Structural basis of motility by dimeric kinesin motor proteins

二聚体驱动蛋白运动的结构基础

• 批准号: 8674409
• 负责人: CHARLES VAUGHN SINDELAR
• 金额: $ 31.64万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8674409
• 关键词: Active Sites; Address; Affinity; Algorithms; Architecture; Binding; Binding Proteins; Biological Assay; Biological Models; Biological Process; Catalytic Domain; Cell division; Cell physiology; Cells; Communication; Complex; Cryoelectron Microscopy; Cytokinesis; DNA Sequence Rearrangement; Data; Defect; Development; Dimerization; Drug Design; Dynein ATPase; Enzymes; Eukaryotic Cell; Failure; Family; Filament; Future; Generations; Head; Heterogeneity; Hydrolysis; Image; Individual; Kinesin; Kinetics; Lead; Left; Malignant Neoplasms; Maps; Methods; Microtubules; Mitosis; Molecular; Molecular Motors; Motor; Motor Activity; Movement; Mutation; Myosin ATPase; Nature; Neurons; Nucleotides; Pathway interactions; Pharmacologic Substance; Play; Power stroke; Property; Proteins; Research; Resolution; Roentgen Rays; Role; Sampling; Series; Site; Site-Directed Mutagenesis; Structure; Techniques; Testing; Therapeutic; Transport Vesicles; Work; X-Ray Crystallography; alpha helix; base; cancer therapy; cell motility; cofactor; density; dimer; empowered; image processing; improved; in vivo; inhibitor/antagonist; innovation; insight; instrumentation; interest; katanin; mutant; nanometer; novel; public health relevance; reconstruction; single molecule; trait

DESCRIPTION (provided by applicant): Kinesin molecular motors move along microtubules by taking alternating steps with a pair of catalytic head domains, where each step is powered by hydrolysis of a single molecule of ATP. This activity plays a key role in numerous cellular functions such as mitosis and neuronal vesicle transport. It is therefore of considerable interest to dissect the molecular details that underlie kinesin's motility functions, not only as a basis fo understanding how this motor's activity may be modulated in vivo by a large variety of regulating factors, but also to aid the development of pharmaceuticals that target these motors for cancer therapy and other therapeutic purposes. Despite intensive study, however, the conformational changes that underlie kinesin's motility cycle remain strongly debated. A particularly elusive question is how dimeric kinesin sustains continuous stepwise movement, because existing methods have not captured the structure of actively stepping kinesin dimers . We have recently made two breakthroughs in our studies of the kinesin motor. First, by using a combination of state of the art cryo-electron microscopy instrumentation together with our own novel image-processing methods, we have solved a new 3D reconstruction of the kinesin-microtubule complex at ~5-6¿ resolution, substantially improving on previous efforts. This map reveals an unanticipated rearrangement of kinesin's active site following microtubule-stimulated ADP release, suggesting a novel mechanism for this key step in the kinesin cycle and also informing the motor's power stroke. Second, we have devised a novel algorithm for producing high-resolution 3D reconstructions from cryo-EM images of imperfectly decorated, heterogeneous assemblies of kinesin with microtubules. This method has allowed us to solve the first 3D reconstruction of a kinesin dimer as it steps along a microtubule. We will combine our new cryo-EM methods with a host of other state of the art structural and functional techniques, including AFM and saturation-transfer EPR, to establish the detailed basis of kinesin motor function. By comparing structure and functional properties of dimeric kinesin in the presence or absence of mutations that cause loss of motor coordination, we will define the structural basis of inter-molecular tension control and other critical properties of kinesin that are enabled by dimerization. We will also apply cryo-EM to structure/function studies of site-directed mutants in the kinesin catalytic domain in order to test hypotheses for how kinesin's activity is regulated by microtubule binding, and how the motor regulates its affinity for the microtubule during its cycle. The methods developed during the course of this research will transform our ability to study many other large and previously intractable filament-binding proteins, including other molecular motor families as well as microtubule severing enzymes.

描述(申请人提供)：动蛋白分子马达通过与一对催化头域交替的步骤沿微管移动，其中每一步由单个三磷酸腺苷分子的水解提供动力。这种活性在许多细胞功能中起着关键作用，如有丝分裂和神经细胞囊泡运输。因此，剖析驱动蛋白运动功能背后的分子细节是相当有意义的，这不仅是了解这种马达的活动如何在体内被各种调节因子调节的基础，而且也有助于开发针对这些马达的药物，用于癌症治疗和其他治疗目的。然而，尽管进行了密集的研究，但是，运动蛋白运动周期背后的构象变化仍然存在激烈的争论。一个特别难以捉摸的问题是，二聚体动蛋白是如何维持连续的步进运动的，因为现有的方法还没有捕捉到主动步进的动蛋白二聚体的结构。最近，我们在运动蛋白运动的研究上取得了两项突破。首先，通过使用最先进的冷冻电子显微镜仪器和我们自己的新的图像处理方法的结合，我们解决了一种新的5-6分辨率的运动蛋白-微管复合体的三维重建，大大改进了以前的工作。这张图揭示了在微管刺激的ADP释放后，Kinesin的活性部位发生了意想不到的重新排列，这表明了Kinesin循环中这一关键步骤的新机制，并通知了马达的功率中风。其次，我们设计了一种新的算法，用于从冷冻-EM图像中产生高分辨率的3D重建，这些图像是不完美装饰的、带有微管的不同种类的肌动蛋白组装。这种方法使我们能够解决运动蛋白二聚体在沿着微管行走时的第一个3D重建。我们将把我们的新冷冻-EM方法与许多其他最先进的结构和功能技术相结合，包括AFM和饱和转移EPR，以建立运动蛋白运动功能的详细基础。通过比较在存在或不存在导致运动协调性丧失的突变的情况下，二聚体Kinesin的结构和功能性质，我们将确定分子间张力控制的结构基础以及由二聚化实现的Kinesin的其他关键性质。我们还将应用冷冻-EM技术研究动蛋白催化域中定点突变体的结构/功能，以验证有关动蛋白活性如何受 微管结合，以及马达如何在其周期中调节其对微管的亲和力。 在这项研究过程中开发的方法将改变我们研究许多其他大型和以前难以处理的细丝结合蛋白的能力，包括其他分子马达家族以及微管切断酶。