Molecular Basis of Multidrug Binding and Transport by the MATE Transporters

描述(由申请人提供)：多药物和毒素挤出(Mate)转运蛋白是一种完整的膜蛋白，它利用预先存在的钠或质子梯度，将结构上无关的亲脂性阳离子转移到细胞膜上。细菌配对转运体通过从细胞质中排出一群抗菌剂而起到多药外排泵的作用，而它们的人类转运体则调节各种细胞毒性代谢物和治疗药物的排泄。鉴于Mate转运蛋白的功能与抗菌药和化疗药物的耐药性有关，Mate转运蛋白的分子结构不仅将揭示它们如何将底物转运到细胞膜上，而且还将揭示它们的转运蛋白活性如何被调节以克服耐药性。我们的目标是利用X射线结晶学来阐明完整的Mate转运蛋白的分子结构。到目前为止，我们已经获得了衍射率高于3.8E分辨率的晶体。基于晶体结构，我们将构建各种配对突变体，将它们重组为脂质体，并利用底物摄取分析来表征它们的转运特性。我们的长期目标是破译多药结合和转运的分子基础。具体地说，我们试图(1)建立有和没有药物底物的Mate转运蛋白的结构；(2)通过脂质体中纯化的Mate转运蛋白的功能重组来探索Mate转运蛋白的转运机制；(3)确定各种Mate突变体的结构和药物结合的特异性。我们的工作将为管理多药结合和运输的一般原则提供新的见解；它还将为针对耐药人类病原体和癌细胞的基于结构的药物设计奠定基础。 公共卫生相关性：多药耐药性是一个广泛而严重的健康问题。拟议的研究试图深入了解多药转运，这是一种关键的、进化上保守的机制，是多药耐药的基础。这项拟议的研究与公共健康相关，因为这些发现通常适用于对抗抗药性病原微生物以及人类癌细胞。

DESCRIPTION (provided by applicant): Multidrug and Toxin Extrusion (MATE) transporters are integral membrane proteins that move structurally unrelated lipophilic cations across the cell membrane by utilizing a preexisting sodium or proton gradient. Bacterial MATE transporters function as multidrug efflux pumps by expelling a cohort of antimicrobial agents from the cytoplasm, whereas their human counterparts mediate the excretion of various cytotoxic metabolites as well as therapeutic drugs. Given their functional relevance to the unwanted resistance to antimicrobials and chemotherapy, molecular structures of the MATE transporters will reveal not only how they transport their substrates across the cell membrane but also how their transport activity can be modulated in order to overcome drug resistance. We aim to elucidate the molecular structure of an intact MATE transporter using X-ray crystallography. To date we have obtained crystals that diffract better than 3.8 E-resolution. Based on the crystal structure, we will construct various MATE mutants, reconstitute them into liposomes and characterize their transport properties utilizing substrate uptake assays. Our long-term objective is to decipher the molecular basis for multidrug binding and transport. Specifically, we seek to (1) establish the structures of a MATE transporter with and without drug substrates; (2) probe the transport mechanism via functional reconstitution of purified MATE transporters in liposomes; (3) determine the structures and drug-binding specificities of various MATE mutants. Our work will provide new insights into the general principles that govern multidrug binding and transport; it will also set the stage for structure-based design of pharmaceuticals targeting drug-resistant human pathogens and cancer cells. PUBLIC HEALTH RELEVANCE: Multidrug resistance is a widespread and serious health issue. The proposed studies seek a deep understanding of multidrug transport, a key and evolutionarily conserved mechanism that underlies multidrug resistance. The proposed research has relevance to public health, because the findings are generally applicable to combating both drug- resistant pathogenic microorganisms as well as human cancer cells.