Abstract 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. Significance Statement The neurotransmitter:sodium symporter (NSS) family plays a crucial role in neurotransmitter reuptake, a sodium-dependent process that transports neurotransmitters from the synapse back into the neuron. This study investigates the bacterial tryptophan transporter MhsT, a homolog of human NSSs, using the deep learning method AlphaFold2 in conjunction with double electron-electron resonance spectroscopy. This combined approach enables us to map the energy landscape that dictates the conformational shifts crucial for MhsT’s function. Furthermore, we reveal how the environment modulates the transporter’s dynamics. From our research, we develop a model of MhsT transport that highlights the extent of mechanistic conservation across the NSS family. Additionally, we introduce a comprehensive framework for exploring the energetic landscapes of transporters, effectively integrating computational and experimental methods.

Alternating Access of a Neurotransmitter:Sodium Symporter Bacterial Homolog Determined from AlphaFold2 Ensembles and DEER Spectroscopy