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

Abstract

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

Serotonin transport in the 21st century

“…5 C (inward-open). These conformational changes are consistent with recent Hydrogen-Deuterium exchange studies of LeuT and SERT (Adhikary et al, 2017;Möller et al, 2019). This movement exposes the same residues predicted by the rocking bundle model to become accessible when the cytoplasmic pathway opens (Krishnamurthy and Gouaux, 2012;Penmatsa and Gouaux, 2014).…”

Mass spectrometry-enabled structural biology of membrane proteins

“…nanodiscs, bicelles or liposomes), the lipids are often removed prior to MS analyses to prevent them interfering with ionization and LC separation [43,198], though more recent work suggests that this is not always necessary [181,[192][193][194][195]199]. HDX-MS in nanodiscs has been used to study propeptide binding to c glutamyl carboxylase [192], and to study the conformational dynamics underlying the alternating access mechanism of LeuT [193]. Bicelles have been used to study the complex of b 2 -adrenergic receptor with a GPCR kinase, combined with data from other methods, including chemical crosslinking [181].…”

HDX-MS reveals nucleotide-regulated, anti-correlated opening and closure of SecA and SecY channels of the bacterial translocon

“…HDX-MS has emerged as a non-invasive and highly sensitive method for interrogating the conformational dynamics of membrane proteins and their complexes [15][16][17][18] . HDX-MS measures the exchange of backbone amide hydrogen to deuterium at peptide-level resolution [19][20][21] .…”

“…For example, the analysis of the structural consequences of protein-ligand/drug interactions, and nucleotide dependent protein dynamics; ATP/GTP versus ADP/GDP 23,24 . Moreover, our group and others have developed and applied HDX-MS to study the conformational diversity of challenging membrane protein systems 15,17,18 .…”