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.

运动蛋白抑制马达蛋白的机制 摘要 细胞器定位的时空调节对于细胞的正常功能是至关重要的，特别是当 细胞对不断变化的环境做出反应。运动蛋白超家族的运动蛋白负责多种 从长距离轴突运输到在细胞过程中协调有丝分裂纺锤体的细胞过程 组织。运动蛋白马达蛋白的调节通过抑制和基于激活的机制发生。 当未与货物结合时，运动蛋白马达蛋白会受到自身抑制。最近，这一发现 运动蛋白结合蛋白(KIFBP)的研究揭示了一种新的运动蛋白抑制形式，KIFBP通过KIFBP与运动蛋白结合 运动域，以阻止微管结合。在这笔赠款中，我们将确定 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。基座 在KIFBP与运动域中两个不同的运动结合的结构研究中，我们开发了一个模型 KIFBP重塑了发动机中的Kinesin，以及KIFBP如何有选择地与电机接合。我们将介绍特定于站点的 基于交联质谱和患者来源突变体的突变以分离动蛋白结合 和KIFBP的改建(目标1)。同时，我们将确定轻链如何调节全长动蛋白 通过自身抑制产生的马达蛋白质。尽管几十年来对全长运动蛋白的调节进行了研究 蛋白质方面，关于抑制运动域中的运动仍然存在相互矛盾的结果。我们已做好准备 通过利用交联质谱学、蛋白质工程和 冷冻-EM，以确定运动蛋白轻链如何稳定紧凑的、受抑制的运动蛋白马达(目标2)。我们会 除了动蛋白-2异源三聚体(KIF3A：3B：KAP)外，还研究两种动蛋白-1异构体(KIF5B：KLC1)，以 比较和对比这些不同的运动蛋白运动复合体是如何自我抑制的。总而言之，这项工作 将扩展我们对Kinesin调控的理解，为建立抑制模式提供一个完整的视角 激动素的活性。