Silverstein Nechama, Ewers David, Forrest Lucy R, Fahlke Christoph, Kanner Baruch I
From the Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada Faculty of Medicine, Hebrew University, Jerusalem 91120, Israel.
the Institute of Complex Systems, Forschungszentrum Jülich, 52425 Jülich, Germany, the Institut für Neurophysiology, Medizinische Hochschule, 30625 Hannover, Germany, and.
J Biol Chem. 2015 Nov 27;290(48):28988-96. doi: 10.1074/jbc.M115.682666. Epub 2015 Oct 16.
Crystal structures of the archaeal homologue GltPh have provided important insights into the molecular mechanism of transport of the excitatory neurotransmitter glutamate. Whereas mammalian glutamate transporters can translocate both glutamate and aspartate, GltPh is only one capable of aspartate transport. Most of the amino acid residues that surround the aspartate substrate in the binding pocket of GltPh are highly conserved. However, in the brain transporters, Thr-352 and Met-362 of the reentrant hairpin loop 2 are replaced by the smaller Ala and Thr, respectively. Therefore, we have studied the effects of T352A and M362T on binding and transport of aspartate and glutamate by GltPh. Substrate-dependent intrinsic fluorescence changes were monitored in transporter constructs containing the L130W mutation. GltPh-L130W/T352A exhibited an ~15-fold higher apparent affinity for l-glutamate than the wild type transporter, and the M362T mutation resulted in an increased affinity of ~40-fold. An even larger increase of the apparent affinity for l-glutamate, around 130-fold higher than that of wild type, was observed with the T352A/M362T double mutant. Radioactive uptake experiments show that GltPh-T352A not only transports aspartate but also l-glutamate. Remarkably, GltPh-M362T exhibited l-aspartate but not l-glutamate transport. The double mutant retained the ability to transport l-glutamate, but its kinetic parameters were very similar to those of GltPh-T352A alone. The differential impact of mutation on binding and transport of glutamate suggests that hairpin loop 2 not only plays a role in the selection of the substrate but also in its translocation.
古菌同源物GltPh的晶体结构为兴奋性神经递质谷氨酸的转运分子机制提供了重要见解。虽然哺乳动物谷氨酸转运体能够转运谷氨酸和天冬氨酸，但GltPh是唯一能够转运天冬氨酸的。GltPh结合口袋中天冬氨酸底物周围的大多数氨基酸残基高度保守。然而，在脑转运体中，折返发夹环2的苏氨酸-352和甲硫氨酸-362分别被较小的丙氨酸和苏氨酸取代。因此，我们研究了T352A和M362T对GltPh结合和转运天冬氨酸及谷氨酸的影响。在含有L130W突变的转运体构建体中监测底物依赖性内在荧光变化。GltPh-L130W/T352A对L-谷氨酸的表观亲和力比野生型转运体高约15倍，而M362T突变导致亲和力增加约40倍。T352A/M362T双突变体对L-谷氨酸的表观亲和力增加甚至更大，比野生型高约130倍。放射性摄取实验表明，GltPh-T352A不仅转运天冬氨酸，还转运L-谷氨酸。值得注意的是，GltPh-M362T表现出转运L-天冬氨酸但不转运L-谷氨酸。双突变体保留了转运L-谷氨酸的能力，但其动力学参数与单独的GltPh-T352A非常相似。突变对谷氨酸结合和转运的不同影响表明，发夹环2不仅在底物选择中起作用，而且在其转运中也起作用。
