Structure and Function of Heterodimeric Kinesin-2 Motor Head Domains

 描述(申请人提供)：本申请提出用三维冷冻电子显微镜(Cryo-EM)对小鼠KIF3AB和KIF3AC的异二聚体Kinesin-2运动域进行结构和功能研究，以确定它们的机械力化学、分子内通信以及与微管晶格的结构关系。与许多其他动蛋白不同的是，关于动蛋白-2的生化和结构方面的数据仍然相对较少。在这里，我们想要研究为什么这些动蛋白使用异二聚体和模块化的运动域，以及这一特征如何在功能上将它们与其他动蛋白区分开来。此外，我们希望将Kinesin-2与我们过去广泛研究的其他异二聚体马达Kar3Vik1和Kar3Cik1进行比较。我们将检验这一假设，即Kinesin-2异二聚体的MT结合模式与异二聚体Kinesin-14家族成员(如Kar3Vik1或Kar3Cik1)具有共同的结构和功能特征，但由于它们的顺向方向性，也与Kinesin-1相似。这个项目将延续苏珊·吉尔伯特的实验室和私家侦探之间的长期合作，这种合作可以追溯到1999年，早在私家侦探S 2006年抵达科罗拉多大学博尔德分校之前。这项提案最重要的技术挑战之一将是分析3-D体积的微管结合的异源二聚体Kinesin-2马达，并明确识别两个头中的哪一个。这比使用单体头部结构要复杂得多。我们通常会找到与微管结合的单体结构，其化学计量比为每个微管蛋白异二聚体一个头。因此，使用螺旋微管模板(即，15个原细丝微管)，单体动蛋白-微管复合体适应螺旋对称性，并可以相应地重建。在这里，我们将使用冷冻电子断层扫描(Cryo-ET)三维重建，然后对从断层图像中提取的子体积进行统计分类和平均。在特定的AIM-1中，我们将使用可克隆的电子密度标记对KIF3A、KIF3B和KIF3C的KIF3A、KIF3B和KIF3C运动素-II运动头结构域进行标记，这将允许通过冷冻电子显微镜对运动微管复合体的头进行明确的分离。这两个磁头很可能在结构上太相似，无法在~1-2 nm分辨率(目前大约是我们用低温超导所能达到的极限)从它们的形状中区分出来。在特定的Aim-2中，我们将用Cryo-EM和Cryo-ET研究异二聚体、标记和天然Kinesin-2运动头结构在不同核苷酸结合条件下与微管相互作用时的三维构型。为此，我们将采用各种可能适合预测条件的三维重建和分析方法。最后，特殊目标-3将使用高分辨率表面阴影来非常直接和畅通无阻地观察Kinesin-2异二聚体马达结构与微管表面的相互作用模式。

 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.