Two Na+ Sites Control Conformational Change in a Neurotransmitter Transporter Homolog

Abstract

In LeuT, a prokaryotic homolog of neurotransmitter transporters, Na ؉ stabilizes outward-open conformational states.We examined how each of the two LeuT Na ؉ binding sites contributes to Na ؉ -dependent closure of the cytoplasmic pathway using biochemical and biophysical assays of conformation. Mutating either of two residues that contribute to the Na2 site completely prevented cytoplasmic closure in response to Na ؉ , suggesting that Na2 is essential for this conformational change, whereas Na1 mutants retained Na ؉ responsiveness. However, mutation of Na1 residues also influenced the Na ؉ -dependent conformational change in ways that varied depending on the position mutated. Computational analyses suggest those mutants influence the ability of Na1 binding to hydrate the substrate pathway and perturb an interaction network leading to the extracellular gate. Overall, the results demonstrate that occupation of Na2 stabilizes outward-facing conformations presumably through a direct interaction between Na ؉ and transmembrane helices 1 and 8, whereas Na ؉ binding at Na1 influences conformational change through a network of intermediary interactions. The results also provide evidence that N-terminal release and helix motions represent distinct steps in cytoplasmic pathway opening.

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

“…We therefore hypothesized that interpretation of these data could be aided by comparison of ensembles of structures generated with MD simulations (43,44) starting with X-ray crystallographic structures of different conformations of LeuT. The outward-and inward-open conformational ensembles of LeuT were mimicked in silico using MD simulations of the following, respectively: the substrate-free, sodiumbound, outward-open structure [Protein Data Bank (PDB) ID code 3TT1] after reversing the Y108F mutation (12); and the substratefree, inward-open, structure (PDB ID code 3TT3) after reversing the modifications at the Na2 site (T354A and S355A) and TM7 (K288A) (10), but maintaining the intracellular gate mutant Y268A. In each case, the protein was embedded in a hydrated lipid bilayer.…”

“…Broadly speaking, transition from the outward-to inwardopen conformation in LeuT involves (i) occlusion of the extracellular vestibule, obstructing access to the substrate-binding site, and (ii) opening of the cytoplasmic pathway, partly facilitated by the untethering of the N-terminal peptide and TM1a from the scaffold (6,12,49). This study provides support for the notion that these global changes, with the same key players, which were previously described predominantly in detergent micelles, also occur in a lipid bilayer.…”

“…LeuT, a nonpolar amino acid transporter (2) and prokaryotic NSS homolog from the hyperthermophilic, chemolithotrophic eubacterium Aquifex aeolicus (3), has provided remarkable mechanistic insight into the molecular determinants of transport activity of its eukaryotic counterparts (4). Such understanding has been facilitated by a combination of high-resolution crystal structures (2,(5)(6)(7), computational modeling (8), and molecular dynamics (MD) simulations (9-11) integrated with substituted cysteine accessibility measurements (SCAM) (12), as well as elegant solution spectroscopic studies such as single-molecule FRET (smFRET) (13,14), electron paramagnetic resonance (EPR), and double electronelectron resonance (DEER) (15,16).…”

“…However, this equilibrium can be shifted to favor one of these conformations by changing the conditions (4). In the presence of Na + and absence of substrate amino acid, for example, LeuT favors an outward-open conformation (6,9,12,16). This conformation is characterized by considerable solvent accessibility to the extracellular vestibule and surrounding residues [transmembrane helix 1b (TM1b), TM3, and EL4] with concomitant closure of the intracellular gate involving interactions between residues R5, Y268, and D369 from TMs 1a, 6b, and 8, respectively (2,6).…”

Control of serotonin transporter phosphorylation by conformational state

“…Phosphorylation was determined under conditions that influence the distribution of SERT between inwardopen and outward-open conformations. Na + is known to stabilize SERT and other transporters in the NSS family in outward-open conformations (21)(22)(23)(24)(25). Fig.…”