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（由适用提供）：了解ATP结合盒（ABC）中的两个核水平结合域（ABC）是否存在基本差距 - 转运蛋白都具有催化活性，以及它们如何协调ATP水解在将化学自由能转化为机械工作中的水解作用。填补这一知识差距可能会通过提供分子级的机制来产生重要的生物医学益处，该机制有助于设计新的治疗策略，以治疗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态如何在ABC转运蛋白中连接的一般原则。这里的目的是通过一种新的多尺度QM/mm仿真方法（称为反应路径力匹配（RP-FM））来确定ABC转运蛋白两个特定成员的ATP水解机理。我们的中心假设是，这些系统的NBD中的两个活动位点在水解ATP中不对称地发挥作用，从而允许其中一个可以一次访问催化能力合理的构型。关于NBDS中详细的酶机制，我们假设两个特定的构成保留，其作用仍然难以捉摸，明确和动态地参与了ATP水解。我们的假设是根据生化研究，晶体结构和我们的初步模拟结果提出的。拟议研究的理由是，一旦确定了针对拟议系统的单个会议状态的精确催化机制，就可以设计和执行进一步的实验和模拟，以检查催化活性，这是沿Transporter循环的多个会议状态的函数。我们计划通过追求两个具体目标来检验中心假设：（1）扩展和验证RP-FM模拟复杂系统的RP-FM方法；（2）阐明毒素转运蛋白Hlyb和麦芽糖转运蛋白中的精确ATP水解机理。在第一个目标下，我们将扩展并验证RP-FM方法，以模拟水溶液中良好的特征化酶突变酶系统和甲基三磷酸甲基水解。在第二个目标下，将使用RP-FM模拟在两个ABC转运蛋白中建立ATPase机理，并确定每个系统中的两个活性位点在催化性上的不同程度。拟议的研究是原始的和创新的，因为QM/MM自由能模拟和RP-FM方法均未应用于任何ABC转运蛋白的ATP水解机制。我们在HLYB系统上的探索性工作代表了此类研究的第一个研究。该项目完成后，我们希望RP-FM方法将作为可靠的酶机制模拟的一般工具，并将获得此处检查的两个ABC转运蛋白的ATP水解机制的详细描述。这些信息不仅在理解两个特定细菌转运蛋白，而且在ABC转运蛋白家族中的其他成员非常有用。

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.

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

Toward Determining ATPase Mechanism in ABC Transporters: Development of the Reaction Path-Force Matching QM/MM Method.

确定 ABC 转运蛋白中的 ATP 酶机制：反应路径力匹配 QM/MM 方法的开发。