Mechanism of microtubule severing enzymes

微管切断酶的机制

• 批准号: 10263056
• 负责人: Antonina Roll-Mecak
• 金额: $ 152.98万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10263056
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Affect; Architecture; Binding; Biogenesis; Biological Process; Cations; Cells; Centrioles; Chromosome Segregation; Cilia; Complex; Coupled; Couples; Crosslinker; Cryoelectron Microscopy; Cytoskeleton; Disease; Electron Microscopy; Enzymes; Excision; Functional disorder; Generations; Glutamates; Hereditary Spastic Paraplegia; Human body; Light; Mechanics; Microcephaly; Microtubules; Missense Mutation; Modification; Molecular; Molecular Conformation; Molecular Machines; Morphology; Movement; Mutate; Mutation; Nature; Neurodegenerative Disorders; Neurodevelopmental Disorder; Neurons; Nucleotides; Pathway interactions; Patients; Phosphorylation; Phototropism; Polymerase; Proteins; Protomer; Reaction; Reporting; Stretching; Structure; Tail; Time; Tubulin; Work; X-Ray Crystallography; arm; design; dimer; grasp; insight; katanin; protein structure function; spastin; support network

Cells constantly assemble and disassemble their microtubule cytoskeleton through the concerted action of microtubule polymerases, depolymerases, crosslinkers and severing enzymes. Microtubule severing enzymes spastin and katanin generate internal breaks in microtubules. They are are critical in a wide range of cell biological processes including biogenesis of neuronal and non-centrosomal microtubule arrays, phototropism, spindle scaling, chromosome segregation, and control of centriole and cilia numbers. Mutations in microtubule severing enzymes cause severe neurodegenerative and neurodevelopmental disorders. The mechanism used by these enzymes to destabilize the microtubule and their effect on microtubule dynamics and the morphology of microtubule networks is still poorly understood. We aim (1) to understand the structural transitions that spastin and katanin undergo during microtubule disassembly; (2) characterize the mechanism of ATP hydrolysis in the katanin and spastin hexamers during the microtubule severing reaction and how they are coupled to the mechanical work of tubulin dimer removal from the microtubule lattice; (3) establish the effects of tubulin modifications on microtubule severing; (4) characterize the effects of microtubule severing enzymes on microtubule dynamics and architecture; (5) develop a comprehensive understanding of how spastin and katanin disease mutations associated with hereditary spastic paraplegia and microcephaly, respectively, affect protein structure and function and (6) identify cellular factors that regulate spastin and katanin. Despite it being a basic mechanism to destabilize microtubules, we know very little about severing, not in small part due to the lack of any structural information. The mechanism of destabilizing microtubules from their ends is far better understood, in large part due to the wealth of structural information on the molecular machines involved, obtained by X-ray crystallography and electron microscopy. A mechanistic approach to the study of microtubule severing enzymes will provide a new framework for analyses and design of cellular studies. Moreover, insights into the mechanism of action of severing enzymes will likely hold implications for AAA ATPase in general, a large class of proteins still poorly understood, despite the fact that every major pathway in the human body contains an AAA ATPase. We have made significant progress in the last year in deciphering the structure and mechanism of both spastin and katanin. We reported the cryo-EM structure of the hereditary spastic paraplegia (HSP) protein spastin in complex with its substrate (Sandate et al., Nature Struct. & Molec. Biol. 2019). This structure revealed for the first time how a severing enzyme engages the tubulin substrate and shed light on how concurrent nucleotide and substrate binding organizes the conserved spastin pore loops into an ordered allosteric network that supports tubulin tail translocation to pull the tubulin dimer out of the microtubule and sever it. The majority of the residues in this allosteric network are mutated in HSP patients, underscoring their importance to spastin function. Our comprehensive structural analysis of all reported HSP-associated spastin missense mutations in its AAA core provides a framework for understanding spastin molecular dysfunction. We also determined recently the cryo-EM structures of katanin complexes with substrate (Zehr et al., Dev Cell 2020). We found that katanin uses two opposing electropositive spirals in its central pore to grip the tubulin tail and that the beta-tail alone is sufficient for microtubule severing. Furthermore, long glutamate stretches in the tubulin tail are critical for katanin ATPase activation and oligomerization, consistent with its stimulation by glutamylation. Each pore spiral couples allosterically to the ATPase and binds alternating, successive residues in the tubulin tail, with consecutive residues coordinated by adjacent protomers. The first spiral is critical for ATPase activation, the second for force generation. Structures in two conformations with different ATP occupancies show that ATP hydrolysis and release uncouples the substrate from the pore loops, suggesting a mechanism for substrate movement that deforms and destabilizes the tubulin subunit and leads to its extraction, and ultimately, microtubule disassembly. Moreover, we identify two cationic regions in the linker arms required for microtubule severing. Aurora B phosphorylation in one of these motifs inhibits severing. Thus, our studies lay bare the complex multivalent interactions that katanin uses to recognize the microtubule and disassemble it.

