协同运输转运蛋白利用离子浓度梯度提供的势能将底物转运到细胞内部。它们在细胞的一侧结合底物，经过构象变化将底物转运进入细胞。协同运输转运蛋白包括同向转运蛋白和反向转运蛋白。大肠杆菌的UraA蛋白是质子和尿嘧啶的同向转运蛋白，AdiC蛋白是精氨酸和胍基丁胺的反向转运蛋白。UraA蛋白属于NAT/NCS2家族，负责同向转运质子和尿嘧啶进入大肠杆菌。人的维生素C转运蛋白SVCT1和SVCT2，以及哺乳动物的尿嘧啶转运蛋白SNBT1也属于这一家族。为了进一步了解UraA和NAT/NCS2家族的工作机制，我们对UraA的三维晶体结构进行了解析。我们得到的分辨率为2.8Å 的UraA结构一共有14次穿膜区域。与其他已知结构的转运蛋白比对和对Dali数据库检索结果显示，Ura的三维结构是一种全新的折叠方式。UraA结构呈现底物尿嘧啶结合的向内开口的构象。UraA结构中的TM3和TM10的穿膜区域上有一段反向平行的β-折叠片结构，这段结构对于底物尿嘧啶的结合和转运有着重要的作用。这也是在转运蛋白中首次报道了β-折叠的存在。接下来，我们对UraA进行了一系列的生化实验研究，验证了转运过程中的重要氨基酸的作用。此外，通过分析分子内部的相互作用，我们将UraA的分为核心结构域和门控结构域，在转运的过程中核心结构域主要负责结合底物，而门控结构域负责整个蛋白构象的变化。这些结构和生化的实验使我们对UraA转运机制的了解有了很大的提高，为整个NAT/NCS2家族的结构以及转运机制的阐明奠定了基础。本文中另外一个反向转运蛋白AdiC属于APC超家族，负责将大肠杆菌胞外的精氨酸运输进入胞内，同时将胍基丁胺运至胞外。AdiC是毒性菌株大肠杆菌的抗酸系统的重要组成成员。我们解析了3.6Å的AdiC的向外开口构象的无底物结合的三维晶体结构，属于LeuT折叠方式。接下来，我们对AdiC蛋白进行了生化研究，从而找到了底物转运过程中起重要作用的氨基酸。经过同组其他成员的努力，我们又解析了N22A突变体AdiC结构，这个结构为精氨酸结合的闭合中间态的构象。综合这两个构象的结构和一系列生化试验，我们确定了AdiC底物精氨酸的结合位点，以及两种底物的转运途径，为整个转运循环通路提供了重要的结构和生化基础。
Secondary active transporters, which can be roughly divided into symporters and antiporters, are responsible for the concentrative transport of substrate across the membrane. UraA is in charge of the symport of proton and uracil into E. coli, while AdiC is an arginine/agmatine antiporter.NAT/NCS2 family are responsible for the permeation of nucleobases in all kingdoms of life and the transport of vitamin C in mammals. In this thesis we determined the crystal structure of a representative NAT protein, the E. coli uracil:H+ symporter UraA, in complex with uracil at 2.9 Å resolution. UraA exhibits a novel structural fold, with 14 transmembrane segments (TMs) divided into two inverted repeats. A pair of anti-parallel β-strands is located in the middle of TM3 and TM10 and plays an important role in the structural organization and substrate recognition. The structure is spatially arranged into a core domain and a gate domain. Uracil, located at the interface between the two domains, is coordinated mainly by residues from the core domain. Structural analysis suggests that alternating-access of the substrate may be achieved through conformational changes of the gate domain.Besides, we also determined the crystal structure of AdiC, the arginine:agmatine antiporter from E. coli O157:H7 and a member of the amino acid/ polyamine/ organocation (APC) superfamily of transporters at 3.6 Å resolution. The overall fold is similar to that of several LeuT fold transporters. AdiC contains 12 transmembrane segments, forms a homodimer, and exists in an outward-facing, open conformation in the crystals. A conserved, acidic pocket opens to the periplasm. Combined with the arginine bound occluded AdiC structure at 3.0 Å resolution, we proposed that three potential gates, involving four aromatic residues and Glu 208, may work in concert to differentially regulate the upload and release of Arg and Agm. Further structural and biochemical analysis reveals the essential ligand-binding residues, defines the transport route, and suggests a conserved mechanism for the antiporter activity.
