Molecular Basis of Multidrug Binding and Transport by the MATE Transporters

描述（由申请人提供）：多药和毒素外排（MATE）转运蛋白是一种整合的膜蛋白，通过利用预先存在的钠或质子梯度，使结构上不相关的亲脂性阳离子穿过细胞膜。细菌MATE转运蛋白通过从细胞质中排出一组抗微生物剂而起多药外排泵的作用，而它们的人类对应物介导各种细胞毒性代谢物以及治疗药物的排泄。考虑到它们与对抗菌剂和化疗的不必要的耐药性的功能相关性，MATE转运蛋白的分子结构不仅将揭示它们如何将其底物转运穿过细胞膜，而且还将揭示它们的转运活性如何被调节以克服耐药性。我们的目的是阐明一个完整的MATE转运蛋白的分子结构，使用X射线晶体学。到目前为止，我们已经获得了晶体，其分辨率优于3.8 E。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，并利用底物摄取测定表征其转运特性。我们的长期目标是破译多药结合和转运的分子基础。具体而言，我们寻求（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转运蛋白的功能重建来探测转运机制;（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转运的一般原则提供新的见解;它还将为靶向耐药人类病原体和癌细胞的药物的基于结构的设计奠定基础。 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。拟议的研究寻求对多药转运的深入理解，这是多药耐药的关键和进化保守机制。拟议的研究与公共卫生有关，因为这些发现通常适用于对抗耐药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.