Exploring the structural and mechanistic basis of H+, Na+, and K+ coupling in secondary active glutamate transporters

离子偶联是次级主动转运蛋白的基本性质。离子耦合的结构和机理基础还没有得到很好的理解，这是本提案的主要主题。谷氨酸转运蛋白非常适合研究离子偶联，原因有二。首先，古细菌转运蛋白Gltph的晶体结构是已知的，并为探索提供了一个框架。第二，谷氨酸转运蛋白家族的不同成员使用不同的偶联离子和底物：偶联离子化学计量。这种多样性将有助于确定钠离子、质子和钾离子选择性的结构基础。将追求Na+偶联转运蛋白Gltph到H+和K+偶联蛋白的结构引导转换。首先，大结构域将在具有不同离子选择性的转运蛋白之间交换，随后嵌合体的序列将被微调。这项工作将导致解开的顺序决定离子耦合。同时，嵌合蛋白将被包括在结晶实验中，这将提供离子耦合的结构基础。一个副项目将集中在时间解决机制的钠和天冬氨酸结合Gltph。通过结合EPR，单分子FRET和停止流测量与不同的结晶试验的运输周期将被解释和改进的功能模型将导致。总之，这项工作将提供在医学上重要的谷氨酸转运蛋白家族的基本见解，更具体地说，在二级转运蛋白的阳离子耦合的结构基础。

Ion coupling is a fundamental property of secondary active transporters. The structural and mechanistic basis of ion coupling is not well understood, and is the main topic of this proposal. Glutamate transporters are exquisitely suited to study ion coupling for two reasons. First, crystal structures of the archaeal transporter Gltph are known and provide a framework for the exploration. Second, different members of the glutamate transporter family use different coupling ions, and substrate:coupling ion stoichiometries. The diversity will facilitate the determination of the structural basis of sodium ion, proton and potassium ion selectivity. A structure-guided conversion of the Na+ coupled transporter Gltph into H+ and K+ coupled proteins will be pursued. First large domains will be exchanged between transporters with different ion selectivity, and subsequently the sequences of the chimeras will be fine-tuned. The work will lead to the unraveling of the sequence determinants for ion coupling. In parallel, the chimeric proteins will be included in crystallization experiments, which will provide the structural basis of ion coupling. A side project will concentrate on the time resolved mechanisms of sodium and aspartate binding in Gltph. By combining EPR, single molecule FRET and stopped flow measurements with different crystallization trial the transport cycle will be explained and an improved functional model will result. In summary, the work will provide basic insights in the medically important family of glutamate transporters, and more specifically in the structural basis of cation coupling in secondary transporters.