什么这些驱动蛋白采用异二聚体和模块化的运动域，以及如何区分功能，从其他驱动蛋白。此外，我们想比较驱动蛋白2与其他异二聚体马达，Kar 3Vik 1和Kar 3Cik 1，我们已经在过去广泛研究。我们将测试的假设，MT结合模式的驱动蛋白-2异二聚体共享共同的结构和功能特征的异二聚体驱动蛋白-14家族成员，如Kar 3Vik 1或Kar 3Cik 1，但也驱动蛋白-1，由于其顺行方向性。这个项目将继续苏珊吉尔伯特的实验室和PI之间的长期合作。这可以追溯到1999年，比私家侦探早得多2006年，他来到位于博尔德的科罗拉多大学。 该提案最重要的技术挑战之一将是分析微管结合的异二聚体驱动蛋白-2马达的3D体积，并明确识别两个头部中的哪一个。这基本上比使用单体头部构建体更复杂。我们通常发现单体结构与微管结合的化学计量比为每个微管蛋白异源二聚体一个头部。因此，使用螺旋微管模板（即15-原丝微管），单体驱动蛋白-微管复合物适应螺旋对称性并且可以相应地重建。在这里，我们将采用冷冻电子断层扫描（cryo-ET）的3-D重建，然后从断层图像中提取的子体积的统计分类和平均值。在特定的目标-1中，我们将应用并改进驱动蛋白-II马达头部结构域KIF 3A、KIF 3B和KIF 3C的标记策略，其具有可克隆的电子致密标记，这将允许通过冷冻电子显微镜在马达微管复合物上明确分离头部。这两个头部很可能在结构上过于相似，以至于无法在约1-2 nm分辨率（目前约为我们使用冷冻ET可以实现的极限）下单独从它们的形状中区分出来。在具体目标-2中，我们将通过cryo-EM和cryo-ET研究异源二聚体，标记和天然驱动蛋白-2马达头结构在各种核苷酸结合条件下与微管相互作用时的3-D构型。为此，我们将采用各种3-D重建和分析方法，可能适合于预测的条件。最后，特定目标-3将采用高分辨率的表面阴影，以获得一个非常直接和畅通无阻的视图上的相互作用模式的驱动蛋白-2异二聚体电机结构与微管表面。

 DESCRIPTION (provided by applicant): This application proposes a structural and functional study by 3-D cryo-electron microscopy (cryo-EM) into the heterodimeric kinesin-2 motor domains of mouse KIF3AB and KIF3AC to define their mechanochemistry, intramolecular communication, and structural relationship with the microtubule lattice. Unlike for many other kinesins there is still comparably little data available on kinesin-2, biochemical as well as structural. Here we would like to investigate why these kinesins employ a heterodimeric and modular motor domain, and how that feature distinguishes them functionally from other kinesins. Furthermore, we would like to compare kinesin-2 to other heterodimeric motors, Kar3Vik1 and Kar3Cik1 that we have studied extensively in the past. We will test the hypothesis that the MT binding pattern of kinesin-2 heterodimers shares common structural and functional features to heterodimeric Kinesin-14 family members such as Kar3Vik1 or Kar3Cik1, but also to kinesin-1 due to their anterograde directionality. This project will continue a long-standing collaboration between the lab of Susan Gilbert and the P.I. that dates back to 1999, much before the P.I.'s arrival at the University of Colorado at Boulder in 2006. One of the most important technical challenges of this proposal will be analyzing 3-D volumes of microtubule-bound, heterodimeric kinesin-2 motors and unambiguously identify which each of the two heads. This is substantially more complex than working with monomeric head constructs. We typically find monomeric constructs to bind to microtubules with a stoichiometry of one head per tubulin heterodimer. Hence, with a helical microtubule template (i.e. a 15-protofilament microtubule) monomeric kinesin- microtubule complexes adapt that helical symmetry and can be reconstructed accordingly. Here we will employ cryo-electron tomography (cryo-ET) 3-D reconstruction followed by statistical classification and averaging of sub-volumes extracted from tomograms. Within specific aim-1 we will apply and refine labeling strategies for kinesin-II motor head domains KIF3A, KIF3B, and KIF3C with clonable, electron- dense labels that will allow for an unambiguous separation of the heads by cryo-electron microscopy on motor-microtubule complexes. The two heads are most likely structurally too similar to be distinguished at ~1-2 nm resolution (currently about the limit that we can achieve with cryo-ET) from their shape alone. In specific aim-2 we will investigate by cryo-EM and cryo-ET the 3-D configuration of heterodimeric, tagged and native kinesin-2 motor head constructs under various nucleotide-binding conditions when interacting with microtubules. To this end we will employ various 3-D reconstruction and analysis methods that may be suitable for the predicted conditions. Finally, specific aim-3 will employ high-resolution surface shadowing to gain a very direct and unobstructed view on the interaction patterns of kinesin-2 heterodimeric motor constructs with the surface of microtubules.