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Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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생물학

基于结构的转录因子蛋白沿DNA从原子级步进到粗晶扩散运动的模拟和采样

Published: March 01, 2022
doi:
10.3791/63406
, , , ,
1Beijing Computational Science Research Center, 2Shenzhen JL Computational Science and Applied Research Institute, 3School of Medical Informatics and Engineering,Xuzhou Medical University, 4Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine,Shanghai Jiao Tong University, 5Department of Physics and Astronomy,University of California, Irvine, 6Department of Chemistry,University of California, Irvine, 7NSF-Simons Center for Multiscale Cell Fate Research,University of California, Irvine

Summary

该协议的目标是揭示蛋白质沿DNA的一维扩散的结构动力学，使用植物转录因子WRKY结构域蛋白作为示例系统。为此，已经实施了原子和粗粒度分子动力学模拟以及广泛的计算采样。

Abstract

转录因子（TF）蛋白沿DNA的一维（1-D）滑动对于促进TF的扩散以定位遗传调控的靶DNA位点至关重要。检测TF滑动或踩踏DNA的碱基对（bp）分辨率在实验上仍然具有挑战性。我们最近进行了全原子分子动力学（MD）模拟，捕获了小的WRKY结构域TF蛋白沿DNA的自发1-bp步进。基于从此类仿真中获得的10 μs WRKY步进路径，此处的协议显示了如何通过构建用于1-bp蛋白质步进的马尔可夫状态模型（MSM）对TF-DNA系统进行更广泛的构象采样，并测试各种数量的微观和宏观状态用于MSM构建。为了检查TF蛋白与结构基础的DNA的加工一维扩散搜索，该方案进一步展示了如何进行粗粒度（CG）MD模拟以采样系统的长期尺度动力学。与全原子模拟揭示的亚微秒到微秒的蛋白质步进运动相比，这种CG建模和模拟对于揭示蛋白质-DNA静电对TF蛋白质在几十微秒以上的过程扩散运动的影响特别有用。

Introduction

转录因子（TF）寻找靶DNA以结合和调节基因转录及相关活性1.除了三维（3D）扩散之外，TF的促进扩散被认为是靶DNA搜索所必需的，其中蛋白质还可以沿着一维（1D）DNA滑动或跳跃，或者在DNA2，3，4，5，6，7上进行节间转移。

在最近的一项研究中，我们对植物TF进行了数十微秒（μs）全原子平衡分子动力学（MD）模拟 – DNA8上的WRKY结构域蛋白。在微秒内捕获了WRKY在多晶A DNA上的完整1-bp步进。已经观察到蛋白质沿DNA槽的运动和氢键（HBs）断裂重整动力学。虽然这样的轨迹代表了一条采样路径，但整体蛋白质步进景观仍然缺乏。在这里，我们展示了如何使用构建的马尔可夫状态模型（MSM）围绕最初捕获的蛋白质步进路径扩展计算采样，该模型已广泛实施，用于模拟涉及实质性构象变化和时间尺度分离的各种生物分子系统9，10，11，12，13，14，15，16，17，18，19.目的是揭示TF蛋白沿DNA扩散的构象集合和亚稳态，用于一个循环步骤。

虽然上述MD模拟揭示了DNA上1 bp的蛋白质运动的原子分辨率，但TF以相同的高分辨率沿DNA的长期过程扩散的结构动力学几乎不容易获得。然而，在残留物水平上进行粗粒度（CG）MD仿真在技术上是可行的。CG模拟时间尺度可以有效地扩展到比原子模拟长20、21、22、23、24、25、26、27、28、29的几十倍或几百倍。在这里，我们展示了通过实施Takada lab30开发的CafeMol软件进行的CG模拟。

在目前的方案中，我们首先介绍了沿多A DNA和MSM构建的WRKY结构域蛋白的原子模拟，其重点是对沿DNA仅1 bp的蛋白质步进运动进行采样。然后，我们提出了同一蛋白质 – DNA系统的CG建模和模拟，该系统将计算采样扩展到蛋白质沿着DNA的数十个bps上的过程扩散。

在这里，我们使用GROMACS31，32，33 软件进行MD模拟，并使用MSMbuilder34 构建用于采样构象快照的MSM，以及使用VMD35 可视化生物分子。该协议要求用户能够安装和实现上述软件。然后，CafeMol30 软件的安装和实施对于进行CG MD模拟是必要的。在VMD中还对轨迹和可视化进行了进一步的分析。

Protocol

1. 从原子MD模拟构建马尔可夫状态模型（MSM） 自发蛋白质步进途径和初始结构收集 使用先前获得的10μs全原子MD轨迹8 从“向前”的1-bp步进路径（即，每纳秒一帧）中均匀地提取10000帧。帧的总数需要足够大，以包括所有代表性构象。 在 VMD 中准备具有 10000 帧的过渡路径，方法是单击 “文件”>“保存坐标”，在“所选原子”框中键…

Representative Results

旋转耦合滑动或 1 bp 步进的 WRKY 从 MSM 结构DNA上的所有蛋白质构象都映射到蛋白质COM沿DNA的纵向运动X和旋转角度（见 图3A）。这两个度的线性偶联表示WRKY结构域蛋白在DNA上的旋转耦合步进。构象可以在 MSM 中进一步聚类为 3 个宏状态（S1、S2 和 S3）。然后，WRKY 的正向步进遵循宏观状态转换 S1->S2->S3。S1是指由建模结构（基于WRKY-DNA复合物40的?…

Discussion

这项工作解决了如何进行基于结构的计算模拟和采样，以揭示转录因子或TF蛋白沿着DNA移动，不仅在步进的原子细节下，而且在过程扩散中，这对于TF在DNA靶标搜索中的促进扩散至关重要。为此，首先构建了小TF结构域蛋白WRKY沿着均匀的poly-A DNA步进1-bp的马尔可夫状态模型或MSM，以便可以揭示DNA上的蛋白质构象集合以及蛋白质 – DNA界面处的集体氢键或HB动力学。为了获得MSM，我们沿着自发蛋白质步?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了NSFC Grant #11775016和#11635002的支持。JY得到了UCI的CMCF通过NSF DMS 1763272和Uci的西蒙斯基金会赠款#594598和启动基金的支持。LTD得到了上海市自然科学基金#20ZR1425400和#21JC1403100。我们也感谢北京计算科学研究中心（CSRC）的计算支持。

Materials

CafeMol Kyoto University coarse-grained (CG) simulations
GROMACS University of Groningen Royal Institute of Technology Uppsala University molecular dynamics simulations software
Matlab MathWorks Numerical calculation software
MSMbuilder Stanford University build MSM
VMD UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN molecular visualization program
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Tags

Structure-based SimulationTranscription Factor ProteinDNA DiffusionMarkov State ModelCoarse-grained SimulationAll-atom MD TrajectoryVMDRMSDRotational DegreeK-means ClusteringAtomistic SystemGROMACSTime-independent Component Analysis

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E, C., Dai, L., Tian, J., Da, L., Yu, J. Structure-Based Simulation and Sampling of Transcription Factor Protein Movements along DNA from Atomic-Scale Stepping to Coarse-Grained Diffusion. J. Vis. Exp. (181), e63406, doi:10.3791/63406 (2022).
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