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

离子耦合是二次活性转运体的基本性质。离子偶联的结构和机理基础尚不清楚，这是本提案的主要主题。谷氨酸转运体非常适合研究离子偶联有两个原因。首先，我们了解了古细菌转运体Gltph的晶体结构，为探索提供了框架。其次，谷氨酸转运体家族的不同成员使用不同的偶联离子和底物：偶联离子化学计量学。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。Na+偶联转运体Gltph将在结构引导下转化为H+和K+偶联蛋白。首先，大结构域将在具有不同离子选择性的转运体之间交换，随后嵌合体的序列将被微调。这项工作将导致解开离子偶联的序列决定因素。同时，嵌合蛋白将被纳入结晶实验，这将为离子耦合提供结构基础。一个副项目将集中于Gltph中钠和天冬氨酸结合的时间分解机制。通过结合EPR，单分子FRET和不同结晶试验的停止流动测量，将解释运输周期并得到改进的功能模型。总之，这项工作将为医学上重要的谷氨酸转运蛋白家族，特别是二级转运蛋白中阳离子偶联的结构基础提供基本的见解。

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.