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The Amino Terminus of LeuT Changes Conformation in an Environment Sensitive Manner.
Neurochemical Research ( IF 3.7 ) Pub Date : 2019-12-19 , DOI: 10.1007/s11064-019-02928-9
Jawad A Khan 1 , Azmat Sohail 1 , Kumaresan Jayaraman 1, 2 , Dániel Szöllősi 1 , Walter Sandtner 1 , Harald H Sitte 1 , Thomas Stockner 1
Affiliation  

Neurotransmitter:sodium symporters are highly expressed in the human brain and catalyze the uptake of substrate through the plasma membrane by using the electrochemical gradient of sodium as the energy source. The bacterial homolog LeuT, a small amino acid transporter isolated from the bacteria Aquifex aeolicus, is the founding member of the family and has been crystallized in three conformations. The N-terminus is structurally well defined and strongly interacts with the transporter core in the outward-facing conformations. However, it could not be resolved in the inward-facing conformation, which indicates enhanced mobility. Here we investigate conformations and dynamics of the N-terminus, by combining molecular dynamics simulations with experimental verification using distance measurements and accessibility studies. We found strongly increased dynamics of the N-terminus, but also that helix TM1A is subject to enhanced mobility. TM1A moves towards the transporter core in the membrane environment, reaching a conformation that is closer to the structure of LeuT with wild type sequence, indicating that the mutation introduced to create the inward-facing structure might have altered the position of helix TM1A. The mobile N-terminus avoids entering the open vestibule of the inward-facing state, as accessibility studies do not show any reduction of quenching by iodide of a fluorophore attached to the N-terminus.

中文翻译:

LeuT 的氨基末端以环境敏感的方式改变构象。

神经递质:钠同向转运体在人脑中高表达,利用钠的电化学梯度作为能量来源,催化质膜对底物的摄取。细菌同源物 LeuT 是从细菌 Aquifex aeolicus 中分离出来的一种小型氨基酸转运蛋白,是该家族的创始成员,已结晶成三种构象。N 末端结构明确,并与面向外的构象中的转运蛋白核心强烈相互作用。然而,它无法在面向内的构象中得到解决,这表明流动性增强。在这里,我们通过将分子动力学模拟与使用距离测量和可达性研究的实验验证相结合来研究 N 末端的构象和动力学。我们发现 N 末端的动态显着增加,而且螺旋 TM1A 的流动性也增强。TM1A 向膜环境中的转运蛋白核心移动,达到更接近野生型序列的 LeuT 结构的构象,表明为创建向内结构而引入的突变可能改变了螺旋 TM1A 的位置。移动的 N 末端避免进入面向内状态的开放前庭,因为可及性研究没有显示连接到 N 末端的荧光团的碘化物对猝灭有任何减少。
更新日期:2019-12-19
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