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Determining ATPase Mechanism of ABC-Transporters by Reaction Path Force Matching

通过反应路径力匹配确定 ABC 转运蛋白的 ATP 酶机制

基本信息

批准号：
8958346
负责人：
Jingzhi Pu
金额：
$ 36.64万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-08-01 至 2019-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8958346
关键词：
ATP phosphohydrolase ATP-Binding Cassette Transporters Active Biological Transport Active Sites Adenosine Triphosphate Bacterial Infections Binding Binding Sites Biochemical Catalysis Catalytic Domain Chemicals Chorismate Mutase Clinical Complex Computer Simulation Coupling Cystic Fibrosis Development Disease Drug resistance Employment Enzymes Family Fingers Free Energy Functional disorder Goals Hydrolysis Knowledge Ligand Binding Domain Literature Maltose Mechanics Medical Methods Molecular Molecular Conformation Molecular Motors Motor Multi-Drug Resistance Nucleotides Outcome Process Protons Public Health Publishing Reaction Research Role Sampling Site Solutions Structure System Testing Time Toxin Transmembrane Domain Work aqueous base cancer therapy computerized tools design dimer enzyme mechanism expectation human disease innovation insight member molecular transporter novel therapeutic intervention novel therapeutics public health relevance research study simulation tool tripolyphosphate

项目摘要

DESCRIPTION (provided by applicant): There is a fundamental gap in understanding whether the two nucleotide binding domains in ATP-binding cassette (ABC)- transporters are both catalytically active and how they orchestrate the action of ATP hydrolysis in converting chemical free energy into mechanical work. Filling this knowledge gap may generate important biomedical benefits by providing a molecular-level mechanism that aids designs of new therapeutic strategies in treating ABC-transporter related human diseases and clinical problems. Our long-term goal is to understand the general principles of how chemical catalysis and conformational dynamics are connected in ABC-transporters. The objective here is to determine the ATP hydrolysis mechanism in two specific members of ABC- transporters, by a new multiscale QM/MM simulation approach called Reaction Path Force Matching (RP-FM). Our central hypothesis is that the two active sites in NBDs of these systems function asymmetrically in hydrolyzing ATP, thereby allowing only one of them to access the catalytic competent configuration at a time. Regarding the detailed enzyme mechanism in NBDs, we hypothesized that two particular conserved residues, whose roles have remained elusive, participate in ATP hydrolysis explicitly and dynamically. Our hypotheses have been formulated based on biochemical studies, crystal structures, and our preliminary simulation results. The rationale for the proposed research is that, once the precise catalytic mechanism is determined for the single conformational state on the proposed systems, further experiments and simulations can be designed and performed to examine the catalytic activity as a function of multiple conformational states along the transporter cycle. We plan to test our central hypothesis by pursuing two Specific Aims: (1) extend and validate the RP-FM method for simulating complex systems; and (2) elucidate the precise ATP hydrolysis mechanism in the toxin transporter HlyB and the maltose transporter. Under the first aim, we will extend and validate the RP-FM approach for simulations of the well-characterized enzyme chorismate mutase system and methyl- triphosphate hydrolysis in aqueous solution. Under the second aim, RP-FM simulations will be employed to establish the ATPase mechanism in the two ABC-transporters and determine the extent to which the two actives sites in each of these systems are catalytically different. The proposed research is original and innovative because neither QM/MM free energy simulations, nor the RP-FM method, have been applied to study ATP hydrolysis mechanisms for any ABC-transporters; our exploratory work on the HlyB system represents the first study of this kind. Upon completion of this project, we expect that the RP-FM method will become available as a general tool for reliable simulations of enzyme mechanisms and a detailed description of ATP hydrolysis mechanism for the two ABC-transporters examined here will be obtained. Such information is extremely useful in understanding not only the two specific bacterial transporters, but also other members in the ABC-transporter family.

描述（由申请人提供）：在理解ATP结合盒（ABC）转运蛋白中的两个核苷酸结合结构域是否都具有催化活性以及它们如何协调ATP水解将化学自由能转化为机械功的作用方面存在根本性的差距。填补这一知识空白可能会产生重要的生物医学效益，提供一个分子水平的机制，帮助设计新的治疗策略，在治疗ABC转运蛋白相关的人类疾病和临床问题。我们的长期目标是了解化学催化和构象动力学如何在ABC转运蛋白连接的一般原则。这里的目标是确定ATP水解机制在两个特定的成员ABC-转运蛋白，通过一个新的多尺度QM/MM模拟方法称为反应路径力匹配（RP-FM）。我们的中心假设是，这些系统的NBD中的两个活性位点在水解ATP时不对称地发挥作用，从而一次只允许其中一个进入催化活性构型。关于NBD中的详细酶机制，我们假设两个特定的保守残基，其作用仍然难以捉摸，明确和动态地参与ATP水解。我们的假设是基于生化研究，晶体结构和我们的初步模拟结果。所提出的研究的基本原理是，一旦精确的催化机制被确定为单一的构象状态上提出的系统，进一步的实验和模拟可以被设计和执行，以检查作为多个构象状态的函数的催化活性沿着转运循环。我们计划通过追求两个特定目标来验证我们的中心假设：（1）扩展和验证RP-FM方法用于模拟复杂系统;（2）阐明毒素转运蛋白HlyB和麦芽糖转运蛋白中的精确ATP水解机制。在第一个目标下，我们将扩展和验证RP-FM方法，用于模拟良好表征的酶分支酸酯水解系统和水溶液中的三磷酸甲酯水解。在第二个目标下，RP-FM模拟将用于建立两个ABC转运蛋白中的ATP酶机制，并确定这些系统中的每一个中的两个活性位点在催化上不同的程度。拟议的研究是原创性和创新性的，因为无论是QM/MM自由能模拟，也没有RP-FM方法，已被应用于研究ATP水解机制的任何ABC转运蛋白;我们的探索性工作HlyB系统代表了这种研究的第一次。在完成这个项目后，我们预计RP-FM方法将成为一个通用的工具，可靠的模拟酶的机制和ATP水解机制的详细描述，这里检查的两个ABC转运蛋白将获得。这些信息不仅对理解两种特定的细菌转运蛋白非常有用，而且对理解ABC转运蛋白家族的其他成员也非常有用。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Mapping Free Energy Pathways for ATP Hydrolysis in the E. coli ABC Transporter HlyB by the String Method.

通过弦方法绘制大肠杆菌ABC转运蛋白Hlyb中ATP水解的自由能途径。

DOI：
10.3390/molecules23102652
发表时间：
2018-10-16
期刊：
Molecules (Basel, Switzerland)
影响因子：
0
作者：
Zhou Y;Ojeda-May P;Nagaraju M;Kim B;Pu J
通讯作者：
Pu J

Reaction Path-Force Matching in Collective Variables: Determining Ab Initio QM/MM Free Energy Profiles by Fitting Mean Force.

DOI：
10.1021/acs.jctc.1c00245
发表时间：
2021-08-10
期刊：
JOURNAL OF CHEMICAL THEORY AND COMPUTATION
影响因子：
5.5
作者：
Kim, Bryant;Snyder, Ryan;Nagaraju, Mulpuri;Zhou, Yan;Ojeda-May, Pedro;Keeton, Seth;Hege, Mellisa;Shao, Yihan;Pu, Jingzhi
通讯作者：
Pu, Jingzhi

Doubly Polarized QM/MM with Machine Learning Chaperone Polarizability.