核酸碱基/抗坏血酸盐转运蛋白（nucleobase/ascorbate transporter proteins， NAT）家族，亦称核酸碱基/阳离子同向转运蛋白-2（nucleobase/cation symporter-2 proteins， NCS2）家族，在不同种类的生物中都负责摄取外源性核酸碱基。同时，在哺乳动物体内，这一类家族蛋白还负责维生素C的跨膜转运。除此之外，该家族蛋白还在某些生物体内履行着转运代谢废物排出细胞的职责。尽管这类家族蛋白的生物学意义如此重要，但在论文工作之前，尚无任何一个细菌、真菌或者哺乳动物的NAT/NCS2家族蛋白的三维晶体结构被解析出来，对其功能特征的描述更是无从谈起。在本论文的工作中，我们报道了NAT/NCS2家族的典型代表，大肠杆菌尿嘧啶/H+同向转运蛋白UraA与底物复合体的分辨率为2.8Å的三维晶体结构。UraA蛋白的结构与以往的预测结果大相径庭：UraA蛋白一共拥有14个跨膜螺旋，在与底物尿嘧啶结合以后呈现出向胞内开口的构象。特别的，在UraA的跨膜区域内，我们第一次发现了反向平行β-折叠片的存在，打破了长久以来人们对膜蛋白跨膜区域二级结构的普遍认识。蛋白结构比对的结果显示，尚无与UraA蛋白结构相似的已知结构。所以，UraA蛋白呈现出一个全新的蛋白质折叠形式。在稍后的生物化学和生物物理的分析实验中，论文详细地讨论了UraA蛋白履行其生物学功能的分子机制。在结构分析的基础上，实验通过对蛋白识别、结合和转运底物的重要氨基酸采用定点突变的研究方法来研究在这一生物学过程中这些氨基酸所起到的作用。在分析了UraA分子内部相互作用关系的情况下，结合得到的生化数据，我们将UraA蛋白划分为核心与门控两个结构域。其中，核心结构域包括TM1-4和TM8-11，主要负责底物的识别和结合，特别是反向平行β-折叠片在这个过程中起了重要作用；门控结构域包括TM5-7和TM12-14，通过自身的构象的改变来完成对底物尿嘧啶的转运功能。在现有的实验数据的支持下，论文提出了UraA蛋白转运底物鸟嘧啶的分子模型。模型中，UraA蛋白通过结合、变构、释放这三个过程完成了对底物的跨膜运输。论文工作为研究NAT/NCS2家族其他蛋白成员的生物学意义提供了有益的借鉴，为利用该家族蛋白解决相关的生理问题奠定了理论的基础。
The nucleobase/ascorbate transporter (NAT) proteins, also known as nucleobase/cation symporter 2 (NCS2) proteins, are responsible for the uptake of nucleobases in all kingdoms of life. In mammals, they are required for the transport of vitamin C inmammals.In some other species, NTA proteins also play key roles in transporting metabolic wastes out of cells. Despite functional characterization of the NAT family members in bacteria, fungi and mammals, detailed structural and molecular information remain unavailable.In the current thesis work, we report the crystal structure of a representative NAT/NCS2 protein, the Escherichia coli uracil/H1 symporter UraA, in complex with uracil at a resolution of 2.8Å . UraA has a novel structural fold, with 14 transmembrane segments (TMs) divided into two inverted repeats, representing an inward open conformation in the presence of uracil binding. Specifically, a pair of antiparallel β-strands, which is located between TM3 and TM10 is first observed within the transmembrane region of a protein. Searching of the Protein Data Bank with Dali indicated that UraA has a previously uncharacterized fold.Further biochemical and biophysical studies reveal the molecular mechanism and biological functions of UraA. Based on structure analysis, key amino acids essential for structural organization and substrate recognition are identified through mutagenesis studies. Combining the biochemical data and spatial arrangement of the structure, UraA is divided into a core domain and a gate domain. The core domain, containing TM1-4 and TM1-8, in which a pair of antiparallel b-strands are unexpectedly crucial,is responsible for recognition and binding of substrate. The conformational changes of gate domain, which consists of TM5-7 and TM12-14, drive the transport of substrate. On the basis of structural and functional analysis, we propose a working model to explain the transport mechanism of UraA. In this model,uracil is transported through steps of uracil binding to UraA, conformational changes of the gate domain and finally releasing uracil to the cytosol. In this work, the structural and biochemical characterizationsof UraA reported here provide an important framework for mechanistic understanding of the NAT family transporters.
