Mechanisms of kinesin motor protein inhibition

驱动蛋白运动蛋白抑制机制

• 批准号: 10518674
• 负责人: Michael Cianfrocco
• 金额: $ 32.12万
• 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/10518674
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Axonal Transport; Binding; Binding Proteins; Biological; Cell division; Cell physiology; Cells; Complement; Complex; Conflict (Psychology); Consensus; Cryoelectron Microscopy; 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; Proteins; Regulation; Reporting; Signal Transduction; Site-Directed Mutagenesis; Specificity; Structure; Tail; Testing; Work; base; 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.

驱动蛋白运动蛋白抑制的机理 概括 细胞器定位的时空调节对于适当的细胞功能至关重要，尤其是 细胞响应不断变化的环境。运动蛋白的运动蛋白超家族负责各种 细胞过程的范围从远程轴突运输到在细胞期间编排有丝分裂主轴的范围 分配。驱动蛋白运动蛋白的调节是通过抑制性和基于激活的机制发生的， 当不绑定货物时，动力蛋白运动蛋白会受到自身抑制。最近，发现 驱动蛋白结合蛋白（KIFBP）揭示了一种新型的抑制作用形式 电机域可阻止微管结合。在这笔赠款中，我们将确定 KIFBP介导的驱动蛋白在反式抑制以及驱动链中如何在驱动蛋白抑制的顺式链中。基于 关于与两个不同驱动蛋白运动结构域结合的KIFBP的结构研究，我们开发了一种模型 KIFBP重塑了运动蛋白电动机以及KIFBP如何选择接合电动机。我们将介绍特定地点的 基于交联的质谱和患者衍生的突变体的诱变，以剖析驱动蛋白结合 并通过KIFBP进行改建（AIM 1）。同时，我们将确定轻链如何调节全长驱动蛋白 运动蛋白通过自身抑制。尽管几十年的工作对全长驱动蛋白电动机进行了调节 蛋白质，关于抑制驱动蛋白运动结构域的结果仍然存在矛盾的结果。我们准备 通过利用交联质谱，蛋白质工程和 冷冻EM确定动力蛋白光链如何稳定紧凑，抑制的动力蛋白电动机（AIM 2）。我们将 研究两个驱动蛋白-1异驱动器（KIF5B：KLC1），除了驱动蛋白-2异三聚体（KIF3A：3B：KAP） 比较和对比这些不同的驱动蛋白运动复合物自身抑制。总之，这项工作 将扩大我们对运动蛋白调节的理解，建立抑制模式以提供完整的观点 动力素活性。