Division of Molecular Medicine, Research Institute, Hospital for Sick Children, Toronto M5G 0A4, Ontario, Canada.
Department of Biochemistry, University of Toronto, Toronto M5S 1A8, Ontario, Canada.
Biochemistry. 2020 Oct 20;59(41):3973-3981. doi: 10.1021/acs.biochem.0c00417. Epub 2020 Oct 7.
Clinically relevant multidrug-resistant bacteria often arise due to overproduction of membrane-embedded efflux proteins that are capable of pumping antibiotics out of the bacterial cell before the drugs can exert their intended toxic effect. The membrane protein AcrB is the archetypal protein utilized for bacterial efflux study because it can extrude a diverse range of antibiotic substrates and has close homologues in many Gram-negative pathogens. Three AcrB subunits, each of which contains 12 transmembrane (TM) helices, are known to trimerize to form the minimal functional unit, stabilized noncovalently by helix-helix interactions between TM1 and TM8. To inhibit the efflux activity of AcrB, we have rationally designed synthetic peptides aimed at destabilizing the AcrB trimerization interface by outcompeting the subunit interaction sites within the membrane. Here we report that peptides mimicking TM1 or TM8, with flanking N-terminal peptoid tags, and C-terminal lysine tags that aid in directing the peptides to their membrane-embedded target, decrease the AcrB-mediated efflux of the fluorescent substrate Nile red and potentiate the effect of the antimicrobials chloramphenicol and ethidium bromide. To further characterize the motif encompassing the interaction between TM1 and TM8, we used Förster resonance energy transfer to demonstrate dimerization. Using the TM1 and TM8 peptides, in conjunction with several selected mutant peptides, we highlight residues that may increase the potency and specificity of the peptide drug candidates. In targeting membrane-embedded protein-protein interactions, this work represents a novel approach to AcrB inhibition and, more broadly, a potential route to a new category of efflux pump inhibitors.
临床上相关的多药耐药菌通常是由于膜嵌入的外排蛋白过度产生而引起的，这些蛋白能够在抗生素发挥其预期的毒性作用之前将抗生素泵出细菌细胞。膜蛋白 AcrB 是用于细菌外排研究的典型蛋白，因为它可以排出多种抗生素底物，并且在许多革兰氏阴性病原体中有密切的同源物。已知三个 AcrB 亚基，每个亚基包含 12 个跨膜（TM）螺旋，三聚体化形成最小功能单位，通过 TM1 和 TM8 之间的螺旋-螺旋相互作用非共价稳定。为了抑制 AcrB 的外排活性，我们已经合理设计了合成肽，旨在通过竞争膜内亚基相互作用位点来破坏 AcrB 的三聚体化界面。在这里，我们报告说，模拟 TM1 或 TM8 的肽，带有侧翼的 N 端肽聚糖标签和 C 端赖氨酸标签，有助于将肽引导到其嵌入膜的靶标，减少 AcrB 介导的荧光底物尼罗红的外排，并增强抗菌药物氯霉素和溴化乙锭的作用。为了进一步表征 TM1 和 TM8 之间相互作用的模体，我们使用Förster 共振能量转移来证明二聚化。使用 TM1 和 TM8 肽，以及几个选定的突变肽，我们突出了可能增加肽药物候选物的效力和特异性的残基。在针对膜嵌入的蛋白-蛋白相互作用时，这项工作代表了一种抑制 AcrB 的新方法，更广泛地说，代表了一类新的外排泵抑制剂的潜在途径。
