葡萄糖是生物体最重要的碳源之一，从原核到真核，从单细胞到多细胞，它在各种细胞生物中都能被广泛利用。作为有氧呼吸及糖基化等胞内生化反应的底物，葡萄糖对于生命体来说无疑是至关重要的。而由于其较强的亲水性，葡萄糖无法自由透过疏水的磷脂双分子层，细胞需要通过跨膜转运来实现对葡萄糖的摄取和利用。所以对葡萄糖跨膜转运的探索对于其代谢调控研究来说是不可或缺的重要部分。 葡萄糖转运蛋白（Glucose transporters, GLUTs）是细胞摄取葡萄糖的重要载体，这类蛋白从属于主要协助转运蛋白超家族（Major Facilitator Superfamily, MFS）。除了负责吸收葡萄糖进入细胞之外， GLUTs的成员亦可以转运如果糖、氨基葡萄糖等其他单糖及衍生物。至今为止，对GLUTs的研究已经由鉴定、克隆和遗传水平的研究过渡到了细胞生物学和生物化学水平的研究。同时GLUTs的功能、性质以及在不同疾病中起到的影响也得到了一定的解释。然而，其行使转运功能的具体分子机制依旧比较模糊。所以在分子水平上研究GLUTs的结构与转运机制具有重要的生物学意义。 在本篇论文中，我们首先利用异源重组表达，得到了性质良好的人源葡萄糖蛋白GLUT1，并解析了其3.2 ?的晶体结构。通过结构分析，初步给出了GLUTs的转运模型，并分析了转运过程中胞外区门控和胞内区ICH结构域的氢键作用网络，同时也给出了GLUTs缺陷突变的可能致病机理。而后我们又利用脂立方相结晶技术，获得了人源GLUT3 1.5 ?高分辨率的底物结合结构，以及结合竞争性抑制剂向外开口和朝向胞外闭合的结构，第一次捕捉到了转运蛋白向外开放的构象，并且完整补充了GLUTs在转运过程中的几个重要状态。结合同位素标记实验，我们完善了葡萄糖转运蛋白“交替开放”的转运模型，并通过各个构象间的结构比对，提出了GLUTs进行底物识别和转运的分子机制。 综上，本研究首次解析了人源葡萄糖转运蛋白GLUT1向胞内开放的晶体结构，以及GLUT3结合底物朝向胞外闭合，结合抑制剂朝向胞外闭合和向外开放三个晶体结构。结合生化实验和结构分析，第一次完整描绘了GLUTs行使功能的具体模型，从分子层面上初步阐明了GLUTs的工作机制。为基于结构的药物研究提供了重要线索。
Glucose is one of the most essential carbon source for creatures, which can be widely used from prokaryotes to eukaryotes, from unicellular to multicellular organisms. As the substrate of respiration and other biochemical reactions in cells like glycosylation, glucose is undoubtedly important small molecule for life beings. However, it can not freely diffuse across the lipid bilayer because of the strong hydrophilicity, so that the uptake and utilize of glucose in cell need facilitating by membrane transport. Therefore, the exploration of glucose transport is significant for studying its metabolism and regulation. Most mammalian cells uptake glucose by glucose transporters, which belong to the sugar porter family in major facilitator superfamily (MFS). So far, the study of GLUTs has been transit to cell biology and biochemistry level, and their function, properties and effects on various diseases have been preliminarily elucidated. Nevertheless, the molecular mechanism of glucose transport by GLUTs remains elusive. So the structural and functional investigations of GLUTs in molecular level make a big difference and have essential biological meanings. In this thesis, we successfully got well behaved human GLUT1 protein by recombinant expression and solved the 3.2 ? crystal structure at first. After structural analysis, we build a primary transport model for GLUTs and describe the hydrogen bond network in intra- and extra- celluar gates. Also, dysfunctional mutations were mapped in the structure to illustrate the possible nosogenesis. Then we got substrate-bound, outward-facing and competitive inhibitor-bound, outward-facing/open structures of GLUT3 by LCP. It makes that possible to complete the important conformations in a transport cycle. Combined with the analysis of isotope assays, substrate recognition and transport mechanism are illustrated. As a brief summary, we solved important structures of human GLUTs and set up the isotope assay. And we firstly described the detailed transport model and interpreted the transport mechanism of GLUTs at molecular level with structural analysis and biochemical experiments, providing structural basis for structure-based drug design.
