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The high‐energy transition state of the glutamate transporter homologue GltPh
The EMBO Journal ( IF 9.4 ) Pub Date : 2020-11-13 , DOI: 10.15252/embj.2020105415
Gerard H M Huysmans 1, 2 , Didar Ciftci 1, 3 , Xiaoyu Wang 1 , Scott C Blanchard 1, 3, 4 , Olga Boudker 1, 3, 5
Affiliation  

Membrane transporters mediate cellular uptake of nutrients, signaling molecules, and drugs. Their overall mechanisms are often well understood, but the structural features setting their rates are mostly unknown. Earlier single‐molecule fluorescence imaging of the archaeal model glutamate transporter homologue GltPh from Pyrococcus horikoshii suggested that the slow conformational transition from the outward‐ to the inward‐facing state, when the bound substrate is translocated from the extracellular to the cytoplasmic side of the membrane, is rate limiting to transport. Here, we provide insight into the structure of the high‐energy transition state of GltPh that limits the rate of the substrate translocation process. Using bioinformatics, we identified GltPh gain‐of‐function mutations in the flexible helical hairpin domain HP2 and applied linear free energy relationship analysis to infer that the transition state structurally resembles the inward‐facing conformation. Based on these analyses, we propose an approach to search for allosteric modulators for transporters.

中文翻译:


谷氨酸转运蛋白同源物 GltPh 的高能过渡态



膜转运蛋白介导细胞对营养物质、信号分子和药物的吸收。它们的整体机制通常很容易理解,但决定其速率的结构特征却大多未知。早期对来自堀越火球菌的古菌模型谷氨酸转运蛋白同源物 Glt Ph 的单分子荧光成像表明,当结合的底物从细胞外转移到细胞质侧时,从外向状态到内向状态的缓慢构象转变膜,是运输的速率限制。在这里,我们深入了解了限制底物易位过程速率的 Glt Ph高能过渡态结构。利用生物信息学,我们鉴定了柔性螺旋发夹结构域 HP2 中的 Glt Ph功能获得突变,并应用线性自由能关系分析来推断过渡态在结构上类似于向内构象。基于这些分析,我们提出了一种寻找转运蛋白变构调节剂的方法。
更新日期:2021-01-04
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