细胞通过微管聚合酶、解聚酶、交联剂和切割酶的协同作用不断地组装和拆卸它们的微管细胞骨架。 微管切断酶spastin和katanin在微管中产生内部断裂。它们在广泛的细胞生物学过程中至关重要，包括神经元和非中心体微管阵列的生物发生、向光性、纺锤体缩放、染色体分离以及中心粒和纤毛数量的控制。 微管切割酶的突变导致严重的神经退行性和神经发育障碍。这些酶使微管不稳定的机制及其对微管动力学和微管网络形态的影响仍然知之甚少。我们的目标是：（1）了解微管分解过程中spastin和katanin的结构转变;（2）描述微管切割反应中katanin和spastin六聚体中ATP水解的机制，以及它们如何与微管蛋白二聚体从微管晶格中去除的机械功相耦合;（3）建立微管蛋白修饰对微管切割的影响;（4）描述微管切割酶对微管动力学和结构的影响;（5）全面了解与遗传性痉挛性截瘫和小头畸形相关的痉挛素和katanin疾病突变如何分别影响蛋白质结构和功能;（6）鉴定调节痉挛素和katanin的细胞因子。 尽管它是破坏微管稳定的基本机制，但我们对切断知之甚少，这在很大程度上是由于缺乏任何结构信息。从微管末端破坏微管稳定的机制已经被更好地理解，这在很大程度上是由于通过X射线晶体学和电子显微镜获得的有关分子机器的丰富结构信息。 微管断裂酶的机理研究将为细胞研究的分析和设计提供一个新的框架。此外，深入了解切断酶的作用机制可能会对AAA ATP酶产生影响，尽管人体中的每一个主要途径都含有AAA ATP酶，但这是一大类仍然知之甚少的蛋白质。 在过去的一年里，我们在破译痉挛素和卡他宁的结构和机制方面取得了重大进展。我们报道了与其底物复合的遗传性痉挛性截瘫（HSP）蛋白spastin的冷冻-EM结构（Sandate等人，自然结构与分子2019）。这种结构首次揭示了切断酶如何与微管蛋白底物结合，并阐明了核苷酸和底物的同时结合如何将保守的痉挛蛋白孔环组织成有序的变构网络，该网络支持微管蛋白尾部易位，将微管蛋白二聚体拉出微管并切断它。这种变构网络中的大多数残基在HSP患者中发生突变，强调了它们对痉挛功能的重要性。我们对所有报道的腹主动脉瘤核心区HSP相关痉挛蛋白错义突变进行了全面的结构分析，为理解痉挛蛋白分子功能障碍提供了一个框架。 我们最近还确定了katanin与底物的复合物的cryo-EM结构（Zehr等人，Dev Cell 2020）。我们发现katanin在其中心孔中使用两个相对的正电性螺旋来抓住微管蛋白尾部，并且单独的β-尾部足以切断微管。此外，微管蛋白尾部的长谷氨酸延伸对于katanin ATP酶激活和寡聚化是至关重要的，与其通过谷氨酰化的刺激一致。每个螺旋孔与ATP酶变构偶联，并结合微管蛋白尾中交替的连续残基，相邻的原聚体协调连续的残基。第一个螺旋是ATP酶激活的关键，第二个是力的产生。在两种构象与不同的ATP occupancy的结构表明，ATP水解和释放解偶联的基板从孔环，这表明一种机制，基板移动变形和不稳定的微管蛋白亚基，并导致其提取，并最终微管解体。此外，我们确定了两个阳离子区域的连接臂所需的微管切断。其中一个基序中的极光B磷酸化抑制切断。因此，我们的研究揭示了卡塔宁用来识别微管并分解它的复杂多价相互作用。