Structure and Function of Heterodimeric Kinesin-2 Motor Head Domains

异二聚体驱动蛋白 2 电机头结构域的结构和功能

• 批准号: 8858369
• 负责人: ANDREAS HOENGER
• 金额: $ 30.34万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-30 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8858369
• 关键词: 3-Dimensional; Alloys; Axon; Back; Binding; Biochemical; Biomedical Research; Chimera organism; Classification; Collaborations; Colorado; Communication; Complex; Computer software; Cryoelectron Microscopy; Data; Dimerization; Electrons; Family member; Figs - dietary; Goals; Head; Health; Human; Investigation; KRP protein; Kinesin; Knowledge; Label; Lead; Light; Link; Maps; Metallothionein; Metals; Methods; Microtubules; Molecular; Molecular Conformation; Molecular Motors; Motor; Mus; Nucleotides; Pathologic; Pattern; Peptides; Property; Resolution; Shadowing (Histology); Shapes; Signal Transduction; Structure; Surface; Tantalum; Tertiary Protein Structure; Testing; Time; Tomogram; Tubulin; Tungsten; Universities; Work; Xenopus laevis; Yang; aptamer; dimer; electron tomography; experience; member; monomer; novel; pre-clinical; public health relevance; reconstruction; stoichiometry; surface coating

 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.

 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 KIF3AB 和 KIF3AC 的异二聚体驱动蛋白-2 运动结构域进行结构和功能研究，以定义它们的机械化学、分子内通讯和与微管晶格的结构关系与许多其他驱动蛋白不同，目前可用的数据仍然相对较少。驱动蛋白-2，生物化学和结构。在这里，我们想研究为什么这些驱动蛋白采用异二聚体和模块化运动结构域，以及该特征如何在功能上将它们与其他驱动蛋白区分开来。此外，我们想将驱动蛋白-2 与其他驱动蛋白进行比较。我们过去主要研究的异二聚体马达 Kar3Vik1 和 Kar3Cik1 我们将检验驱动蛋白 2 异二聚体的 MT 结合模式具有共同结构的假设。以及异二聚体 Kinesin-14 家族成员（例如 Kar3Vik1 或 Kar3Cik1）的功能特征，以及由于其顺行方向性而导致的 kinesin-1 的功能特征。该项目将继续 Susan Gilbert 实验室与 P.I. 之间的长期合作。 1999 年，早在 2006 年 P.I. 到达科罗拉多大学博尔德分校之前。该提案最重要的技术挑战之一是分析微管结合的异二聚体驱动蛋白 2 马达的 3D 体积，并明确识别两个头中的每一个，这比使用单体头构建体要复杂得多，我们通常发现与化学计量为 1 的微管结合的单体构建体。因此，具有螺旋微管模板（即 15 个原丝微管）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）3D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。我们将应用和完善驱动蛋白-II 运动头域 KIF3A、KIF3B 和 KIF3C 的标记策略，使用可克隆的电子密集标签，这将允许通过冷冻电子显微镜对运动微管复合体进行明确的分离，这两个头部很可能在结构上过于相似，无法在约 1-2 nm 的分辨率（目前大约是我们使用冷冻电子断层扫描所能达到的极限）下进行区分。在特定的 goal-2 中，我们将通过冷冻电镜和冷冻电子发射技术研究异二聚体、标记的和天然驱动蛋白-2 运动头结构在各种核苷酸结合下的 3-D 构型。为此，我们将采用可能适合预测条件的各种 3-D 重建和分析方法。驱动蛋白 2 异二聚体运动结构与微管表面的相互作用模式。