Ababou Abdessamad, Koronakis Vassilis
Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, United Kingdom.
PLoS One. 2016 Jul 12;11(7):e0159154. doi: 10.1371/journal.pone.0159154. eCollection 2016.
Gram-negative bacteria such as E. coli use tripartite efflux pumps such as AcrAB-TolC to expel antibiotics and noxious compounds. A key feature of the inner membrane transporter component, AcrB, is a short stretch of residues known as the gate/switch loop that divides the proximal and distal substrate binding pockets. Amino acid substitutions of the gate loop are known to decrease antibiotic resistance conferred by AcrB. Here we present two new AcrB gate loop variants, the first stripped of its bulky side chains, and a second in which the gate loop is removed entirely. By determining the crystal structures of the variant AcrB proteins in the presence and absence of erythromycin and assessing their ability to confer erythromycin tolerance, we demonstrate that the gate loop is important for AcrB export activity but is not required for erythromycin binding.
革兰氏阴性菌，如大肠杆菌，利用三联外排泵，如AcrAB-TolC，来排出抗生素和有害化合物。内膜转运蛋白组分AcrB的一个关键特征是一小段被称为门/开关环的残基，它将近端和远端底物结合口袋分开。已知门环的氨基酸取代会降低AcrB赋予的抗生素抗性。在这里，我们展示了两种新的AcrB门环变体，第一种去除了其庞大的侧链，第二种则完全去除了门环。通过确定变体AcrB蛋白在有和没有红霉素存在时的晶体结构，并评估它们赋予红霉素耐受性的能力，我们证明门环对AcrB的输出活性很重要，但对红霉素结合不是必需的。
