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; 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（由应用程序提供）：本应用程序提出了3-D低温电子显微镜（Cryo-EM）进行的结构和功能研究，用于小鼠KIF3AB和KIF3AC的杂二聚体运动蛋白-2运动结构域，以定义其机械化学，分子内通信，以及与微管曲霉的结构关系。与许多其他驱动蛋白不同，在驱动蛋白2，生化和结构上，可用的数据相当少。在这里，我们想调查为什么这些动力蛋白-2异二聚体与异二聚体运动蛋白-14家族成员（例如Kar3Vik1或Kar3Cik1）共享共同的结构和功能特征，以及由于它们的顺序方向而与Kinesin-1共同。该项目将继续在苏珊·吉尔伯特（Susan Gilbert）和P.I.实验室之间进行长期合作。这可以追溯到1999年，在P.I.于2006年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将是分析3-D量的微管结合，杂型型动力蛋白2电机，并毫不含糊地识别两个头部的哪个。这比使用单体头构建体工作要复杂得多。通常，我们发现单体构建体以每个微管蛋白异二聚体的化学计量法结合微管结合。因此，具有螺旋微管模板（即15个细节微管）单体动力学微管复合物适应了螺旋形对称性，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）3-D重建，然后进行统计分类和从横线图中提取的子体积的平均。在特定的目标1中，我们将使用可克隆的，电子的标签应用和完善标记策略KIF3A，KIF3B和KIF3C，这将允许通过在摩托微管群上的冷冻电子显微镜通过冷冻电子显微镜将头部明确分开。这两个头部最有可能在结构上太相似，以至于仅在〜1-2 nm的分辨率（目前大约是我们用冰冻-ET实现的极限）中区分了它们的形状。在特定的AIM-2中，我们将通过Cryo-EM和Cryo-ET调查异二聚体，标记和天然驱动蛋白-2运动头构建体在各种核苷酸结合条件下的3-D构型，当时与微管相互作用。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。最后，特定的AIM-3将采用高分辨率的表面阴影，以获得与微管表面的驱动蛋白-2异二聚体电动机构建体的相互作用模式的非常直接且毫无结构的视图。