Alternating access of a bacterial homolog of neurotransmitter: sodium symporters determined from AlphaFold2 ensembles and DEER spectroscopy

• 成果类型: Article
• 署名作者: Schwartz, Alexandra C.; Stein, Richard A.; Gil-Iturbe, Eva; Quick, Matthias; Mchaourab, Hassane S.
• 署名单位: Vanderbilt University; Vanderbilt University; Vanderbilt University; NewYork-Presbyterian Hospital; Columbia University; Columbia University; NewYork-Presbyterian Hospital
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-10646
• DOI: 10.1073/pnas.2406063121
• 发表日期: 2024-10-01
• 关键词: human serotonin transporter; x-ray structures; conformational dynamics; molecular-mechanism; structural basis; leut; substrate; RECOGNITION; release; prody

摘要

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 understand the structural motifs of alternating access in NSSs and the extent of conservation of these motifs across the family. Here, we apply a computational/experimental methodology to illuminate the conformational 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 movements of multiple structural motifs that underpin MhsT's conformational cycle. Remarkably, comparative DEER analysis in detergent micelles and lipid nanodiscs highlights 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 an approach that can be applied across a diverse spectrum of transporters to describe their conformational landscapes.