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

 描述(由申请人提供)：在理解ATP结合盒(ABC)转运体中的两个核苷酸结合域是否都具有催化活性以及它们如何协调将化学自由能转化为机械功的ATP水解作用方面，存在着一个根本的空白。填补这一知识空白可能会产生重要的生物医学益处，因为它提供了一种分子水平的机制，有助于设计新的治疗策略，治疗与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ABC转运蛋白中化学催化和构象动力学如何联系的一般原理。本文的目的是通过一种新的多尺度QM/MM模拟方法，即反应路径力匹配(RP-FM)，来确定ABC转运蛋白中两个特定成员的ATP水解机制。我们的中心假设是，这些系统的NBD中的两个活性部位在水解三磷酸腺苷的过程中起着不对称的作用，因此一次只允许其中一个进入催化活性构型。关于NBDS中详细的酶机制，我们假设有两个特定的保守残基明确地和动态地参与了ATP的水解，它们的作用仍然难以捉摸。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而形成的。这项研究的理论基础是，一旦确定了所建议系统上单一构象状态的精确催化机理，就可以设计和进行进一步的实验和模拟，以考察催化活性作为沿转运体循环的多个构象状态的函数。我们计划通过追求两个特定的目标来验证我们的中心假设：(1)扩展和验证用于模拟复杂系统的RP-FM方法；(2)阐明毒素转运体HlyB和麦芽糖转运体中精确的ATP水解机制。在第一个目标下，我们将扩展和验证RP-FM方法，用于模拟具有良好特性的分支酸变位酶体系和三磷酸甲酯在水溶液中的水解。在第二个目标下，将使用RP-FM模拟来建立两个ABC转运体中的ATPase机制，并确定这两个系统中两个活性部位在催化方面的不同程度。这项研究具有原创性和创新性，因为无论是QM/MM自由能模拟，还是RP-FM方法，都没有被用来研究任何ABC转运蛋白的ATP水解机制；我们在HlyB系统上的探索性工作是这类研究的第一次。在这个项目完成后，我们预计RP-FM方法将成为可靠的酶机制模拟的通用工具，并将获得这里研究的两个ABC转运蛋白的ATP水解机制的详细描述。这些信息不仅对了解这两种特定的细菌转运蛋白非常有用，而且对了解ABC转运蛋白家族的其他成员也非常有用。

 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.