Mechanisms of kinesin motor protein inhibition

驱动蛋白运动蛋白抑制机制

• 批准号: 10686209
• 负责人: Michael Cianfrocco
• 金额: $ 32.09万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10686209
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Axonal Transport; Binding; Binding Proteins; Biological; Cell division; Cell physiology; Cells; Complement; Complex; Consensus; Cryoelectron Microscopy; Cytoskeleton; Data; Elements; Ensure; Environment; Fluorescence; Grant; Individual; Kinesin; Lead; Length; Light; Mass Spectrum Analysis; Mediating; Microtubules; Mitotic; Mitotic spindle; Modeling; Molecular; Molecular Conformation; Motor; Mutagenesis; Mutation; Organelles; Patients; Peptides; Positioning Attribute; Property; Protein Engineering; Protein Family; Protein Inhibition; Protein Region; Proteins; Regulation; Reporting; Signal Transduction; Site-Directed Mutagenesis; Specificity; Structure; Tail; Testing; Work; cell motility; crosslink; experimental study; mutant; novel; organelle movement; prevent; spatiotemporal

MECHANISMS OF KINESIN MOTOR PROTEIN INHIBITION SUMMARY The spatiotemporal regulation of organelle positioning is critical for proper cellular function, especially as the cell responds to a changing environment. The kinesin superfamily of motor proteins is responsible for various cellular processes that range from long-range axonal transport to orchestrating the mitotic spindle during cell division. The regulation of kinesin motor proteins occurs via inhibitory and activation-based mechanisms, where kinesin motor proteins are subject to autoinhibition when not bound to a cargo. Recently, the discovery of kinesin-binding protein (KIFBP) revealed a novel form of kinesin inhibition whereby KIFBP binds to kinesin motor domains to block microtubule-binding. In this grant, we will determine the molecular basis for KIFBP-mediated kinesin inhibition in trans and how kinesin light chains in cis-lead to kinesin inhibition. Based on structural studies of KIFBP bound to two different kinesin motor domains, we developed a model of how KIFBP remodels kinesin motors and how KIFBP selectively engages motors. We will introduce site-specific mutagenesis based on crosslinking mass spectrometry and patient-derived mutants to dissect kinesin binding and remodeling by KIFBP (Aim 1). In parallel, we will determine how light chains regulate full-length kinesin motor proteins via autoinhibition. Despite decades of work into the regulation of full-length kinesin motor proteins, there remain conflicting results regarding inhibition of kinesin motor domains. We are poised to answer this question by exploiting a combination of crosslinking mass spectrometry, protein engineering, and cryo-EM to determine how kinesin light chains stabilize a compact, inhibited kinesin motor (Aim 2). We will study both kinesin-1 heterotetramers (KIF5B:KLC1) in addition to kinesin-2 heterotrimers (KIF3A:3B:KAP) to compare and contrast how these different kinesin motor complexes are autoinhibited. Taken together, this work will expand our understanding of kinesin regulation, establishing modes of inhibition to provide a complete view of kinesin activity.

运动蛋白抑制的机制 总结 细胞器定位的时空调节对于适当的细胞功能是至关重要的，特别是当细胞器定位时。 细胞对变化的环境作出反应。运动蛋白的驱动蛋白超家族负责各种 细胞过程的范围从远距离轴突运输到细胞过程中协调有丝分裂纺锤体 师.驱动蛋白马达蛋白的调节通过基于抑制和激活的机制发生， 其中驱动蛋白马达蛋白在不与货物结合时受到自抑制。最近， 驱动蛋白结合蛋白（KIFBP）的研究揭示了一种新的驱动蛋白抑制形式， 马达域阻断微管结合。在这项资助中，我们将确定 KIFBP介导的驱动蛋白反式抑制以及驱动蛋白轻链顺式如何导致驱动蛋白抑制。基于 在KIFBP与两个不同驱动蛋白运动域结合的结构研究中，我们开发了一个模型来说明如何结合 KIFBP重塑驱动蛋白马达以及KIFBP如何选择性地接合马达。我们将介绍特定地点 基于交联质谱的诱变和患者衍生的突变体以剖析驱动蛋白结合 和KIFBP的重塑（Aim 1）。同时，我们将确定轻链如何调节全长驱动蛋白 马达蛋白通过自身抑制。尽管对全长驱动蛋白运动的调节进行了数十年的研究， 蛋白质，仍然存在关于抑制驱动蛋白马达结构域的相互矛盾的结果。我们准备 通过利用交联质谱、蛋白质工程和 cryo-EM以确定驱动蛋白轻链如何稳定紧凑的、受抑制的驱动蛋白马达（Aim 2）。我们将 除了研究驱动蛋白-2异源三聚体（KIF 3A：3B：KAP）之外，还研究了驱动蛋白-1异源四聚体（KIF 5 B：KLC 1）， 比较和对比这些不同的驱动蛋白运动复合体是如何被自我抑制的。总体而言，这项工作 将扩大我们对驱动蛋白调节的理解，建立抑制模式，提供一个完整的视图 驱动蛋白的活性。