Computational Structural Biology Group, The Max Planck Institute of Biophysics, Max-von-Laue-Strasse 3, 60438 Frankfurt am Main, Germany.
Proc Natl Acad Sci U S A. 2009 Dec 8;106(49):20752-7. doi: 10.1073/pnas.0908570106. Epub 2009 Nov 19.
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 A apart in the crystal structures of GltPh, but approximately = 10 A 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-beta-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.
谷氨酸转运体通过将其通量与钠离子和其他阳离子的通量偶联来调节这种神经递质的突触浓度。这些转运体的古菌同源物 GltPh 的可用晶体结构类似于面向细胞外的状态，其中结合的底物仅被称为 HP2 的小螺旋发夹片段所阻塞。然而，膜的细胞质侧的途径并不明显。我们之前仅基于拓扑倒置结构重复的存在，为神经递质：钠协同转运体家族的转运体构建了另一种状态的模型，该模型具有完全不同的折叠。在 GltPh 中，我们鉴定了两组不同的拓扑倒置重复，并使用这些重复来模拟该蛋白的内向构象。为了测试该模型，我们在神经元谷氨酸转运体 EAAC1 中引入了一对半胱氨酸，其位置在 GltPh 的晶体结构中相隔> 27Å，但在内向构象模型中大约相隔 10Å。这些突变体的转运可通过用还原剂二硫苏糖醇预处理来激活。随后用氧化剂铜(II)(1,10-邻菲啰啉)(3)处理可消除这种激活。在被认为促进转运体内向构象的条件下，转运的抑制作用增强。相比之下，在非转运类似物 D,L-threo-beta-苄氧琥珀酸的存在下，抑制作用降低，这有利于外向构象。其他构象敏感的可及性测量也被我们的内向构象模型所适应。这些结果表明，在转运体中包含拓扑倒置重复可能为单个蛋白中两个对称相关状态的要求提供了一种通用的解决方案。
