Computational Investigations of the Mechanisms Behind Microtubule Catastrophe

微管灾难背后机制的计算研究

基本信息

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
项目类别：
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

项目摘要

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、细胞运动和有丝分裂期间的细胞分裂。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。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。 β-微管蛋白结合位点处的 GDP（二磷酸鸟苷）被重新捕获，以在 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 开始剥离并发生灾难，GTP 水解和聚合之间的滞后创建了一个结构。称为GTP帽：MT末端的一组亚基，尚未水解GTP，释放产物无机磷酸盐（Pi），或经历了结构转变，最近的研究引起了人们的怀疑。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。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详细的损失视图。另外，GTP 上限和导致灾难的步骤，我将与两位领先的实验家合作。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 MT 掺杂了纺锤体毒物，这将使我能够将药物的作用整合到 CG 模型中并研究它们的影响如何沿着 MT 传播。这些结果和开发的模型将大大推进。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

期刊论文数量（1）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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.