Neurotransmitter:sodium symporters (NSSs) play critical roles in neural signaling by regulating neurotransmitter uptake into cells powered by sodium electrochemical gradients. Bacterial NSSs orthologs, including MhsT from Bacillus halodurans, have emerged as model systems to understands the structural motifs of alternating access in NSSs and the extent of conservation of these motifs across the family. Here, we apply a novel computational/experimental methodology to illuminate the energy landscape of MhsT alternating access. Capitalizing on our recently developed method, Sampling Protein Ensembles and Conformational Heterogeneity with AlphaFold2 (SPEACH_AF), we derived clusters of MhsT models spanning the transition from inward-facing to outward-facing conformations. Systematic application of double electron-electron resonance (DEER) spectroscopy revealed ligand-dependent movement of multiple structural motifs that underpins MhsT’s conformational cycle. Remarkably, comparative DEER analysis in detergent micelles and lipid nanodiscs highlight the profound effect of the environment on the energetics of conformational changes. Through experimentally-derived selection of collective variables, we present a model of ion and substrate powered transport by MhsT consistent with the conformational cycle derived from DEER. Our findings not only advance the understanding of MhsT’s function but also uncover motifs of conformational dynamics conserved within the broader context of the NSS family and within the LeuT-fold class of transporters. Importantly, our methodological blueprint introduces a novel approach that can be applied across a diverse spectrum of transporters to describe their energy landscapes.

中文翻译：

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神经递质的交替访问：通过 AlphaFold2 整体和 DEER 光谱测定钠同源转运蛋白细菌同源物

神经递质：钠同向转运体 (NSS) 通过调节钠电化学梯度驱动的细胞对神经递质的摄取，在神经信号传导中发挥关键作用。细菌 NSS 直系同源物，包括来自嗜盐芽孢杆菌的 MhsT ，已成为了解 NSS 中交替访问的结构基序以及这些基序在整个家族中的保守程度的模型系统。在这里，我们应用一种新颖的计算/实验方法来阐明 MhsT 交替访问的能量景观。利用我们最近开发的方法“使用 AlphaFold2 进行蛋白质整体采样和构象异质性”(SPEACH_AF)，我们衍生出了涵盖从内向构象到外向构象转变的 MhsT 模型簇。双电子-电子共振 (DEER) 光谱的系统应用揭示了支撑 MhsT 构象循环的多个结构基序的配体依赖性运动。值得注意的是，洗涤剂胶束和脂质纳米盘的比较 DEER 分析强调了环境对构象变化能量学的深远影响。通过实验衍生的集体变量选择，我们提出了 MhsT 的离子和底物动力运输模型，与源自 DEER 的构象循环一致。我们的研究结果不仅增进了对 MhsT 功能的理解，而且还揭示了 NSS 家族和 LeuT 折叠类转运蛋白的更广泛背景下保守的构象动力学基序。重要的是，我们的方法蓝图引入了一种新颖的方法，可以应用于各种运输者来描述其能源景观。