Proton transfer and substrate recognition in SLC17 transporters

囊泡型谷氨酸转运蛋白(VGLUT)属于SLC17有机阴离子转运蛋白家族。它们在突触小泡中积累谷氨酸，因此是兴奋性突触突触强度的主要决定因素。最近对同源细菌D-半乳糖转运体(DgoT)结构的确定为了解底物跨该家族的转运机制开辟了道路。这里将结合多尺度分子模拟、定点突变、异源表达、细胞电生理学和荧光光谱来研究两个重要的功能方面。首先，我们将深入了解实验测量的SLC17家族转运的pH依赖性的分子基础。量子力学/分子力学模拟将揭示必需酸性残基Glu133(在DgoT和VGLUT中保守)和Asp46(仅存在于DgoT中)的作用。这些模拟充分利用了Jülich可用的并行计算能力，将精确定位负责质子转移的残基，这些残基在DgoT和VGLUT中的作用将在实验中进一步仔细研究。此外，我们还将研究SLC17家族中底物选择性的重要方面。具体地说，对上述DgoT结构的模拟，以及对VGLUT同源模型的实验指导的模拟，以及对两个转运体的实验，将为底物选择性的分子决定因素提供线索。

Vesicular glutamate transporters (VGLUTs) belong to the SLC17 family of organic anion transporters. They accumulate glutamate in synaptic vesicles and thus represent a major determinant of synaptic strength in excitatory synapses. The recent determination of the structure of the homologous bacterial D-galactonate transporter (DgoT) has opened the way to understand the mechanisms of substrate transport across this family. Two important functional aspects will be investigated here, by a combination of multi-scale molecular simulations, site-directed mutagenesis, heterologous expression, cellular electrophysiology and fluorescence spectroscopy. First, we will provide insights into the molecular basis of the experimentally measured pH dependence of transport in the SLC17 family. Quantum mechanics/molecular mechanics simulations will shed light on the role of the essential acidic residues Glu133 (conserved across DgoT and VGLUTs) and Asp46 (only present in DgoT). These simulations, which fully exploit the parallel computing power available in Jülich, will pinpoint the residues responsible for proton transfer, and the role of these residues in both DgoT and VGLUTs will be further scrutinized in experiments. In addition, we will study important aspects of substrate selectivity in the SLC17 family. Specifically, simulations on the aforementioned DgoT structure, together with experimentally-guided simulations on VGLUT homology models and experiments on both transporters will provide hints on the molecular determinants of substrate selectivity.