Computational Investigations of the Mechanisms Behind Microtubule Catastrophe

微管灾难背后机制的计算研究

• 批准号: 10515664
• 负责人: Daniel Beckett
• 金额: $ 3.59万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10515664
• 关键词: Active Sites; Affect; Behavior; Binding; Binding Sites; Catalysis; Cell division; Collaborations; Communities; Computational Technique; Coupling; Cryoelectron Microscopy; Cytoskeleton; Data; Development; Drug Targeting; Eukaryotic Cell; Feedback; Free Energy; Future; Geometry; Grain; Guanosine Diphosphate; Guanosine Triphosphate; Head; Hybrids; Hydrolysis; Intracellular Transport; Investigation; Knowledge; Lead; London; Maps; Methodology; Methods; Microtubule-Associated Proteins; Microtubules; Mitosis; Mitotic spindle; Modeling; Molecular; Molecular Conformation; Mutagenesis; Nature; Paclitaxel; Pathway interactions; Pharmaceutical Preparations; Poison; Polymers; Process; Property; Reaction; Recombinants; Research Personnel; Resolution; Rice; Role; Route; Sampling; Series; Site; Stimulus; Stress; Structure; Surface; Tail; Techniques; Testing; Texas; Therapeutic; Tube; Tubulin; Uncertainty; Universities; Vinblastine; Work; alpha Tubulin; beta Tubulin; cell motility; chemotherapeutic agent; computing resources; design; experience; experimental study; inorganic phosphate; microscopic imaging; molecular mechanics; mutant; novel; polymerization; quantum; reaction rate; response; simulation; targeted treatment; theories

PROJECT SUMMARY Microtubules (MTs) constitute the largest components of the eukaryotic cytoskeleton and facilitate a plethora of diverse functions including intracellular transport, cellular motility, and, cell division. During mitosis, MTs aggregate to form the mitotic spindle, making them a potent drug target for many successful chemotherapeutic agents, including paclitaxel and vinblastine, known as spindle poisons. MT-targeting drugs operate by interfering with dynamic instability (DI): the ability of MTs to rapidly switch from polymerizing to depolymerizing (referred to as catastrophe) and vice-versa. Paclitaxel operates by decreasing catastrophe rate while vinblastine encourages catastrophe and inhibits polymerization. A full understanding of MT catastrophe will greatly aid in the design of spindle poisons with fewer off-target effects, as well as greatly advance general understanding of DI. Each MT is composed of αβ-tubulin heterodimers, stacked head-to-tail in protofilaments (PFs) which are aligned laterally to form a hollow tube. Both α- and β-tubulin bind guanosine triphosphate (GTP) and hydrolysis of GTP to GDP (guanosine diphosphate) at the β-tubulin binding site is hypothesized to induce stress on the MT lattice. This stress gradually builds until the subunits at the MT end undergo GTP hydrolysis, at which point PFs begin to peel apart and catastrophe has occurred. Lag between GTP hydrolysis and polymerization creates a construct referred to as the GTP cap: a group of subunits at the MT end that have yet to hydrolyze GTP, release the product inorganic phosphate (Pi), or undergo a structural transition. Recent studies have caused doubt in the field on the nature of this transition and an atomistic understanding of the underlying mechanisms will lead to a full understanding of catastrophe. I propose to computationally resolve three key aspects of catastrophe: the mechanism of GTP hydrolysis, the release of Pi, and the structural coupling between PFs leading to catastrophe. First, I will use enhanced sampling methodology to uncover the enzymatic mechanism of GTP hydrolysis, with emphasis placed on potential catalytic residues belonging to α-tubulin, which sits atop β-tubulin upon polymerization to form the active site. Subsequently, I will develop novel computational techniques to determine the pathway of Pi release post-hydrolysis and examine the potential for structural change upon release. Lastly, I will develop a coarse-grained (CG) model of a full MT, using rates determined from the previous studies, able to undergo catastrophe to examine how hydrolysis and Pi release in neighboring subunits affects the potential for these reactions to occur in a particular subunit. This will give an unprecedentedly detailed view of the loss of the GTP cap and the steps leading to catastrophe. Additionally, I will collaborate with two leading experimentalists in the MT community to develop mutants that specifically test my hypotheses and to obtain lattice parameters of MTs doped with spindle poisons. This will allow me to integrate the effects of drugs into the CG model and examine how their effects propagate along an MT. These results and the developed models will greatly advance the understanding of DI and hopefully lead to the development of gentler MT-targeting therapies in the future.

项目摘要 微管（MTS）构成了真核细胞骨架的最大成分，并促进了很多 各种功能，包括细胞内转运，细胞运动和细胞分裂。在有丝分裂期间，MTS 骨料形成有丝分裂主轴，使其成为许多成功化学治疗的潜在药物靶标 包括紫杉醇和长黄质包括纺锤体毒物在内的特工。 MT靶向药物通过干扰 动态不稳定性（DI）：MT迅速从聚合转换为解聚的能力（参考 作为灾难​​），反之亦然。紫杉醇通过降低灾难率而运作，而vinblastine则鼓励 灾难并抑制聚合。对MT灾难的充分理解将极大地帮助设计 主轴毒药具有更少的脱靶效应，并且对DI的一般性了解。 每个MT都由αβ-微管蛋白异二聚体组成，在原丝（PFS）中堆叠成尾巴，它们是对齐的 横向形成一个空心管。 α-和β-微蛋白蛋白都结合三磷酸鸟苷（GTP）和GTP的水解 假设在β-微管蛋白结合位点上的GDP（鸟苷二磷酸）以诱导MT晶格的应力。 这种压力逐渐建立，直到MT端的亚基进行GTP水解，此时PFS开始 分开剥皮并发生灾难。 GTP水解和聚合之间的滞后会产生一个构建体 称为GTP帽：MT端的一组亚基，尚未水解GTP，释放 产物无机磷酸盐（PI）或进行结构过渡。最近的研究引起了怀疑 关于这种过渡的性质和对基本机制的原子理解的领域将导致 对灾难的充分理解。我建议通过计算解决灾难的三个关键方面： GTP水解的机理，PI的释放以及PFS导致灾难的结构耦合。 首先，我将使用增强的抽样方法来揭示GTP水解的酶促机理，并使用 重点放在属于α-微管蛋白的潜在催化残留物上，该残留蛋白位于β-微管蛋白上 聚合以形成活性位点。随后，我将开发新颖的计算技术来确定 释放后PI释放的途径并检查释放后结构变化的潜力。最后，我 将使用从先前的研究确定的速率开发完整MT的粗粒（CG）模型，能够 发生灾难以检查邻近亚基中的水解和PI如何影响 这些反应发生在特定的亚基中。这将为丢失的遗失提供前所未有的详细观点 GTP帽和导致灾难的步骤。此外，我将与两位领先的实验学家合作 在MT社区中，开发突变体，这些突变体专门检验我的假设并获得晶格参数 MTS掺有纺锤毒物。这将使我能够将药物的影响整合到CG模型中，并 检查其效果如何沿MT传播。这些结果和开发的模型将大大推动 对DI的理解并希望可以导致未来更温和的MT靶向疗法的发展。

项目成果

Unveiling the catalytic mechanism of GTP hydrolysis in microtubules.

• DOI: 10.1073/pnas.2305899120
• 发表时间: 2023-07-04
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Beckett, Daniel;Voth, Gregory A.
• 通讯作者: Voth, Gregory A.