Energy landscape of LeuT from molecular simulations

Abstract

The bacterial sodium-coupled leucine transporter (LeuT) has been broadly used as a structural model for understanding the structure-dynamics-function of mammalian neurotransmitter transporters as well as other solute carriers that share the same fold (LeuT fold), as the first member of the family crystallographically resolved in multiple states: outward-facing open, outward-facing occluded, and inward-facing open. Yet, a complete picture of the energy landscape of (sub)states visited along the LeuT transport cycle has been elusive. In an attempt to visualize the conformational spectrum of LeuT, we performed extensive simulations of LeuT dimer dynamics in the presence of substrate (Ala or Leu) and co-transported Na + ions, in explicit membrane and water. We used both conventional molecular dynamics (MD) simulations (with Anton supercomputing machine) and a recently introduced method, collective MD, that takes advantage of collective modes of motions predicted by the anisotropic network model. Free energy landscapes constructed based on ∼40 µs trajectories reveal multiple substates occluded to the extracellular (EC) and/or intracellular (IC) media, varying in the levels of exposure of LeuT to EC or IC vestibules. The IC-facing transmembrane (TM) helical segment TM1a shows an opening, albeit to a smaller extent and in a slightly different direction than that observed in the inward-facing open crystal structure. The study provides insights into the spectrum of conformational substates and paths accessible to LeuT and highlights the differences between Ala-and Leu-bound substates. C 2015 AIP Publishing LLC. [http://dx

The Environment Shapes the Inner Vestibule of LeuT

“…The conformation of TM1A has been investigated in two recent simulation reports. TM1A was shown to have a high probability to partially close the inner vestibule in a 1-palmitoyl-2-oleyl-phosphatidylethanolamine (POPE) membrane [42]. The opposing result was reported earlier, as TM1A was found to be very stable when residing in the hydrophobic core of a POPC membrane (stabilized by ~20 kJ/mol) [36].…”

Conformational dynamics of a neurotransmitter:sodium symporter in a lipid bilayer

“…We note that, to the best of our knowledge, the simulations of the inward-facing conformation of LeuT-Y268A presented here are unique, because all previous studies either examined the inward-facing conformation after restoring the missing Tyr at position 268 (10,22,45,49) or considered the allosteric effect of the Y268A modification in the outward-occluded conformation (11). Although the available evidence shows that the Y268A modification strongly affects the ability for LeuT to stabilize outward-facing conformations and to allosterically detect ligand binding in those conformations (11), comparison of our simulation data with that reported for previous studies (10,22) revealed no obvious difference due to the Y268A modification in the nature of the inward-facing conformations, presumably because position 268 was entirely solvent exposed, and therefore unable to mediate direct interactions.…”

“…6A). Indeed, the extent of TM1a movement has been a source of contention in the field given that the crystal conditions contained mostly detergent and only a minute quantity of lipid [1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE)], and that biophysical and computational studies have indicated a more restrained tilt of the helix (10,15,22,45). Our HDX-MS data demonstrate that TM1a (amino acids 17-28) as well as the attached N-terminal peptide (amino acids 1-16) were less protected against deuterium exchange under the inward-favoring conditions and thus apparently free to sample a larger conformational space with greater solvent accessibility than was available under the outward-favoring conditions (Figs.…”

Effect of Dimerization on the Dynamics of Neurotransmitter:Sodium Symporters

“…39 No dimeric structure has been resolved to date for IF LeuT dimer. However, comparison of LeuT crystal structures shows that the interfacial helices TM9 (K376-F395) and TM12 (V483-R507) undergo minimal movements, if any, between the OF and IF states.…”

“…Among them, our previous studies 39,40 were (to the best of our knowledge) the only simulations of LeuT wild-type (WT) dimer dynamics. The IF o dimer conformation was generated therein using the dimerization interface observed in the OF o crystal structure as a template.…”