Molecular Basis of Substrate Translocation in the Drug/H+ Antiporter 1 Family

药物/H+逆向转运蛋白1家族底物转运的分子基础 总结 被称为多药转运蛋白的整合膜蛋白挤出多种治疗药物， 细胞膜上的化学结构，阻碍人类癌症的治疗，传染性 疾病和神经系统疾病。目前，我们缺乏一个深刻的和机械的理解， 这些蛋白质输出药物或如何阻止它们。我们将研究其结构和机制 来自大肠杆菌的模型多药物转运蛋白MdfA，其将H+的流入与 它是多种抗菌剂的外排，属于普遍存在的药物/H+反向转运蛋白1（DHA1）家族。 MdfA同源基因存在于许多病原微生物中，并且E.杆菌 MdfA可导致临床患者的抗菌药物耐药性。因此，MdfA代表重要的 用于治疗开发以克服多药耐药性靶点。此外，SLC18 反向转运蛋白是DHA1家族中MdfA的人类对应物， 单胺神经递质、多胺神经调质和神经毒素的囊泡转运。 人SLC18反向转运蛋白是脑功能所必需的，并且是有希望的治疗靶点。 与酗酒、自闭症谱系障碍、躁郁症、亨廷顿病、重度抑郁症作斗争 帕金森氏症、精神分裂症和抽动秽语综合征。我们长远的目标是 了解DHA1多药转运蛋白和人SLC18反向转运蛋白如何转运其 底物以及如何调节它们的功能以获得潜在的治疗益处。值得注意的是， 生物化学研究表明，MdfA通过非典型的 机制利用这些数据和我们在膜蛋白结构生物学方面的经验，我们 将实现两个目标：（1）阐明两个同时易位的分子基础 单阳离子底物的DHA1;（2）揭示非典型的结构机制，DHA1- 介导的双阳离子治疗剂的挤出。通过结合晶体学和生物化学研究， 我们将获得有关DHA1如何同时转移两种底物、DHA1如何同时转移两种底物的新见解 抑制剂与底物不同，以及DHA1如何在两个连续的 还有不同的去质子化/质子化循环。新的概念框架和DHA1结构 从这项研究中获得的信息将作为设计新策略的垫脚石， 抑制临床相关的多药转运蛋白，这可能会挽救对 多药耐药细胞，并阻止无法治愈的感染的传播。此外，我们的工作将提供 人类SLC18反向转运蛋白的机制研究的跳板，这将揭示新的光 它们如何利用电化学H+梯度来转运单胺神经递质， 神经毒素或多胺神经调节剂，穿过囊泡膜。

Molecular Basis of Substrate Translocation in the Drug/H+ Antiporter 1 Family Summary Integral membrane proteins known as multidrug transporters extrude therapeutic drugs of diverse chemical structures across cell membranes, impeding the treatment of human cancers, infectious diseases, and neurological disorders. Currently we lack a deep and mechanistic understanding of how these proteins export drugs or how they can be thwarted. We will study the structure and mechanism of a model multidrug transporter, MdfA from Escherichia coli, which couples the influx of H+ to the efflux of various antimicrobials and belongs to the ubiquitous Drug/H+ Antiporter 1 (DHA1) family. MdfA orthologues are present in many pathogenic microorganisms, and the overexpression of E. coli MdfA can lead to antimicrobial resistance in clinical patients. Thus, MdfA represents an important target for therapeutic exploitation to overcome multidrug resistance. Furthermore, the SLC18 antiporters, which are the human counterparts of MdfA in the DHA1 family, conduct the H+-dependent vesicular transport of monoamine neurotransmitters, polyamine neuromodulators, and neurotoxins. The human SLC18 antiporters are essential for brain function and promising therapeutic targets for battling alcoholism, autism spectrum disorders, bipolar disorder, Huntington disease, major depressive disorder, Parkinson’s disease, schizophrenia, and Tourette syndrome. Our long-term objective is to understand how the DHA1 multidrug transporters and human SLC18 antiporters translocate their substrates and how their function can be modulated for potential therapeutic benefit. Notably, prior biochemical studies have suggested that MdfA translocates certain substrates via a non-canonical mechanism. Drawing upon these data and our experience in membrane protein structural biology, we will accomplish two aims: (1) to elucidate the molecular basis for simultaneous translocation of two mono-cationic substrates by a DHA1; (2) to reveal the structural mechanism for non-canonical, DHA1- mediated extrusion of di-cationic therapeutics. By combining crystallographic and biochemical studies, we will acquire new insights into how a DHA1 translocates two substrates concurrently, how a DHA1 inhibitor differs from the substrate, and how a DHA1 exports a therapeutic drug in two consecutive and yet different deprotonation/protonation cycles. The new conceptual framework and DHA1 structures obtained from this study will serve as a stepping-stone toward devising novel strategies to evade or inhibit the clinically relevant multidrug transporters, which may rescue therapeutic efficacy against multidrug-resistant cells and halt the spread of untreatable infections. Furthermore, our work will offer a springboard for the mechanistic studies of human SLC18 antiporters, which will shed new light on how they utilize the electrochemical H+ gradient to translocate monoamine neurotransmitters, neurotoxins, or polyamine neuromodulators, across vesicular membranes.