Integrated AlphaFold2 and DEER investigation of the conformational dynamics of a pH-dependent APC antiporter.
Integrated AlphaFold2 and DEER investigation of the conformational dynamics of a pH-dependent APC antiporter.
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DOI：
10.1073/pnas.2206129119
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发表时间：
2022-08-23
影响因子：
11.1
通讯作者：
中科院分区：
文献类型：
--
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转运蛋白GadC通过使用称为交替进入的机制交换两种底物谷氨酸和γ-氨基丁酸（GABA）来促进细菌病原体的耐酸性。在这项研究中，交替访问的构象动力学进行了研究，使用光谱学和计算建模相结合。构象不同的合奏模型，使用AlphaFold 2生成，指导双电子-电子共振光谱实验的设计和解释。我们发现，而GadC是不活跃的，在中性pH值的构象均匀，低pH值诱导两种构象之间的异构化。从我们的综合计算/实验研究出现了一个运输模型，可能是相关的真核同源物参与其他细胞过程。氨基酸-多胺-有机定位（APC）转运蛋白GadC通过将细胞外谷氨酸交换为细胞内γ-氨基丁酸（GABA）来促进病原菌在极端酸胁迫下的存活。它的结构，确定在一个面向内的构象在碱性pH值，由经典的LeuT折叠与保守的五螺旋反向重复，从而类似于功能不同的转运蛋白，如血清素转运蛋白SERT和葡萄糖钠同向转运蛋白SGLT 1。然而，尽管这种结构相似性，目前还不清楚，如果反向转运蛋白，如GadC的构象动力学遵循这些或其他LeuT折叠转运蛋白的蓝图。在这里，我们使用双电子-电子共振（DEER）光谱监测GadC在脂质双层中的构象动力学响应酸化和底物结合。为了指导实验设计和促进DEER数据的解释，我们使用最近引入的AlphaFold 2修改生成了多种构象的结构模型的集合。我们的实验结果揭示了酸诱导的构象变化，从渗透途径中驱逐C末端，再加上螺旋重排，使向内和向外的状态之间的异构化。底物谷氨酸，但不是GABA，调节细胞外薄门的动态，而不改变内向和外向构象之间的平衡。除了引入一个综合的方法探测运输构象动力学，一致性的DEER数据与模式的结构重排推导出的AlphaFold 2模型的合奏阐明了GadC支撑运输的构象周期，并暴露了另一个例子的分歧之间的动态不同的家庭在亮氨酸折叠。
The transporter GadC contributes to acid resistance in bacterial pathogens by exchanging two substrates, glutamate and γ-aminobutyric acid (GABA), using a mechanism termed alternating access. In this study, the conformational dynamics underlying alternating access were studied using a combination of spectroscopy and computational modeling. A conformationally diverse ensemble of models, generated using AlphaFold2, guided the design and interpretation of double electron-electron resonance spectroscopy experiments. We found that whereas GadC was inactive and conformationally homogeneous at neutral pH, low pH induced isomerization between two conformations. From our integrated computational/experimental investigation emerges a transport model that may be relevant to eukaryotic homologs that are involved in other cellular processes. The Amino Acid–Polyamine-Organocation (APC) transporter GadC contributes to the survival of pathogenic bacteria under extreme acid stress by exchanging extracellular glutamate for intracellular γ-aminobutyric acid (GABA). Its structure, determined in an inward-facing conformation at alkaline pH, consists of the canonical LeuT-fold with a conserved five-helix inverted repeat, thereby resembling functionally divergent transporters such as the serotonin transporter SERT and the glucose-sodium symporter SGLT1. However, despite this structural similarity, it is unclear if the conformational dynamics of antiporters such as GadC follow the blueprint of these or other LeuT-fold transporters. Here, we used double electron-electron resonance (DEER) spectroscopy to monitor the conformational dynamics of GadC in lipid bilayers in response to acidification and substrate binding. To guide experimental design and facilitate the interpretation of the DEER data, we generated an ensemble of structural models in multiple conformations using a recently introduced modification of AlphaFold2 . Our experimental results reveal acid-induced conformational changes that dislodge the Cterminus from the permeation pathway coupled with rearrangement of helices that enables isomerization between inward- and outward-facing states. The substrate glutamate, but not GABA, modulates the dynamics of an extracellular thin gate without shifting the equilibrium between inward- and outward-facing conformations. In addition to introducing an integrated methodology for probing transporter conformational dynamics, the congruence of the DEER data with patterns of structural rearrangements deduced from ensembles of AlphaFold2 models illuminates the conformational cycle of GadC underpinning transport and exposes yet another example of the divergence between the dynamics of different families in the LeuT-fold.
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2.9
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