Outward-facing State Research Articles

Hydrogen-deuterium exchange mass spectrometry captures distinct dynamics upon substrate and inhibitor binding to a transporter

Proton-coupled transporters use transmembrane proton gradients to power active transport of nutrients inside the cell. High-resolution structures often fail to capture the coupling between proton and ligand binding, and conformational changes associated with transport. We combine HDX-MS with mutagenesis and MD simulations to dissect the molecular mechanism of the prototypical transporter XylE. We show that protonation of a conserved aspartate triggers conformational transition from outward-facing to inward-facing state. This transition only occurs in the presence of substrate xylose, while the inhibitor glucose locks the transporter in the outward-facing state. MD simulations corroborate the experiments by showing that only the combination of protonation and xylose binding, and not glucose, sets up the transporter for conformational switch. Overall, we demonstrate the unique ability of HDX-MS to distinguish between the conformational dynamics of inhibitor and substrate binding, and show that a specific allosteric coupling between substrate binding and protonation is a key step to initiate transport.

Na+-dependent gate dynamics and electrostatic attraction ensure substrate coupling in glutamate transporters.

Excitatory amino acid transporters (EAATs) harness [Na+], [K+], and [H+] gradients for fast and efficient glutamate removal from the synaptic cleft. Since each glutamate is cotransported with three Na+ ions, [Na+] gradients are the predominant driving force for glutamate uptake. We combined all-atom molecular dynamics simulations, fluorescence spectroscopy, and x-ray crystallography to study Na+:substrate coupling in the EAAT homolog GltPh A lipidic cubic phase x-ray crystal structure of wild-type, Na+-only bound GltPh at 2.5-Å resolution revealed the fully open, outward-facing state primed for subsequent substrate binding. Simulations and kinetic experiments established that only the binding of two Na+ ions to the Na1 and Na3 sites ensures complete HP2 gate opening via a conformational selection-like mechanism and enables high-affinity substrate binding via electrostatic attraction. The combination of Na+-stabilized gate opening and electrostatic coupling of aspartate to Na+ binding provides a constant Na+:substrate transport stoichiometry over a broad range of neurotransmitter concentrations.

What monomeric nucleotide binding domains can teach us about dimeric ABC proteins

The classic conceptualization of ATP binding cassette (ABC) transporter function is an ATP-dependent conformational change coupled to transport of a substrate across a biological membrane via the transmembrane domains (TMDs). The binding of two ATP molecules within the transporter's two nucleotide binding domains (NBDs) induces their dimerization. Despite retaining the ability to bind nucleotides, isolated NBDs frequently fail to dimerize. ABC proteins without a TMD, for example ABCE and ABCF, have NBDs tethered via elaborate linkers, further supporting that NBD dimerization does not readily occur for isolated NBDs. Intriguingly, even in full-length transporters, the NBD-dimerized, outward-facing state is not as frequently observed as might be expected. This leads to questions regarding what drives NBD interaction and the role of the TMDs or linkers. Understanding the NBD-nucleotide interaction and the subsequent NBD dimerization is thus pivotal for understanding ABC transporter activity in general. Here, we hope to provide new insights into ABC protein function by discussing the perplexing issue of (missing) NBD dimerization in isolation and in the context of full-length ABC proteins.

A structural framework for unidirectional transport by a bacterial ABC exporter

The ATP-binding cassette (ABC) transporter of mitochondria (Atm1) mediates iron homeostasis in eukaryotes, while the prokaryotic homolog from Novosphingobium aromaticivorans (NaAtm1) can export glutathione derivatives and confer protection against heavy-metal toxicity. To establish the structural framework underlying the NaAtm1 transport mechanism, we determined eight structures by X-ray crystallography and single-particle cryo-electron microscopy in distinct conformational states, stabilized by individual disulfide crosslinks and nucleotides. As NaAtm1 progresses through the transport cycle, conformational changes in transmembrane helix 6 (TM6) alter the glutathione-binding site and the associated substrate-binding cavity. Significantly, kinking of TM6 in the post-ATP hydrolysis state stabilized by MgADPVO4 eliminates this cavity, precluding uptake of glutathione derivatives. The presence of this cavity during the transition from the inward-facing to outward-facing conformational states, and its absence in the reverse direction, thereby provide an elegant and conceptually simple mechanism for enforcing the export directionality of transport by NaAtm1. One of the disulfide crosslinked NaAtm1 variants characterized in this work retains significant glutathione transport activity, suggesting that ATP hydrolysis and substrate transport by Atm1 may involve a limited set of conformational states with minimal separation of the nucleotide-binding domains in the inward-facing conformation.

Expanding the membrane-protein NMR toolkit.

An NMR method to monitor conformational states of challenging large protein targets is described. The method, which can be used to evaluate distances between two labels and to measure conformational exchange rates, revealed an unanticipated outward-facing state in a glutamate transporter.

Use of paramagnetic 19F NMR to monitor domain movement in a glutamate transporter homolog.

In proteins where conformational changes are functionally important, the number of accessible states and their dynamics are often difficult to establish. Here we describe a novel 19F-NMR spectroscopy approach to probe dynamics of large membrane proteins. We labeled a glutamate transporter homologue with a 19F probe via cysteine chemistry and with a Ni2+ ion via chelation by a di-histidine motif. We used distance-dependent enhancement of the longitudinal relaxation of 19F nuclei by the paramagnetic metal to assign the observed resonances. We identified two outward- and one inward-facing states of the transporter, in which the substrate-binding site is near the extracellular and intracellular solutions, respectively. We then resolved the structure of the unanticipated second outward-facing state by Cryo-EM. Finally, we showed that the rates of the conformational exchange are accessible from measurements of the metal-enhanced longitudinal relaxation of 19F nuclei.

