多重药物外排泵AcrB的底物转运机制的计算模拟研究

Multidrug resistance (MDR) is becoming a serious problem in these years. It can render a bacterial infection untreatable by a wide range of chemically unrelated antibiotics and drugs. Employment of multidrug efflux pumps is among the most common mechanisms when bacteria resist antibiotics. One of the best characterized multidrug efflux pumps is AcrAB-TolC. The pump is utilized by E. coli for clinical levels of resistance against β-lactams, macrolides, oxazolidinones, fluoroquinolones and tetracyclines etc. As a tripartite complex, it is consisted of TolC, a passive outer membrane channel, AcrB, an inner membrane protein, and AcrA, an adaptor linking AcrB and TolC. AcrB is responsible for the uptake and selectivity of the substrates, and also powers the complex by proton-motive force. The crystal structures of AcrB indicate that it is a trimer of 1049-residue protomers. Three domains are defined in each protomer: the transmembrane domain, the pore domain and the TolC docking domain. Inside the pore domain, there are channels though which substrates are pumped to the periplasm. Two multidrug binding sites have been identified inside the pore domain: a distal pocket consisted of a phenylalanine cluster and a proximal pocket separated from the former by the switch loop. The sites may identify the substrates of AcrB and facilitate the translocation process. Though the molecular basis of AcrB has been established, the translocation mechanism of the protein is still obscure. A functionally rotating mechanism has been proposed, suggesting that the conformational changes of the intramolecular channels would facilitate the translocation of substrates, but no further expatiation is available. Computational simulation is a powerful tool in studying the conformational changes of proteins. Molecular dynamics (MD), as an example, can start with a high-resolution structure and give a full-scale description of the movement. Our project aims to provide a better understanding of the translocation mechanism from a thermodynamic view using computational methods. The adaptive biasing force (ABF) method will be employed to calculate the potential of mean force (PMF) of the translocation process. Targeted MD will also be used to unveil the correlation between the substrates during their translocation process. These methods will explain how the substrates enter the binding sites and go through the channels on an atomistic level. We hope that our work will shed light on the researches on multidrug efflux pumps and establish a theoretical foundation to cope with multidrug resistance.

多重药物外排泵是细菌对抗抗生素、产生抗药性的常用手段。这些蛋白能够将多种抗生素泵出细菌，使细菌免受伤害。广布于革兰氏阴性菌中的AcrAB-TolC三联体能够转运多种抗生素，是目前研究最多的多重药物外排泵体系之一。其中的AcrB是整个体系的核心，它能够帮助底物穿越细胞内膜，并为底物转运提供能量。目前，人们已经获得了AcrB的原子结构，但对于底物与结合位点的相互作用方式，底物在孔道中转运的过程依然存在很多争议。本项目中，我们将综合运用自适应偏置力和靶向分子动力学等计算模拟手段，在原子水平上对底物的结合和转运进行详细研究。从获得的底物转运轨迹和平均力势曲线中，我们将讨论底物的结合方式，底物的转运过程以及多底物转运的协同效应，为探索AcrB的底物转运机制、应对多重抗药性提供理论支持和实践依据。

抗生素在人类战胜众多疾病的过程中起着极为重要的作用。然而，具有广谱抗药性的“超级细菌”在近年来逐步出现，使得人类再次面临无药可用的危险。本项目重点针对广泛存在于革兰氏阴性菌中的多重药物外排泵AcrB，通过计算模拟在原子水平上解析AcrB的功能机制。.我们的主要结果之一是通过自适应偏置力方法获得了药物分子阿霉素在AcrB内部通道中转移时的自由能（也称平均力势）。平均力势曲线清晰地显示，阿霉素分子在转运过程中会经历两个能量相近的低谷区，分别对应于它结合在近端结合位点和远端结合位点。当药物分子结合在不同的位置时，两个结合位点自身也会发生构象变化，以便配合药物分子的移动。根据这些结果，我们提出了一个新的底物转运机制。之前的机理认为两个结合位点中只有一个会在转运时发挥功能，而我们的机理则着重强调了近端和远端结合位点都会参与底物转运的过程，并且很可能对AcrB的底物选择性都有重要的贡献。这一结果为抗菌药物的设计和研发提供了重要的理论参考。.我们的另一个主要结果是通过靶向分子动力学模拟的方法首次在原子水平上证实了AcrB内部的多条通道之间存在着正向协同性。当相邻的通道内存在第二个阿霉素分子时，AcrB的构象变化会被加快，推动第一个阿霉素分子加速脱离远端结合位点。这一发现进一步加深了人们对于AcrB的底物转运机制的理解和认识。.除了对底物转运机制的研究之外，我们也对AcrB抑制剂的抑制机理进行了分析。这些抑制剂能够抑制AcrB的外排泵活性，因此有潜力成为应对多重抗药性的药物。我们首次在原子水平上观察了抑制剂D13-9001的排出过程，并与药物阿霉素的情况进行了比较。我们发现AcrB的远端结合位点的疏水凹陷和通道末端的出口区域是阻碍抑制剂外排、使之发挥功效的两个最重要的区域。这一发现让人们更深入地了解了AcrB的外排泵活性的抑制机理，为改进已有的AcrB抑制剂提供了新的思路。.这些主要成果都为破解细菌抗药性难题提供了重要的理论支持和实践依据。

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