Mechanism of microtubule severing enzymes

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。 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的六聚体的cryo-EM重建（Zehr等人，自然结构与分子2017）。 这些揭示了AAA结构域由微管切断酶特有的结构元件介导的意外的不对称排列，所述微管切断酶对其功能至关重要。 我们的cryo-EM重建在4.4和6分辨率的卡他宁六聚体揭示了一个开放的螺旋和封闭的环的AAA核心的构象，这取决于一个门控原聚体，关闭一个40宽的门在卡他宁六聚体的核苷酸占有率。 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，其中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，并与C-微管蛋白的末端尾部通过保守的孔环，通过在开放螺旋和闭合AAA之间循环，逐渐将微管蛋白二聚体拉出微管晶格环状构象 最近，我们还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。 我的实验室还发现，切断酶spastin和katanin可以通过促进沿着微管轴的微管蛋白二聚体的交换来调节微管动力学（Vemu等人，Science 2018）。结合单分子TIRF和电子显微镜，我们发现，痉挛素和katanin通过主动提取微管蛋白异源二聚体，通过GTP-微管蛋白掺入自发修复，在整个微管中引入纳米级损伤。结果，微管轴被GTP-微管蛋白岛再生，所述GTP-微管蛋白岛使其稳定以防止解聚，并且新切断的末端出现高密度的GTP-微管蛋白，所述GTP-微管蛋白防止解聚。新切断的正末端的稳定性和较高的拯救率协同作用以增加微管的数量和质量。因此，我们的工作确定了微管切断酶spastin和katanin作为负责微管内GTP-微管蛋白岛的产生的生物制剂，并证明了单独的微管切断酶可以通过促进GTP-微管蛋白掺入微管轴中来扩大微管的数量和质量，远离长期被认为是微管蛋白交换的唯一位点的动态末端。这种基于微管的扩增机制在不存在成核因子的情况下可以解释为什么spastin和katanin的损失导致依赖于非中心体微管生成的系统中微管质量的损失。

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.