CNR-IOM, Unità SLACS, S.P. Monserrato-Sestu Km 0.700, I-09042 Monserrato (CA), Italy.
J Am Chem Soc. 2011 Jul 20;133(28):10704-7. doi: 10.1021/ja202666x. Epub 2011 Jun 27.
The tripartite efflux pump AcrAB-TolC is responsible for the intrinsic and acquired multidrug resistance in Escherichia coli. Its active part, the homotrimeric transporter AcrB, is in charge of the selective binding of substrates and energy transduction. The mutation F610A has been shown to significantly reduce the minimum inhibitory concentration of doxorubicin and many other substrates, although F610 does not appear to interact strongly with them. Biochemical study of transport kinetics in AcrB is not yet possible, except for some β-lactams, and other techniques should supply this important information. Therefore, in this work, we assess the impact of the F610A mutation on the functionality of AcrB by means of computational techniques, using doxorubicin as substrate. We found that the compound slides deeply inside the binding pocket after mutation, increasing the strength of the interaction. During subsequent conformational alterations of the transporter, doxorubicin was either not extruded from the binding site or displaced along a direction other than the one associated with extrusion. Our study indicates how subtle interactions determine the functionality of multidrug transporters, since decreased transport might not be simplistically correlated to decreased substrate binding affinity.
三组分外排泵 AcrAB-TolC 负责大肠杆菌的固有和获得性多药耐药性。其活性部分，同源三聚体转运蛋白 AcrB，负责底物的选择性结合和能量转导。已经表明 F610A 突变显著降低了多柔比星和许多其他底物的最小抑菌浓度，尽管 F610 似乎与它们没有强烈相互作用。除了一些β-内酰胺类药物外，AcrB 的转运动力学的生化研究尚不可行，其他技术应该提供这些重要信息。因此，在这项工作中，我们使用计算技术评估了 F610A 突变对 AcrB 功能的影响，以多柔比星为底物。我们发现，该化合物在突变后深入结合口袋滑动，增加了相互作用的强度。在转运蛋白随后的构象改变过程中，多柔比星要么没有从结合位点中排出，要么沿着与排出方向不同的方向位移。我们的研究表明，微妙的相互作用如何决定多药转运蛋白的功能，因为转运减少可能与底物结合亲和力降低并不简单相关。
