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.

项目概要 微管 (MT) 构成真核细胞骨架的最大组成部分，并促进大量的 多种功能，包括细胞内运输、细胞运动和细胞分裂。有丝分裂期间，MT 聚集形成有丝分裂纺锤体，使它们成为许多成功化疗的有效药物靶点 药物，包括紫杉醇和长春花碱，被称为纺锤体毒物。 MT靶向药物通过干扰发挥作用 具有动态不稳定性（DI）：MT从聚合快速切换到解聚的能力（称为 作为灾难​​），反之亦然。紫杉醇通过降低灾难发生率来发挥作用，而长春碱则鼓励 灾难并抑制聚合。对 MT 灾难的充分理解将极大地有助于设计 纺锤体毒物具有较少的脱靶效应，并大大增进了对 DI 的一般理解。 每个 MT 由 αβ-微管蛋白异二聚体组成，头尾相连地堆叠在排列整齐的原丝 (PF) 中 横向形成空心管。 α-和 β-微管蛋白均结合三磷酸鸟苷 (GTP) 并水解 GTP 假设 β-微管蛋白结合位点上的 GDP（二磷酸鸟苷）会在 MT 晶格上产生应力。 这种压力逐渐增加，直到 MT 末端的亚基发生 GTP 水解，此时 PF 开始 剥落，灾难就发生了。 GTP 水解和聚合之间的滞后创建了一个构建体 称为GTP帽：MT末端的一组亚基，尚未水解GTP，释放 产品无机磷酸盐（Pi），或经历结构转变。最近的研究引起了人们的质疑 关于这种转变本质的领域以及对基本机制的原子理解将导致 对灾难的充分认识。我建议通过计算解决灾难的三个关键方面： GTP 水解、Pi 释放以及 PF 之间的结构耦合导致灾难的机制。 首先，我将使用增强采样方法来揭示 GTP 水解的酶促机制，其中 重点放在属于 α-微管蛋白的潜在催化残基，它位于 β-微管蛋白之上 聚合形成活性位点。随后，我将开发新的计算技术来确定 水解后 Pi 释放的途径，并检查释放后结构变化的可能性。最后，我 将使用先前研究确定的速率开发完整 MT 的粗粒度 (CG) 模型，能够 经历灾难来检查相邻亚基中的水解和 Pi 释放如何影响潜在的 这些反应发生在特定的亚基中。这将为我们提供前所未有的关于损失的详细视图。 GTP 上限和导致灾难的步骤。此外，我将与两位领先的实验学家合作 在 MT 社区开发突变体来专门测试我的假设并获得晶格参数 MTs 掺杂纺锤体毒物。这将使我能够将药物的作用整合到 CG 模型中 检查它们的影响如何沿着 MT 传播。这些结果和开发的模型将极大地推进 对 DI 的理解并有望在未来引领更温和的 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.