Mechanism of microtubule severing enzymes

细胞通过微管聚合酶、解聚酶、交联剂和裂解酶的协同作用，不断地组装和分解其微管细胞骨架。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起着关键作用，包括神经元和非中心体微管阵列的生物发生、向光性、纺锤体伸缩、染色体分离以及中心粒和纤毛数量的控制。微管切断酶的突变会导致严重的神经退行性和神经发育障碍。这些酶用来破坏微管稳定性的机制以及它们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)了解微管拆解过程中spastin和katanin经历的结构转变；(2)表征微管切断反应过程中katanin和spastin六聚体中ATP的水解机制，以及它们如何与微管晶格中微管蛋白二聚体的机械作用相耦合；(3)建立微管蛋白修饰对微管切断的影响；(4)表征微管切断酶对微管动力学和结构的影响；(5)全面了解与遗传性痉挛截瘫和小头畸形相关的疾病突变如何影响蛋白质结构和功能；以及(6)确定调节spastin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，这在很大程度上是因为缺乏任何结构信息。微管从其末端破坏稳定的机制被更好地理解，这在很大程度上是由于所涉及的分子机器的丰富的结构信息，这些信息是通过X射线结晶学和电子显微镜获得的。研究微管切断酶的机械方法将为细胞研究的分析和设计提供一个新的框架。此外，对切断酶的作用机制的深入了解可能会对AAA ATPase产生影响，AAA ATPase是一大类蛋白质，尽管人体内的每一条主要途径都含有AAA ATPase，但对AAA ATPase的了解仍然很少。 我们报道了单体AAA katanin模块的第一个X射线结构和两种构象中六聚体的冷冻EM重建(Zehr等人，自然结构。&Molec.比奥尔。2017年)。这些揭示了AAA结构域的意外不对称排列，这些结构元件是由微管切断酶特有的结构元件介导的，这些结构元件对它们的功能至关重要。我们在4.4和6分辨率下对katanin六角体进行的冷冻-EM重建显示了AAA核心的开放螺旋和闭合环状构象，这取决于关闭katanin六角体中40宽的门的门控原始体的核苷酸占有率。结合溶液小角X射线散射(SAXS)重建，我们的综合结构研究使我们能够提出一个模型，即katanin通过其AAA核心、灵活的MIT结构域和新定义的连接元件与微管进行多价相互作用，并通过保守的孔环与微管蛋白的C末端结合，通过开放的螺旋和闭合的AAA环之间的循环逐渐将微管蛋白二聚体从微管晶格中拉出。 最近，我们还报道了遗传性痉挛截瘫(HSP)蛋白spastin与其底物的复合体的冷冻-EM结构(Sandate等人，自然结构。&Molec.比奥尔。2019年)。这种结构首次揭示了切断酶如何与微管蛋白底物结合，并揭示了同时的核苷酸和底物结合如何将保守的spastin孢子环组织成一个有序的变构网络，该网络支持微管蛋白尾部移位，将微管蛋白二聚体从微管中拉出并切断。在HSP患者中，这个变构网络中的大多数残基都发生了突变，这突显了它们对痉挛蛋白功能的重要性。我们对所有已报道的与热休克蛋白相关的spastin AAA核心错义突变的结构分析为理解spastin分子功能障碍提供了一个框架。 我的实验室还发现，切断酶spastin和katanin通过促进微管轴上微管蛋白二聚体的交换来调节微管动力学(Vemu等人。科学2018年)。结合单分子TIRF和电子显微镜，我们发现，spastin和katanin通过主动提取微管蛋白异源二聚体，并通过GTP-微管蛋白掺入自发修复，从而在微管中引入纳米级的损伤。结果，微管轴被GTP-微管蛋白岛恢复活力，以稳定其防止解聚，新切断的末端出现高密度的GTP-微管蛋白，以防止解聚。新切断的正端的稳定和更高的救援率协同作用，扩大了微管的数量和质量。因此，我们的工作确定了微管切断酶spastin和katanin是负责在微管内产生GTP-微管蛋白岛的生物制剂，并证明了单独的微管切断酶可以通过促进微管轴中的GTP-微管蛋白的掺入来放大微管的数量和质量，而远离长期以来被认为是微管蛋白交换的唯一位置的动态末端。在缺乏成核因子的情况下，这种基于微管的放大机制可以解释为什么在依赖于非中心体微管生成的系统中，spastin和katanin的丢失会导致微管质量的损失。

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 reported the first X-ray structure of the monomeric AAA katanin module and cryo-EM reconstructions of the hexamer in two conformations (Zehr et al., Nature Struct. & Molec. Biol. 2017). These revealed an unexpected asymmetric arrangement of the AAA domains mediated by structural elements unique to microtubule severing enzymes that are critical for their function. Our cryo-EM reconstructions at 4.4 and 6 resolution of the katanin hexamer revealed an open spiral and a closed ring conformations of the AAA core, depending on the nucleotide occupancy of a gating protomer that closes a 40 wide gate in the katanin hexamer. Together with solution small-angle X-ray scattering (SAXS) reconstructions, our integrated structural study allowed us to advance a model whereby katanin makes multivalent interactions with the microtubule through its AAA core, flexible MIT domains and a newly defined linker element that crowns the AAA ring, and engages the C-terminal tails of tubulin through conserved pore loops that gradually pull tubulin dimers out of the microtubule lattice by cycling between open spiral and closed AAA ring conformations. More recently, we also 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. My laboratory also discovered that severing enzymes spastin and katanin regulate microtubule dynamics by promoting the exchange of tubulin dimers along the microtubule shaft (Vemu et al. Science 2018). Combining single-molecule TIRF and electron microscopy we showed that spastin and katanin introduce nanoscale damage throughout the microtubule by active extraction of tubulin heterodimers that is repaired spontaneously by GTP-tubulin incorporation. As a result, the microtubule shaft is rejuvenated with GTP-tubulin islands that stabilize it against depolymerization and newly severed ends emerge with a high-density of GTP-tubulin that protects against depolymerization. The stabilization of the newly severed plus-ends and the higher rescue rates synergize to amplify microtubule number and mass. Thus, our work identified microtubule-severing enzymes spastin and katanin as biological agents responsible for the generation of GTP- tubulin islands within microtubules and demonstrated that microtubule-severing enzymes alone can amplify microtubule number and mass by promoting GTP-tubulin incorporation in the microtubule shaft, away from the dynamic ends long thought to be the sole locus of tubulin exchange. This microtubule-based amplification mechanism in the absence of a nucleating factor can explain why loss of spastin and katanin results in loss of microtubule mass in systems that are dependent on non-centrosomal microtubule generation.