Sohail Azmat, Jayaraman Kumaresan, Venkatesan Santhoshkannan, Gotfryd Kamil, Daerr Markus, Gether Ulrik, Loland Claus J, Wanner Klaus T, Freissmuth Michael, Sitte Harald H, Sandtner Walter, Stockner Thomas
Medical University of Vienna, Center for Physiology and Pharmacology, Institute of Pharmacology, Vienna, Austria.
University of Copenhagen, Faculty of Health and Medical Sciences Denmark, Department of Neuroscience and Pharmacology, Copenhagen, Denmark.
PLoS Comput Biol. 2016 Nov 11;12(11):e1005197. doi: 10.1371/journal.pcbi.1005197. eCollection 2016 Nov.
Human neurotransmitter transporters are found in the nervous system terminating synaptic signals by rapid removal of neurotransmitter molecules from the synaptic cleft. The homologous transporter LeuT, found in Aquifex aeolicus, was crystallized in different conformations. Here, we investigated the inward-open state of LeuT. We compared LeuT in membranes and micelles using molecular dynamics simulations and lanthanide-based resonance energy transfer (LRET). Simulations of micelle-solubilized LeuT revealed a stable and widely open inward-facing conformation. However, this conformation was unstable in a membrane environment. The helix dipole and the charged amino acid of the first transmembrane helix (TM1A) partitioned out of the hydrophobic membrane core. Free energy calculations showed that movement of TM1A by 0.30 nm was driven by a free energy difference of ~15 kJ/mol. Distance measurements by LRET showed TM1A movements, consistent with the simulations, confirming a substantially different inward-open conformation in lipid bilayer from that inferred from the crystal structure.
人类神经递质转运体存在于神经系统中，通过快速从突触间隙清除神经递质分子来终止突触信号。在嗜热栖热菌中发现的同源转运体LeuT以不同构象结晶。在此，我们研究了LeuT的内向开放状态。我们使用分子动力学模拟和基于镧系元素的共振能量转移（LRET）比较了膜和胶束中的LeuT。胶束增溶的LeuT模拟显示出一种稳定且广泛开放的内向构象。然而，这种构象在膜环境中不稳定。第一个跨膜螺旋（TM1A）的螺旋偶极和带电荷氨基酸从疏水膜核心中分离出来。自由能计算表明，TM1A移动0.30 nm是由约15 kJ/mol的自由能差驱动的。LRET的距离测量显示了TM1A的移动，与模拟结果一致，证实了脂质双分子层中的内向开放构象与晶体结构推断的构象有很大不同。
