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机制 概括 细胞器定位的时空调节对于正常的细胞功能至关重要，特别是当 细胞对不断变化的环境做出反应。运动蛋白的驱动蛋白超家族负责各种 细胞过程，范围从长程轴突运输到在细胞过程中协调有丝分裂纺锤体 分配。驱动蛋白运动蛋白的调节通过基于抑制和激活的机制发生， 其中驱动蛋白运动蛋白在未与货物结合时会受到自动抑制。近日，发现 驱动蛋白结合蛋白（KIFBP）的研究揭示了一种新型的驱动蛋白抑制作用，其中 KIFBP 与驱动蛋白结合 运动结构域阻止微管结合。在这笔赠款中，我们将确定以下分子基础： KIFBP 介导的反式驱动蛋白抑制以及顺式驱动蛋白轻链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 KIFBP 重塑了驱动蛋白马达以及 KIFBP 如何选择性地接合马达。我们将针对具体地点进行介绍 基于交联质谱和患者衍生突变体的诱变来剖析驱动蛋白结合 并由 KIFBP 进行改造（目标 1）。同时，我们将确定轻链如何调节全长驱动蛋白 通过自抑制的运动蛋白。尽管对全长驱动蛋白马达的调节进行了数十年的研究 蛋白质，关于驱动蛋白运动结构域的抑制仍然存在相互矛盾的结果。我们准备 通过利用交联质谱、蛋白质工程和 冷冻电镜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（目标 2）。我们将 除了驱动蛋白 2 异源三聚体 (KIF3A:3B:KAP) 之外，还研究驱动蛋白 1 异源四聚体 (KIF5B:KLC1)，以 比较和对比这些不同的驱动蛋白运动复合物如何被自动抑制。综合起来，这部作品 将扩大我们对驱动蛋白调节的理解，建立抑制模式以提供完整的观点 驱动蛋白活性。