Department of Physics, University of Cagliari, s.p. 8, Cittadella Universitaria, 09042 Monserrato (CA), Italy.
Department of Physics, University of Cagliari, s.p. 8, Cittadella Universitaria, 09042 Monserrato (CA), Italy.
Biochim Biophys Acta Gen Subj. 2018 Apr;1862(4):836-845. doi: 10.1016/j.bbagen.2018.01.010. Epub 2018 Jan 13.
Efflux pumps of the Resistance-Nodulation-cell Division superfamily confer multi-drug resistance to Gram-negative bacteria. The most-studied polyspecific transporter belonging to this class is the inner-membrane trimeric antiporter AcrB of Escherichia coli. In previous studies, a functional rotation mechanism was proposed for its functioning, according to which the three monomers undergo concerted conformational changes facilitating the extrusion of substrates. However, the molecular determinants and the energetics of this mechanism still remain unknown, so its feasibility must be proven mechanistically.
A computational protocol able to mimic the functional rotation mechanism in AcrB was developed. By using multi-bias molecular dynamics simulations we characterized the translocation of the substrate doxorubicin driven by conformational changes of the protein. In addition, we estimated for the first time the free energy profile associated to this process.
We provided a molecular view of the process in agreement with experimental data. Moreover, we showed that the conformational changes occurring in AcrB enable the formation of a layer of structured waters on the internal surface of the transport channel. This water layer, in turn, allows for a fairly constant hydration of the substrate, facilitating its diffusion over a smooth free energy profile.
Our findings reveal a new molecular mechanism of polyspecific transport whereby water contributes by screening potentially strong substrate-protein interactions.
We provided a mechanistic understanding of a fundamental process related to multi-drug transport. Our results can help rationalizing the behavior of other polyspecific transporters and designing compounds avoiding extrusion or inhibitors of efflux pumps.
抗性-调节-细胞分裂超家族的外排泵赋予革兰氏阴性细菌多重耐药性。 属于该类的研究最多的多特异性转运蛋白是大肠杆菌的内膜三聚体反向转运蛋白 AcrB。 在以前的研究中，提出了一种针对其功能的功能旋转机制，根据该机制，三个单体协同发生构象变化，有利于底物的外排。 然而，该机制的分子决定因素和能量仍然未知，因此必须从机械上证明其可行性。
开发了一种能够模拟 AcrB 中功能旋转机制的计算方案。 通过使用多偏压分子动力学模拟，我们描述了由蛋白质构象变化驱动的阿霉素底物的转运。 此外，我们首次估计了与该过程相关的自由能曲线。
我们提供了与实验数据一致的过程的分子视图。 此外，我们表明，AcrB 中发生的构象变化使转运通道内部表面形成一层结构化的水层。 反过来，这个水层允许底物相当恒定的水合作用，从而促进其在平滑的自由能曲线上扩散。
我们的发现揭示了一种多特异性转运的新分子机制，其中水通过屏蔽潜在的强底物-蛋白相互作用来贡献。
我们提供了与多药转运相关的基本过程的机械理解。 我们的结果可以帮助合理化其他多特异性转运蛋白的行为，并设计避免外排或外排泵抑制剂的化合物。
