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Inward-facing conformation of glutamate transporters as revealed by their inverted-topology structural repeats

机译：谷氨酸转运蛋白的向内构象，如其反向拓扑结构重复所示

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Glutamate transporters regulate synaptic concentrations of this neurotransmitter by coupling its flux to that of sodium and other cations. Available crystal structures of an archeal homologue of these transporters, GltPh, resemble an extracellular-facing state, in which the bound substrate is occluded only by a small helical hairpin segment called HP2. However, a pathway to the cytoplasmic side of the membrane is not clearly apparent. We previously modeled an alternate state of a transporter from the neurotransmitter:sodium symporter family, which has an entirely different fold, solely on the presence of inverted-topology structural repeats. In GltPh, we identified two distinct sets of inverted-topology repeats and used these repeats to model an inward-facing conformation of the protein. To test this model, we introduced pairs of cysteines into the neuronal glutamate transporter EAAC1, at positions that are >27 Å apart in the crystal structures of GltPh, but ≈10 Å apart in the inward-facing model. Transport by these mutants was activated by pretreatment with the reducing agent dithithreitol. Subsequent treatment with the oxidizing agent copper(II)(1,10-phenantroline)3 abolished this activation. The inhibition of transport was potentiated under conditions thought to promote the inward-facing conformation of the transporter. By contrast, the inhibition was reduced in the presence of the nontransportable substrate analogue d,l-threo-β-benzyloxyaspartate, which favors the outward-facing conformation. Other conformation-sensitive accessibility measurements are also accommodated by our inward-facing model. These results suggest that the inclusion of inverted-topology repeats in transporters may provide a general solution to the requirement for two symmetry-related states in a single protein.
机译：谷氨酸转运蛋白通过将其通量与钠和其他阳离子的通量耦合来调节该神经递质的突触浓度。这些转运蛋白的古细菌同源物的可用晶体结构GltPh类似于细胞外状态，其中结合的底物仅被称为HP2的小螺旋发夹片段封闭。然而，通往膜细胞质侧的途径尚不清楚。我们以前从神经递质：钠同向转运蛋白家族建模了转运蛋白的替代状态，其折叠方式完全不同，仅在存在反向拓扑结构重复的情况下。在GltPh中，我们确定了两个不同的反向拓扑重复集，并使用这些重复来建模蛋白质的向内构象。为了测试该模型，我们在神经元谷氨酸转运蛋白EAAC1中引入了半胱氨酸对，它们的位置在GltPh的晶体结构中相距> 27 ,，而在向内模型中相距≈10Å。这些突变体的转运通过用还原剂二硫苏糖醇预处理而被激活。随后用氧化剂铜（II）（1,10-菲咯啉）3处理消除了这种活化。在认为促进转运蛋白向内构象的条件下加强了对转运的抑制。相反，在不可运输的底物类似物d，1-苏-β-苄氧基天冬氨酸的存在下，抑制作用降低，这有利于向外构象。我们的向内模型也包含其他对构象敏感的可访问性度量。这些结果表明，在转运蛋白中包​​含反向拓扑重复序列可以为单一蛋白质中两个对称相关状态的需求提供一般解决方案。

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