Conformational dynamics of ligand-dependent alternating access in LeuT.

Abstract

The leucine transporter (LeuT) from Aquifex aeolicus is a bacterial homolog of neurotransmitter:sodium symporters (NSS) that catalyze reuptake of neurotransmitters at the synapse. Crystal structures of wild type (WT) 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 utilized 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 novel conformational changes reported here present a dynamic picture of the alternating access mechanism of LeuT and NSS that is different to the inferences reached from currently available crystal structures.