Mechanism of microtubule severing enzymes

细胞通过微管聚合酶、解聚合酶、交联剂和切断酶的协同作用，不断地组装和拆卸其微管细胞骨架。微管切断酶痉挛蛋白和角朊蛋白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至关重要的作用，包括神经元和非中心体微管阵列的生物发生、向光性、纺锤体缩放、染色体分离以及中心粒和纤毛数量的控制。微管切断酶的突变导致严重的神经退行性和神经发育障碍。这些酶破坏微管稳定性的机制及其对微管动力学和微管网络形态的影响尚不清楚。我们的目标是(1)了解spastin和katanin在微管分解过程中所经历的结构转变；(2)表征了微管切断反应中角朊蛋白和肌蛋白酶六聚体中ATP水解的机制，以及它们如何与微管晶格中微管蛋白二聚体去除的机械功耦合；(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性截瘫和小头畸形相关的spastin和katanin疾病突变如何分别影响蛋白质结构和功能；(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，这在很大程度上是由于缺乏任何结构信息。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，在很大程度上，人们对微管从末端破坏稳定的机制有了更好的理解。研究微管切断酶的机制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 atp酶产生影响，尽管人体的每个主要途径都含有AAA atp酶，但对这一大类蛋白质仍知之甚少。

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.