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Operation Mechanism of CLCF Fluoride/Proton Antiporter

CLCF氟化物/质子逆向转运蛋白的运行机制

基本信息

批准号：
10201208
负责人：
Hai Lin
金额：
$ 45.96万
依托单位：
UNIVERSITY OF COLORADO DENVER
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：
2021-05-01 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10201208
关键词：
ATP phosphohydrolase Algorithms Anions Architecture Bacteria Binding Binding Sites Biomedical Computing CLC Gene Caries prevention Carrier Proteins Cell membrane Cells Charge Chlorides Complex Computer Models Coupling Dental caries Electrostatics Environment Enzymes Exhibits Fluoride Ion Fluorides Free Energy Goals Growth Hydrogen Bonding Ion Transport Ions Knowledge Light Metabolic Molecular Mutation Pathway interactions Population Prevention Protein Conformation Proteins Protons Quantum Mechanics Radial Research Research Project Grants Resistance Rosaniline Dyes Rotation Sampling Series Side Streptococcus mutans Structure Students Techniques Testing Urease Water Work antiporter bacterial resistance base design enolase experimental study extracellular innovation insight migration models and simulation molecular dynamics molecular mechanics molecular modeling mutant novel novel therapeutic intervention novel therapeutics operation oral bacteria oral care oral microbial community prevent simulation student training training opportunity undergraduate student

项目摘要

Project Summary/Abstract Widely applied to prevent dental caries, F− ion can inhibit bacterial growth. However, many bacterial strains have evolved to be resistant to F–, utilizing exporters situated in the bacterial cell membranes that quickly reduce the intracellular F– concentration. Our long-term goal is to unlock the molecular details of these F– exporters. Our objective in this proposal is to elucidate the operation mechanism of a prototypical CLCF F–/H+ antiporter from E. casseliflavus, which regulates F– efflux and H+ influx with a 1:1 stoichiometric ratio. Our central hypothesis is that multiple structural and energetic factors, including ionic size, electrostatic interactions, hydrogen-bonding, and anion-H+ coupling, are fine-tuned to determine the F– selection and transport in CLCF. We have formulated the hypothesis based on experimental studies of CLCF by Miller and coworkers and on our and others' previous computational work on the homologous canonical CLC Cl–/H+ antiporters. We will carry a series of steered molecular dynamics and umbrella sampling simulations to answer the questions in three specific aims: (1) How are H+ and anions transported in CLCF-Eca? It has been hypothesized that the H+ gate E118 undergoes rotation, carrying H+ from the extracellular to intracellular solutions and propelling F– through the pore. We also hypothesize that F– passes more easily than Cl– due to its smaller radius. We will identify the two protein conformations that are currently missing in the transport cycle, and we will quantify the translocation free-energy barriers for both anions. (2) Why is F– permeation decreased when E318, a residue near the anion binding site, is neutralized? The working hypothesis is that neutralizing the negatively charged E318 in the E318Q and E318A mutants reduces the electrostatic forces that displace F– from the binding site. We will compare the barriers for anion displacements among WT, E318Q, and E318A. (3) What causes the switch in anion selectivity from F– to Cl– in the E118Q and E118A mutants, in which the H+ pathway is abolished? We hypothesize that H+ concentration gradients drive H+ into the anion pore to protonate E318 and that the H+ sharing between F– (but not Cl–) and E318, which have similar pKa values, traps F– in the pore, disrupting the transport cycle. We will simulate water wires formation and subsequent H+ migration. We will estimate free-energy barriers for anion permeation in E118Q and E118Q/E318A; we predict that the double mutant reduces permeation for both anions but retains the F–-over-Cl– selectivity, which can be experimentally tested. The study is innovative because it will (a) shift the current research paradigm for CLCF by including insights from the computational perspective and (b) use novel adaptive-partitioning quantum- mechanics/molecular-mechanics algorithms to simulate explicit H+ transport. The research is significant in that it will (i) provide critical insights into F– resistance in the oral microbial community, (ii) deepen our understanding of the homologous canonical CLC Cl–/H+ and other transport proteins, and (iii) enhance the research environment at CU-Denver Downtown Campus and promote undergraduate student research.

项目总结/摘要 F −离子被广泛应用于预防龋齿，可以抑制细菌生长。然而，许多细菌菌株已经进化到对F-有抵抗力，利用位于细菌细胞膜中的出口商，降低细胞内F-浓度。我们的长期目标是解开这些F- 出口商。我们的目标是阐明一个典型的CLCF F-/H+的运作机制反向转运蛋白E. casseliflavus，其以1：1的化学计量比调节F-流出和H+流入。我们中心假设是，多种结构和能量因素，包括离子大小，静电相互作用，氢键，和阴离子-H+耦合，微调，以确定F-选择和在CLCF的交通。我们根据米勒对CLCF的实验研究提出了这一假设，以及我们和其他人以前对同源规范CLC Cl-/H+的计算工作反向转运体我们将进行一系列转向分子动力学和伞式采样模拟来回答（1）H+和阴离子在CLCF-Eca中是如何转运的？已经假设H+门E118经历旋转，将H+从细胞外运送到细胞内溶液和推进F-通过孔隙。我们还假设F-比Cl-更容易通过，因为它半径较小。我们将确定目前在运输周期中缺失的两种蛋白质构象，并且我们将量化两种阴离子的移位自由能势垒。(2)为什么氟渗透减少当E318，阴离子结合位点附近的残基，被中和？目前的假设是， E318Q和E318A突变体中带负电荷的E318降低了取代F-的静电力。从结合位点。我们将比较WT、E318Q和E318A之间阴离子置换的势垒。 (3)是什么导致E118Q和E118A突变体中阴离子选择性从F-转换为Cl-，其中H + 路被废除了吗？我们假设，H+浓度梯度驱动H+进入阴离子孔，质子化E318，F-（而不是Cl-）和E318之间的H+共享，具有相似的pKa值，在孔隙中捕获F-，破坏运输循环。我们将模拟水线的形成和随后的H + 迁移我们将估计E118 Q和E118 Q/E318 A中阴离子渗透的自由能垒;我们预测双突变体降低了两种阴离子的渗透，但保留了F-对Cl-的选择性，这可以实验测试。该研究具有创新性，因为它将（a）改变CLCF目前的研究范式通过包括从计算角度的见解和（B）使用新的自适应分割量子，力学/分子力学算法来模拟明确的H+运输。该研究在以下方面具有重要意义：它将（i）提供对口腔微生物群落中耐氟性的重要见解，（ii）加深我们对了解同源的典型CLC Cl-/H+和其他转运蛋白，和（iii）增强研究环境在CU-Denver Downtown Campus和促进本科生的研究。