Transport domain unlocking sets the uptake rate of an aspartate transporter

摘要

在这篇论文中，作者采用单分子荧光共振能量转移成像、X-射线晶体学和分子动态模拟方法来研究GIt_(Ph)(来自古菌Pyrococcus horikoshii的一个天冬氨酸转运蛋白)和这种酶的一个双突变体形式（它对基质亲和力较低、但对基质转运速度较大）的动态。GIt_(Ph)是谷氨酸转运蛋白的一个同源物，这些蛋白通过在大脑突触内维持低浓度的神经传输物而在神经传输中扮演一个重要角色，而两个突变使得这种酶的作用更接近人类相应的酶。这些结构显示，GIt_(Ph)的截然不同的转运和支架域之间界面的开启（与朝外和朝内两个状态之间的转变有关)与基质转运速度直接相关联。%Glutamate transporters terminate neurotransmission by clearing synaptically released glutamate from the extracellular space, allowing repeated rounds of signalling and preventing glutamate-mediated excitotoxicity. Crystallographic studies of a glutamate transporter homologue from the archaeon Pyrococcus horikoshii, Glt_(ph), showed that distinct transport domains translocate substrates into the cytoplasm by moving across the membrane within a central tri-merization scaffold. Here we report direct observations of these 'elevator-like, transport domain motions in the context of reconstituted proteoliposomes and physiological ion gradients using single-molecule fluorescence resonance energy transfer (smFRET) imaging. We show that Glt_(ph) bearing two mutations introduced to impart characteristics of the human transporter exhibits markedly increased transport domain dynamics, which parallels an increased rate of substrate transport, thereby establishing a direct temporal relationship between transport domain motion and substrate uptake. Crystallographic and computational investigations corroborated these findings by revealing that the 'humanizing' mutations favour structurally 'unlocked' intermediate states in the transport cycle exhibiting increased solvent occupancy at the interface between the transport domain and the trimeric scaffold.