铜绿假单胞菌,一种以抗菌素耐药性闻名的强大病原体,对免疫受损的个体构成重大威胁。在这方面,MexAB-OprM外排泵通过从细菌细胞中挤出抗菌剂而充当关键的防线。内膜同三聚体蛋白MexB捕获抗生素并将它们转运到通过MexA衔接蛋白连接的外膜OprM通道蛋白中。尽管付出了广泛的努力,针对MexB蛋白的紧密(T)前体的竞争性抑制剂尚未获得FDA批准用于医疗用途。在过去的几年里,变构抑制剂已成为流行的替代经典的竞争性抑制剂为基础的方法,因为它们的特异性较高,较低的剂量,减少毒理学影响。因此,在这项研究中,我们揭示了MexB跨膜变构结合袋的存在,其灵感来自于最近发现的一种重要的变构抑制剂,BDM88855,为同源AcrB卵白。虽然重新利用BDM88855被证明在控制MexB松散(L)质子方面无效,我们的调查确定了一个有希望的替代方案:DB08385的含氯变体(2-ClDB08385或变体1).分子动力学模拟,包括结合非均相介电隐式膜模型(隐式膜MM/PBSA)的结合自由能估计,相互作用熵(IE)分析和平均力势能(PMF)计算,证明了变体1对跨膜袋的优异结合亲和力,在配体解结合过程中显示出最高的能障。为了阐明MexB的跨膜和转运结构域之间的变构串扰,我们在从蛋白质相关网络获得的线性互信息中采用了“特征向量中心性”度量。值得注意的是,这项研究证实了MexBL启动子中变构跨膜位点的存在。除此之外,变体1是变构串扰的有效调节剂,在MexBL原型器中诱导\'O-L中间状态\'。这种诱发状态可能具有减少进入进入通道袋中的底物摄入的潜力,导致抗生素的无效外排。
Pseudomonas aeruginosa, a formidable pathogen renowned for its antimicrobial resistance, poses a significant threat to immunocompromised individuals. In this regard, the MexAB-OprM efflux pump acts as a pivotal line of defense by extruding antimicrobials from bacterial cells. The inner membrane homotrimeric protein MexB captures antibiotics and translocates them into the outer membrane OprM channel protein connected through the MexA adaptor protein. Despite extensive efforts, competitive inhibitors targeting the tight (T) protomer of the MexB protein have not received FDA approval for medical use. Over the past few years, allosteric inhibitors have become popular as alternatives to the classical competitive inhibitor-based approach because of their higher specificity, lower dosage, and reduced toxicological effects. Hence, in this study, we unveiled the existence of a transmembrane allosteric binding pocket of MexB inspired by the recent discovery of an important allosteric inhibitor, BDM88855, for the homolog AcrB protein. While repurposing BDM88855 proved ineffective in controlling the MexB loose (L) protomer, our investigation identified a promising alternative: a chlorine-containing variant of DB08385 (2-Cl DB08385 or Variant 1). Molecular dynamics simulations, including binding free energy estimation coupled with heterogeneous dielectric implicit membrane model (implicit-membrane MM/PBSA), interaction entropy (IE) analysis and potential of mean force (PMF) calculation, demonstrated Variant 1\'s superior binding affinity to the transmembrane pocket, displaying the highest energy barrier in the ligand unbinding process. To elucidate the allosteric crosstalk between the transmembrane and porter domain of MexB, we employed the \'eigenvector centrality\' measure in the linear mutual information obtained from the protein correlation network. Notably, this study confirmed the presence of an allosteric transmembrane site in the MexB L protomer. In addition to this, Variant 1 emerged as a potent regulator of allosteric crosstalk, inducing an \'O-L intermediate state\' in the MexB L protomer. This induced state might hold the potential to diminish substrate intake into the access pocket, leading to the ineffective efflux of antibiotics.