Mechanisms of substrate transport by the major facilitator superfamily multidrug transporter LmrP

耐药性已成为全球卫生保健的威胁。对缺乏有效的抗微生物药物的关切越来越频繁地在媒体上传播。病原微生物已经进化出几种方式，通过它们可以抵抗抗生素和消毒剂的毒性作用。一种非常强大的机制是基于抗微生物剂通过膜转运蛋白（也称为药物外排泵）从细胞内部转运（外排）。由于这些外排泵的活性，药物只能在细胞内达到亚毒性浓度，使抗微生物剂在治疗感染性疾病中无效。关于药物外排泵，有许多医学相关和学术上有趣的问题需要回答。一个问题涉及多药外排泵识别非常广泛的抗菌药物的能力。由于外排泵需要在药物外排过程中进行渗透功，另一个问题涉及细胞可以为这种外排活动提供代谢能量的方式。代谢能的一种形式是基于跨转运蛋白所在的质膜的离子梯度的存在。药物外排泵含有一条通道，这些离子可以沿着该通道沿着沿其梯度向下移动进入细胞，但这种移动只有在与药物沿相反方向的移动（=外排）相耦合时才能发生。因此，这些药物外排泵可以通过介导药物-质子反向转运反应将代谢能偶联到药物外排。该项目将集中在一个充分研究的药物质子反向转运蛋白称为LmrP从革兰氏阳性菌乳酸乳球菌。已知LmrP含有底物和质子可以结合的口袋。在本项目中，我们将测试这样的假设：在运输反应的不同阶段，这些口袋的表面性质从吸引水到排斥水发生变化，以便基质可以结合，而质子被释放，反之亦然。几种生物化学和生物物理方法将用于测量口袋的表面性质，同时LmrP保持在结合口袋暴露于细胞内部（面向内的状态）或细胞外部（面向外的状态）的状态。这项研究将增加我们对LmrP和其他药物外排泵工作机制的理解。这些结果将使我们在未来产生新的药物，阻止或绕过膜转运蛋白的外排活动

Drug resistance has become a global threat to health care. Concerns about the lack of effective antimicrobial drugs are communicated in the media with increased frequency. Pathogenic microorganisms have evolved several ways by which they can resist the toxic effects of antibiotics and disinfectants. One very powerful mechanism is based on the transport (efflux) of antimicrobial agents from the cell's interior by membrane transport proteins, also referred to as drug efflux pumps. Due to the activity of these efflux pumps, the drugs only reach subtoxic concentrations inside the cell, making the antimicrobial agents ineffective in the treatment of infectious diseases. There are many medically relevant and academically interesting questions that need to be answered about drug efflux pumps. One question concerns the ability of multidrug efflux pumps to recognize a very wide range of antimicrobial agents. As efflux pumps need to perform osmotic work during drug efflux,another question concerns the ways by which the cell can provide metabolic energy for this efflux activity. One form of metabolic energy is based on the existence of ion gradients across the plasma membrane where transport proteins reside. The drug efflux pumps contain a pathway along which these ions can move down their gradients into the cell, but this movement can only occur if it is coupled to the movement of drugs in the opposite direction (= efflux). Thus, these drug efflux pumps can couple to metabolic energy to drug efflux by mediating a drug-proton antiport reaction. This project will focus on a well-studied drug-proton antiporter termed LmrP from the Gram-positive bacterium Lactococcus lactis. LmrP is known to contain pockets in which substrates and protons can bind. In this project, we will test the hypothesis that the surface properties in these pockets change from attracting water to repelling water at different stages of the transport reaction so thatsubstrates can bind while protons are released, and vice versa. Several biochemical and biophysical approaches will be used to measure surface properties of the pockets while LmrP is kept in a state where the binding pockets are exposed to the interior of the cell (inward-facing state) or exterior of the cell (outward-facing state). This research will increase our understanding of the mechanisms by which LmrP and other drug efflux pumps work. The results will allow us in the future to generate new drugs that block or bypass the efflux activity of membrane transporters