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.

Neurotransmitter:sodium symporters (NSSs) are integral membrane proteins responsible for the sodium-dependent reuptake of smallmolecule neurotransmitters from the synaptic cleft. The symporters for the biogenic amines serotonin (SERT), dopamine (DAT), and norepinephrine (NET) are targets of multiple psychoactive agents, and their dysfunction has been implicated in numerous neuropsychiatric ailments. LeuT, a thermostable eubacterial NSS homolog, has been exploited as a model protein for NSS members to canvass the conformational mechanism of transport with a combination of X-ray crystallography, cysteine accessibility, and solution spectroscopy. Despite yielding remarkable insights, these studies have primarily been conducted with protein in the detergent-solubilized state rather than embedded in a membrane mimic. In addition, solution spectroscopy has required site-specific labeling of nonnative cysteines, a labor-intensive process occasionally resulting in diminished transport and/or binding activity. Here, we overcome these limitations by reconstituting unlabeled LeuT in phospholipid bilayer nanodiscs, subjecting them to hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS), and facilitating interpretation of the data with molecular dynamics simulations. The data point to changes of accessibility and dynamics of structural elements previously implicated in the transport mechanism, in particular transmembrane helices (TMs) 1a and 7 as well as extracellular loops (ELs) 2 and 4. The results therefore illuminate the value of this strategy for interrogating the conformational mechanism of the more clinically significant mammalian membrane proteins including SERT and DAT, neither of which tolerates complete removal of endogenous cysteines, and whose activity is heavily influenced by neighboring lipids.nanodisc | hydrogen-deuterium exchange mass spectrometry | conformational dynamics | neurotransmitter symporter | molecular dynamics simulations

Huntington's disease (HD) is caused by the presence of an extended polyglutamine (polyQ) region at the N-terminus of the huntingtin (htt) protein. The presence of flanking sequences adjacent to the polyQ region has been reported to modulate the effects of potentially toxic protein-membrane interactions. In this study, we consider four peptide systems with various combinations of flanking sequences (KKQ35KK, KKQ35P11KK, N17Q35KK, N17Q35P11KK) and use atomistic molecular dynamics simulations to study the interactions with a DOPC lipid bilayer. We observe significant membrane thinning, disorderliness of lipid molecules, and compensation effects between the top and the bottom leaflets of the bilayer depending on the presence of particular flanking sequences. Overall, we find that the presence of the N-17 flanking sequence is crucial for membrane interactions. Polyproline decreases the interaction with the membrane in the absence of N-17, but enhances it when present along N-17.

The serotonin transporter (SERT) is an integral membrane protein that exploits preexisting sodium-, chloride-, and potassium ion gradients to catalyze the thermodynamically unfavorable movement of synaptic serotonin into the presynaptic neuron. SERT has garnered significant clinical attention partly because it is the target of multiple psychoactive agents, including the antidepressant paroxetine (Paxil), the most potent selective serotonin reuptake inhibitor known. However, the binding site and orientation of paroxetine in SERT remain controversial. To provide molecular insight, we constructed SERT homology models based on the Drosophila melanogaster dopamine transporter and docked paroxetine to these models. We tested the predicted binding configurations with a combination of radioligand binding and flux assays on wild-type and mutant SERTs. Our data suggest that the orientation of paroxetine, specifically its fluorophenyl ring, in SERT’s substrate binding site directly depends on this pocket’s charge distribution, and thereby provide an avenue toward understanding and enhancing high-affinity antidepressant activity.