Subnanoscale mechanics of microtubule dynamic instability

真核微管（MTs）是形成有丝分裂纺锤体的细胞细丝，决定轴突和树突的形状，并为细胞内运输提供通道。微管蛋白二聚体（构成微管蛋白晶格的构建块）水解GTP核苷酸，使微管蛋白在生长和收缩阶段之间进行随机切换。MT的这种半稳定行为，也称为动态不稳定，对MT函数至关重要。GTP结合和水解控制MT组装和拆卸的详细机制仍然知之甚少，其中有两个方面仍然没有得到解决：(a)微管蛋白结合GTP与其构象之间的缺失环节，以及(b) GTP水解使MT晶格不稳定的机制。因此，该项目旨在通过完全原子模拟来表征(a)依赖于其核苷酸状态的单个微管蛋白二聚体的力学和能量学，以及(b)响应GTP水解的完整MT复合物。利用在准备阶段获得的初步数据，我们将首先采用蛋白质结构分析来评估GTP结合诱导的微管蛋白构象变化。原子自由能计算将被用来表征沿着这条构象途径的微管蛋白二聚体的能量学。获得的自由能图将使我们能够判断目前可用的基于实验的MT装配假设中哪一个在力学方面是最合理的。其次，我们建议使用最新的高分辨率低温电子显微镜（cro - em）数据开发和应用完整MT的新原子模型，以研究GTP水解时MT晶格的结构转变。通过使用自由能计算，我们打算定量地了解晶格中的单个二聚体如何以及在多大程度上促进了这种转变的能量学。所获得的能量势垒将使我们能够在原子水平上解释MT晶格的极端状态：预水解和完全水解之间的差异。考虑到仅在过去两年中对微管蛋白和mt的结构研究数量迅速增长，高分辨率的结构数据有望在拟议项目结束时可用，从而可以评估我们结果的全部潜力。我们还与Eva Nogales（美国加州大学伯克利分校）的实验实验室建立了合作关系，她目前正在利用高端冷冻电镜技术揭示MT晶格的中间（水解后）状态以及细胞因子对其的调制。此次合作将为验证我们的结果提供坚实的基础，并使我们能够在MT晶格中水解驱动跃迁的动力学和能量学方面提供新的原子细节，以补充MT的低温电镜分析。

Eukaryotic microtubules (MTs) are cellular filaments that form the mitotic spindle, define the shape of axons and dendrites, and provide tracks for intracellular transport. MTs undergo stochastic switching between phases of growth and shrinkage driven by the hydrolysis of GTP nucleotides by tubulin dimers, building blocks that constitute the MT lattice. This semistable behavior of MTs, also known as dynamic instability, is crucial for MT function. The detailed mechanism by which GTP binding and hydrolysis control the MT assembly and disassembly is still poorly understood, with two aspects standing out as essentially unresolved: (a) the missing link between GTP binding by tubulin and its conformation, and (b) the mechanism of destabilization of the MT lattice by GTP hydrolysis.This project therefore aims at characterizing, by fully atomistic simulations, the mechanics and energetics of (a) a single tubulin dimer depending on its nucleotide state and (b) a complete MT complex in response to GTP hydrolysis. Using preliminary data obtained during the preparatory phase, we will first employ protein structural analysis to assess what conformational changes in tubulin are induced by GTP binding. Atomistic free energy calculations will then be used to characterize the energetics of the tubulin dimer along this conformational pathway. The obtained free energy landscapes will enable us to judge which of the currently available experiment-based hypotheses of MT assembly is most plausible in terms of mechanics. Second, we propose to develop and apply new, atomistic models of complete MTs using the most recent high-resolution cryo-electron microscopy (cryo-EM) data to study the structural transitions that the MT lattice undergoes upon GTP hydrolysis. By using free energy calculations, we intend to obtain a quantitative understanding of how and how much single dimers in the lattice contribute to the energetics of this transition. The obtained energy barriers will enable us to interpret at the atomic level experimentally observable differences between the extreme states of the MT lattice: pre-hydrolysis and fully hydrolyzed.Considering the rapidly growing number of structural studies of tubulin and MTs only over the past 2 years, high-resolution structural data are expected to be available toward the end of the proposed project such that the full potential of our results can be assessed. We have also established a collaboration with the experimental lab of Eva Nogales (University of California, Berkeley, USA) who is currently working on revealing intermediate (post-hydrolysis) states of the MT lattice and their modulation by cellular factors using high-end cryo-EM. The collaboration would provide a solid basis for validating our results and allow us to complement the cryo-EM analysis of MTs with new atomistic details on the dynamics and energetics of hydrolysis-driven transitions in the MT lattice.