Exploring the Structural Dynamics of LeuT Using EPR Spectroscopy: A Focus on Transmembrane Helix 10

Abstract

The amino-acid transporter LeuT from Aquifex aeolicus is a well-studied bacterial homologue of the neurotransmitter: sodium symporters (NSS), especially the solute carrier 6 (SLC6) family. Within the nervous system, SLC6 transporters play a vital role in the termination of synaptic transmission, and their dysfunction leads to severe neurological conditions, rendering them key pharmacological targets. LeuT was the first SLC6 homologue to be crystallised and remains the main reference transporter to develop transport cycle models for its eukaryotic counterparts. Here, we aim to probe LeuT and investigate mechanistically important conformational changes using a combination of Site-Directed Spin Labelling (SDSL) and Electron Paramagnetic Resonance (EPR) spectroscopic techniques in detergent solubilised micelles and proteoliposomes. We focus, primarily, on ‘subtle’ structural, molecular motions occurring at the extracellular region of transmembrane helix (TM) 10, which cannot be resolved using conventional high-resolution crystallographic techniques. We observe similar but not identical ion/ligand-dependent conformational changes of LeuT on the extracellular domain of TM10 in detergent micelles and proteoliposomes. Close agreement is also observed between in silico analysis of existing static structural models and the experimental data acquired here in the form of coarse-grained accessibility restraints, demonstrating that such subtle movements can be important for understanding both function and mechanism. The observed differences for the dynamics of LeuT in different environments underpin future work, which aims to explore ‘more native’ reconstituted proteoliposome conditions more thoroughly using pulsed EPR methods before generalised conclusions can be drawn on the physiological relevance of such structural changes and whether they can provide novel insights on the molecular events underlying the transport cycle of LeuT.