Identification of the Substrate Recognition and Transport Pathway in a Eukaryotic Member of the Nucleobase-Ascorbate Transporter (NAT) Family

Using the crystal structure of the uracil transporter UraA of Escherichia coli, we constructed a 3D model of the Aspergillus nidulans uric acid-xanthine/H+ symporter UapA, which is a prototype member of the Nucleobase-Ascorbate Transporter (NAT) family. The model consists of 14 transmembrane segments (TMSs) divided into a core and a gate domain, the later being distinctly different from that of UraA. By implementing Molecular Mechanics (MM) simulations and quantitative structure-activity relationship (SAR) approaches, we propose a model for the xanthine-UapA complex where the substrate binding site is formed by the polar side chains of residues E356 (TMS8) and Q408 (TMS10) and the backbones of A407 (TMS10) and F155 (TMS3). In addition, our model shows several polar interactions between TMS1-TMS10, TMS1-TMS3, TMS8-TMS10, which seem critical for UapA transport activity. Using extensive docking calculations we identify a cytoplasm-facing substrate trajectory (D360, A363, G411, T416, R417, V463 and A469) connecting the proposed substrate binding site with the cytoplasm, as well as, a possible outward-facing gate leading towards the substrate major binding site. Most importantly, re-evaluation of the plethora of available and analysis of a number of herein constructed UapA mutations strongly supports the UapA structural model. Furthermore, modeling and docking approaches with mammalian NAT homologues provided a molecular rationale on how specificity in this family of carriers might be determined, and further support the importance of selectivity gates acting independently from the major central substrate binding site.本研究以大肠杆菌（Escherichia coli）尿嘧啶转运蛋白UraA的晶体结构为模板，构建了构巢曲霉（Aspergillus nidulans）尿酸-黄嘌呤/H+同向转运蛋白UapA的三维模型——该蛋白是碱基-抗坏血酸转运蛋白（Nucleobase-Ascorbate Transporter, NAT）家族的原型成员。该模型包含14个跨膜结构域（transmembrane segments, TMSs），分为核心结构域与门控结构域两部分，其中门控结构域与UraA的门控结构域存在显著差异。通过分子力学（Molecular Mechanics, MM）模拟与定量构效关系（quantitative structure-activity relationship, SAR）分析方法，本研究提出了黄嘌呤-UapA复合物的结构模型：底物结合位点由残基E356（位于TMS8）与Q408（位于TMS10）的极性侧链，以及A407（位于TMS10）与F155（位于TMS3）的主链共同构成。此外，本模型显示TMS1-TMS10、TMS1-TMS3以及TMS8-TMS10之间存在多处极性相互作用，这些相互作用对UapA的转运活性至关重要。通过大规模分子对接计算，本研究鉴定出一条连接底物结合位点与细胞质的面向细胞质的底物转运通路（包含D360、A363、G411、T416、R417、V463与A469残基），同时还发现了一条可能的面向胞外的门控通路，可通往主要底物结合位点。最为关键的是，对大量已有的公开实验数据进行重新评估，并对本研究中构建的多株UapA突变体开展分析，结果有力验证了该UapA结构模型的合理性。此外，针对哺乳动物NAT同源蛋白的建模与分子对接分析，为该转运蛋白家族的底物特异性分子决定机制提供了理论依据，进一步佐证了独立于核心底物结合位点的选择性门控结构的重要性。