Operation Mechanism of CLCF Fluoride/Proton Antiporter

项目摘要/摘要 F-−离子广泛应用于预防龋齿，能抑制细菌生长。然而，许多细菌菌株 已经进化到对F-产生抗药性，利用位于细菌细胞膜上的输出体 降低细胞内F-浓度。我们的长期目标是解开这些F-的分子细节 出口商。我们在这个方案中的目标是阐明一个原型CLCFF-/H+的运行机制 它以1：1的化学计量比调节F-外流和H+内流。我们的 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+偶联被微调以确定F-选择和 CLCF中的运输。我们根据Miller和他的CLCF实验研究提出了假设 同事和我们等人以前对同系正则ClC-/H+的计算工作 反搬运者。我们将进行一系列分子动力学和伞形抽样模拟来回答 三个具体目的的问题：(1)H+和阴离子在CLCF-ECA中是如何运输的？一直以来 假设H+门E118经历旋转，将H+从细胞外运送到细胞内 溶液和推动F-穿过毛孔。我们还假设F-比Cl-更容易通过，因为它的 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， 我们将量化这两种阴离子的移位自由能垒。(2)为什么氟渗透率下降？ 当阴离子结合部位附近的残基E318被中和时？工作假设是，中和 E318Q和E318A突变体中带负电荷的E318减少了取代F-的静电力。 从结合部位。我们将比较WT、E318Q和E318A之间的阴离子位移势垒。 (3)在E118Q和E118A突变体中，是什么原因导致阴离子选择性从F-切换到Cl-，其中H+ 途径被废除了吗？我们假设H+浓度梯度驱使H+进入阴离子孔道 质子化E318以及具有相似pKa值的F-(但不是Cl-)和E318之间的H+共享， 把F-困在毛孔里，扰乱了运输周期。我们将模拟水线的形成和随后的H+ 迁移。我们将估计阴离子在E118Q和E118Q/E318A中渗透的自由能势垒；我们预测 双突变体减少了对两个阴离子的渗透，但保留了F-对-Cl-的选择性，这可以是 经过实验测试。这项研究具有创新性，因为它将(A)改变目前CLCF的研究范式 通过包括从计算角度的见解和(B)使用新的自适应划分量子- 用于模拟显式H+传输的力学/分子力学算法。这项研究具有重要的意义 它将(I)为口腔微生物群落中的氟抗性提供关键的见解，(Ii)加深我们的 了解同源的典范ClC、Cl-/H+和其他转运蛋白，以及(Iii)增强 科罗拉多大学丹佛市中心校区的研究环境，并促进本科生的研究。

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.