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.

Conformational cycle and ion-coupling mechanism of the Na + /hydantoin transporter Mhp1

“…This methodology has been successfully applied to define coupled conformational cycles for a number of transporter classes (13,(26)(27)(28)(29)(30)(31)(32). We find that patterns of distance distributions between pairs of spin labels monitoring the intra-and extracellular sides of Mhp1 are consistent with isomerization between the crystallographic inward-and outward-facing conformations.…”

“…The results presented here describe the ligand-dependent equilibrium between these conformations, reveal that substrate but not Na + binding changes the energetics of these conformations, and indicate that the bundle and scaffold motifs are not strictly rigid bodies. In conjunction with a previous investigation of LeuT (13), this work illuminates commonalities and differences in the structural mechanism of alternating access in the LeuT-fold class of ioncoupled transporters.…”

“…Typically, transporter crystal structures are classified as inward-facing, outwardfacing, or occluded on the basis of the accessibility of the substrate binding site (7)(8)(9)(10)(11). In a recent spectroscopic analysis of LeuT, we demonstrated that detergent selection and mutations of conserved residues appeared to stabilize conformations that were not detected in the wild-type (WT) LeuT and concurrently inhibited movement of structural elements involved in ligand-dependent alternating access (13). Therefore, although crystal structures define the structural context and identify plausible pathways of substrate binding and release, development of transport models requires confirming or assigning the mechanistic identity of these structures and framing them into ligand-dependent equilibria (14).…”

“…simulations (20,21) and electron paramagnetic resonance (EPR) analysis (13,22) of LeuT demonstrated that Na + binding favors an outward-facing conformation although it is unclear which Na + site (or both) is responsible for triggering this conformational transition. Similarly, a role for Na + in conformational switching has been uncovered in putative human LeuT-fold transporters, including hSGLT (23).…”

Energy landscape of LeuT from molecular simulations

“…The degree of this opening and its direction has been debated ever since, as it probably lifts TM1a into the membrane. Kazmier et al 40 have recently performed measurements between spin-label pairs in LeuT and observed significantly smaller degree of openings of the IF vestibule for WT LeuT compared to the IFo crystal structure. However, in the Y268A and R5A mutants considerably larger openings were observed compared with WT LeuT.…”