Conformational dynamics of ligand-dependent alternating access in LeuT

• Published in: Nature structural & molecular biology Vol. 21; no. 5; pp. 472 - 479
• Main Authors: Kazmier, Kelli, Sharma, Shruti, Quick, Matthias, Islam, Shahidul M, Roux, Benoît, Weinstein, Harel, Javitch, Jonathan A, Mchaourab, Hassane S
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.05.2014; Nature Publishing Group
• Subjects: 631/535; 631/57/2272; 82; Bacterial proteins; Bacterial Proteins - chemistry; Bacterial Proteins - physiology; Biochemistry; Biological Microscopy; Catalysis; Cell research; Crystal structure; Ion transport; Life Sciences; Ligands; Membrane Biology; Models, Molecular; Molecular biology; Neural transmission; Neurotransmitter Transport Proteins - chemistry; Neurotransmitters; Physiological aspects; Protein Structure; Protein Structure, Tertiary; Sodium; Spin Labels; Structural models; Symporters - chemistry; Symporters - physiology; Testing
• ISSN: 1545-9993; 1545-9985; 1545-9985
• DOI: 10.1038/nsmb.2816

LeuT is a Na + -coupled amino acid transporter that is similar in sequence and function to eukaryotic neurotransmitter/sodium symporters, which are active in reuptake of neurotransmitters from the synapse. Distance measurements between spin-label pairs are used to identify ligand-dependent structural transitions in LeuT. The leucine transporter (LeuT) from Aquifex aeolicus is a bacterial homolog of neurotransmitter/sodium symporters (NSSs) that catalyze reuptake of neurotransmitters at the synapse. Crystal structures of wild-type and mutants of LeuT have been interpreted as conformational states in the coupled transport cycle. However, the mechanistic identities inferred from these structures have not been validated, and the ligand-dependent conformational equilibrium of LeuT has not been defined. Here, we used distance measurements between spin-label pairs to elucidate Na + - and leucine-dependent conformational changes on the intracellular and extracellular sides of the transporter. The results identify structural motifs that underlie the isomerization of LeuT between outward-facing, inward-facing and occluded states. The conformational changes reported here present a dynamic picture of the alternating-access mechanism of LeuT and NSSs that is different from the inferences reached from currently available structural models.