ECF转运蛋白的结构与转运机制

Membrane transporters are responsible for the uptake of nutrients and elimination of metabolic wastes,and therefore are essential for the cell survival.ATP-binding cassette (ABC) transporters form one of the biggest protein superfamilies that transport a variety of substrates across the membrane by using the energy of ATP hydrolysis.Recently, a new class of ABC transporters-ECF(energy-coupling factor) transporters have been discovered widely distributed in bacteria. They are characterized to be responsible for vitamins and micronutrients uptake. Each ECF transporter is composed of four different components, namely the transmembrane T protein which together with the cytoplasmic nucleotide-binding proteins A and A' constitute the energy coupling module, and another transmembrane S protein which is responsible for substrate-specific binding. The ECF transporters are classified into two groups: those encoded by linked genes for ecfS and an ecfS-specific ecfA-ecfA'-ecfT module (group I), and those with a common ecfA-ecfA'-ecfT module shared by up to 12 different S components in the same bacterial species (group II).Therefore, ECF transporters exhibit unique structural and functional properties in contrast to canonical ABC transporters.To elucidate the underlying substrate recoganizing and transport mechanisms of ECF transporters, we plan to investigate and characterize the structures of different S proteins and ECF transporter complexes at different conformations. Moreover, ECF transporters are mainly existed in prokaryotes which include numerous human pathogens that are dependent of the uptake of specific substrates for survival, structural and mechanistic understanding of ECF transporters may open a new road for new antibiotics development.

细胞对营养物质的摄取和对代谢废物的排出依赖于细胞膜上的转运蛋白，因此，转运蛋白对细胞乃至生物体的生存和生长至关重要。ABC转运蛋白是一大类重要的跨膜转运蛋白复合体，它依靠分解ATP产生的能量驱动物质的跨膜转运。近年来，人们成功鉴定了一类新型ABC转运蛋白-ECF转运蛋白，它存在于多种生物体中负责维生素和微量营养元素的摄入。ECF 转运蛋白在结构上包括细胞膜上底物特异性结合蛋白S、以及由膜结合蛋白T、胞内ATP 结合蛋白A/A'形成的能量耦合模块组成。根据能量耦合模块的使用情况，将ECF转运蛋白分成耦合模块专有型和共享型两类。我们成功解析了能量耦合模块共享型S蛋白与底物核黄素以及叶酸ECF转运复合体（inward-facing 状态）的结构，在此基础上，本研究拟通过解析不同S 蛋白和不同状态转运复合体的晶体结构以及相关的功能分析，阐释ECF转运蛋白对底物特异性识别以及跨膜转运的分子机制。

本项目“ECF转运蛋白的结构与转运机制”研究目标为通过对 ECF 转运蛋白及其突变体的结构与功能分析，揭示底物特异性识别的分子机制、负责底物转运的通道，阐释组成底物转运通道的S、T、A、A’蛋白在转运过程中的功能，理解底物通过不同ECF 转运蛋白跨膜转运的分子机制。项目执行四年，研究团队紧紧围绕原定研究内容和计划，开展了ECF转运蛋白的结构与机理的研究。取得的研究成果包括：1) 解析了泛酸（维生素B5）ECF转运蛋白的晶体结构，揭示了ECF转运蛋白模块共享的结构基础，论文发表于PNAS（2014）。2) 解析了叶酸ECF转运蛋白复合体中底物结合蛋白FolT叶酸结合状态的晶体结构，揭示了叶酸特异性结合的分子基础和底物诱导的构象变化，论文发表于Nature Commun（2015）。3）受Trends Microbiol（2013）邀请撰写了ECF转运复合体的进展综述，提出了ECF型ABC转运蛋白的工作模型。项目在研期间发表研究论文3篇，3名参与研究的研究生毕业并获得博士学位，项目负责人张鹏入选国家“万人计划”—领军人才。圆满完成了研究项目的各项任务和目标。