Molecular Basis of Multidrug Binding and Transport by the MATE Transporters

描述（由申请人提供）：多药和毒素挤出（MATE）转运蛋白是一种完整的膜蛋白，利用预先存在的钠或质子梯度将结构无关的亲脂性阳离子穿过细胞膜。细菌的MATE转运体作为多药物外排泵，将一系列抗菌药物从细胞质中排出，而人类的MATE转运体则介导各种细胞毒性代谢物和治疗药物的排泄。考虑到它们与抗微生物药物和化疗药物不必要的耐药性的功能相关性，MATE转运体的分子结构不仅将揭示它们如何在细胞膜上运输底物，而且还将揭示它们的运输活性如何被调节以克服耐药性。我们的目的是利用x射线晶体学阐明完整的MATE转运体的分子结构。到目前为止，我们已经获得了衍射分辨率高于3.8 e的晶体。基于晶体结构，我们将构建各种MATE突变体，将其重组为脂质体，并利用底物摄取试验表征其运输特性。我们的长期目标是破译多药结合和转运的分子基础。具体来说，我们寻求(1)建立有和没有药物底物的MATE转运体的结构；(2)通过脂质体中纯化的MATE转运体的功能重构探索转运机制；(3)确定各种MATE突变体的结构和药物结合特异性。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；它还将为基于结构设计针对耐药人类病原体和癌细胞的药物奠定基础。

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.