Partial Unwinding of Transmembrane Helices Facilitates Alternating Access in the Neurotransmitter:Sodium Symporter LeuT

The prokaryotic neurotransmitter:sodium symporter (NSS) LeuT from Aquifex aeolicus is an established structural model for mammalian NSS counterparts. Here, we investigate the substrate translocation mechanism of LeuT by measuring the solution-phase structural dynamics of the transporter in distinct functional states by hydrogen/deuterium exchange mass spectrometry (HDX-MS). Our HDX-MS data pinpoint LeuT segments involved in substrate transport and reveal for the first time a comprehensive and detailed view of the solution-phase dynamics associated with transition of the transporter between outward- and inwardfacing configurations in a Na+- as well as K+-dependent manner. The results suggest that partial unwinding of transmembrane helices 1/5/6/7 drive LeuT from a substrate-bound, outward-facing occluded conformation towards an inward-facing open state. We thereby envisage that substrate release is facilitated by formation of two distinct solvent pathways, which mediate access to the Na+ and substrate binding sites. The neurotransmitter:sodium symporter (NSS) family includes prokaryotic and eukaryotic integral membrane proteins that harness the energy stored in the Na+ concentration gradient to transport solutes across the cell membrane (1, 2). Mammalian NSS proteins play an important role in terminating the neurotransmission in the central nervous system by rapid uptake of neurotransmitters against their concentration gradient into the presynaptic neuron or neighboring glial cells (3-5). Numerous neuropsychiatric conditions are associated with altered function of NSS transporters or low neurotransmitter concentrations in the synaptic cleft (2). Many NSS proteins therefore represent favorable targets for therapeutic drugs that act as potent transport inhibitors to increase neurotransmitter levels at the synaptic junction (2, 6). Despite the importance of mammalian NSS proteins in neurobiology and pharmacology, the molecular mechanisms underlying the transport function of mammalian NSS family members have proven difficult to elucidate by traditional methods as the target proteins are unstable in detergent-solubilized form (7) and difficult to purify in required quantity and purity. The prokaryotic NSS protein LeuT from Aquifex aeolicus has emerged as an important structural model to investigate the structure-function relationship of mammalian NSS counterparts (7-9). High-resolution crystal structures of LeuT in distinct functional states (10-13) have established a structural framework and given rise to mechanistic models depicting the substrate transport mechanism (14, 15). LeuT comprises 12 transmembrane segments (TMs) that are interconnected by relatively short extracellular (EL) and intracellular (IL) loops. The first TMs 1-5 share a similar structural repeat with the following TMs 6-10 but are inverted in the membrane plane (10). The specific arrangement of TM domains, also referred to as the ‘5+5’ or LeuT fold (10), has been observed in other NSS proteins (16-18), but also in transporters without sequence homology to LeuT (19, 20), suggesting the possibility of a conserved structural scaffold for many secondary active transporters (8). The primary binding site for the substrate (S1) and residues involved in coordination of two sodium ions (Na1 and Na2) are located approximately halfway across the membrane bilayer in the core of the transporter (10). The characteristic unwound regions of TMs 1 and 6 (providing both TMs with an a- and a b-section) fulfill a dual role in coordinating the sodium ions and forming interactions with the substrate molecule. Individual amino acid residues of TMs 3 and 8 as well as the sodium occupancy in the Na1 site complete the S1 binding pocket. According to the widely embraced ‘alternating access’ model (21, 22), secondary active transporters isomerize between distinct functional states in a substrate-dependent manner. That is, the substrate binding site is alternatively exposed to either the intracellular or extracellular aqueous environment. X-ray crystallography provided structures of LeuT in ‘outward-facing open’ (11, 12), ‘outward-facing occluded’ (10), and ‘inwardfacing open’ (11) conformations and led to the identification of external and internal gating residues in LeuT and related transporters (23-26). Based on these structural snapshots, it has been hypothesized that local and large-scale structural rearrangements are required to regulate the molecular gates and the outward-to-inward transition of the transporter, respectively (11). Combined evidence from crystallographic, functional, and simulation studies suggest that the underlying allosteric couplings are essential for LeuT to function as a symporter (27). Several key aspects of the transport cycle, in particular the molecular mechanism related to the transition of LeuT to the inward-facing open state, have remained controversial and are the subject of extensive debate (8, 28-30). Here, we have studied the substrate translocation mechanism of LeuT by measuring the structural dynamics of the protein in solution as a function of time and substrate/ion composition (e.g. leucine, Na+, K+, and Cs+) by local hydrogen/deuterium exchange mass spectrometry (HDX-MS). The exchange of hydrogen to deuterium (HDX) of backbone amides in a protein is dependent on the presence and stability of hydrogen bonds and thus provides a sensitive probe for higher-order structure and dynamics of the target protein in solution (31, 32). HDX-MS is a non-perturbing technique that allows the collection of structural dynamics data along the entire protein backbone in a coherent manner without the need for sequence alterations or changes to the covalent structure of the protein for labeling (33). Briefly, the target protein is diluted into deuterated buffer and labeled for various time intervals. The isotopic exchange reaction is quenched by lowering pH and temperature to approximately 2.5 and 0 °C, respectively, and the protein subsequently digested using an acid-stable protease (e.g. pepsin). Chromatographic separation and mass analysis of these peptides, in turn, reveal the shift in mass over time (i.e., deuterium uptake) of individual regions of the target protein, which is commonly referred to as local HDX analysis. A more detailed background on the HDX-MS technique and its applications in protein science can be found in several reviews (34-37). Our HDX-MS measurements provide a detailed map of LeuT regions involved in conformational changes during substrate transport (TMs 1a/1b/2/5/6a/6b/7 and interconnecting loops IL1/EL2/EL3/EL4b) and allow for the first time an unperturbed global view on the structural dynamics associated with the outward-to-inward transition of the wild-type transporter in solution. Of special interest, our acquired HDX data suggest that several helices (TMs 1a/5/6/7 and EL4b) are partially unwound in the course of substrate transport and that these unfolding events are dynamically coupled between individual helices that form the substrate binding site and the cytoplasmic gate. Addition of Na+ or the combination of Na+ and leucine destabilized discrete structural motifs on the extracellular side, stabilized the inner gate of LeuT, and substantially reduced the rate of unfolding in individual TM helices relative to the K+-bound state. We envisage that partial unwinding of TM helices accompanies the outward-to-inward isomerization in LeuT and that the same concept might be relevant to related transporters bearing the LeuT fold, hence extending the general model of NSS transport mechanism. Moreover, we provide additional experimental evidence for a potential role of K+ in the transport cycle as K+ selectively shifted the conformational equilibrium of LeuT in a dose-dependent manner towards an inwardfacing state under physiologically relevant concentrations.