In vivo FRET analyses reveal a role of ATP hydrolysis–associated conformational changes in human P-glycoprotein

P-glycoprotein (P-gp; also known as MDR1 or ABCB1) is an ATP-driven multidrug transporter that extrudes various hydrophobic toxic compounds to the extracellular space. P-gp consists of two transmembrane domains (TMDs) that form the substrate translocation pathway and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. At least two P-gp states are required for transport. In the inward-facing (pre-drug transport) conformation, the two NBDs are separated, and the two TMDs are open to the intracellular side; in the outward-facing (post-drug transport) conformation, the NBDs are dimerized, and the TMDs are slightly open to the extracellular side. ATP binding and hydrolysis cause conformational changes between the inward-facing and the outward-facing conformations, and these changes help translocate substrates across the membrane. However, how ATP hydrolysis is coupled to these conformational changes remains unclear. In this study, we used a new FRET sensor that detects conformational changes in P-gp to investigate the role of ATP binding and hydrolysis during the conformational changes of human P-gp in living HEK293 cells. We show that ATP binding causes the conformational change to the outward-facing state and that ATP hydrolysis and subsequent release of γ-phosphate from both NBDs allow the outward-facing state to return to the original inward-facing state. The findings of our study underscore the utility of using FRET analysis in living cells to elucidate the function of membrane proteins such as multidrug transporters.

Microscopic View of Structural Transitions of Glucose Transporters

Glucose transporters (GLUTs) facilitate the diffusion of glucose across cellular membranes, thereby playing an essential role in cell metabolism. In this context, GLUT3 has been drawing special attention due to its reported over-expression in tumors (due to their increased demand for glucose) and, therefore, as a potential drug target in cancer therapy. The transport cycle of GLUT3 consists of large-scale structural changes between the outward-facing (OF) and inward-facing (IF) states. However, the molecular details of these structural changes are still unknown.

A Kinetic Description of Cytosolic K+ Binding to the Human Sertotonin Transporter Under Turnover Conditions

The human serotonin transporter (hSERT) belongs to the neurotransmitter:sodium symporter (NSS) family, which regulate neurotransmission by re-uptaking the released neurotransmitters into the presynaptic neuron. During the transport cycle, the transporter alternates between the outward-facing (OF) and inward-facing (IF) states to translocate the substrate across the membrane. Central to the cycle in hSERT is the OF-to-IF state transition, during which 5-HT (serotonin) is inwardly symported in a Na+- and Cl--dependent manner, followed by cytosolic-K+-binding-triggered returning to the OF state. K+ binding might act as a “kinetic decision point” by frustrating the outward transport of 5-HT, namely, the reverse of the physiological direction. However, the mechanism of such K+ regulation remains elusive. Moreover, the conventional transport stoichiometry (15-HTin:1Na+in:1Cl−in:1K+out) has been challenged by recent studies showing that Cl- might remain bound to hSERT during the entire cycle. To explore the role of cytosolic K+ binding to IF hSERT, we have constructed a Markov State Model from an extensive set of molecular dynamics simulations. Starting with the IF state, the system is modeled under turnover conditions, i.e., post-5-HT-release and exposed to cytosolic K+ concentration, resulting in 50 independent, 200-ns replicas. More than half of the trajectories capture spontaneous K+ binding to the Na2 site, a conserved Na+ binding site within NSSs deemed to control the transporter conformational changes and substrate release. Furthermore, consistent with recent experimental results, Cl- was found to remain bound in the majority of the ensembles, questioning the contribution of the Cl- gradient to the substrate transport. Together, these results characterize the previously unknown K+ site in hSERT and present a quantitative kinetic description of K+ binding, shedding light on the mechanism of the ion-dependence of the IF-to-OF transition that resets the transport cycle.

Spin-labeled nanobodies as protein conformational reporters for electron paramagnetic resonance in cellular membranes

Nanobodies are emerging tools in a variety of fields such as structural biology, cell imaging, and drug discovery. Here we pioneer the use of their spin-labeled variants as reporters of conformational dynamics of membrane proteins using DEER spectroscopy. At the example of the bacterial ABC transporter TM287/288, we show that two gadolinium-labeled nanobodies allow us to quantify, via analysis of the modulation depth of DEER traces, the fraction of transporters adopting the outward-facing state under different experimental conditions. Additionally, we quantitatively follow the interconversion from the outward- to the inward-facing state in the conformational ensemble under ATP turnover conditions. We finally show that the specificity of the nanobodies for the target protein allows the direct attainment of structural information on the wild-type TM287/288 expressed in cellular membranes without the need to purify or label the investigated membrane protein.

Mechanistic basis of the inhibition of SLC11/NRAMP-mediated metal ion transport by bis-isothiourea substituted compounds.

In humans, the divalent metal ion transporter-1 (DMT1) mediates the transport of ferrous iron across the apical membrane of enterocytes. Hence, its inhibition could be beneficial for the treatment of iron overload disorders. Here we characterize the interaction of aromatic bis-isothiourea-substituted compounds with human DMT1 and its prokaryotic homologue EcoDMT. Both transporters are inhibited by a common competitive mechanism with potencies in the low micromolar range. The crystal structure of EcoDMT in complex with a brominated derivative defines the binding of the inhibitor to an extracellular pocket of the transporter in direct contact with residues of the metal ion coordination site, thereby interfering with substrate loading and locking the transporter in its outward-facing state. Mutagenesis and structure-activity relationships further support the observed interaction mode and reveal species-dependent differences between pro- and eukaryotic transporters. Together, our data provide the first detailed mechanistic insight into the pharmacology of SLC11/NRAMP transporters.

Locking Two Rigid-body Bundles in an Outward-Facing Conformation: The Ion-coupling Mechanism in a LeuT-fold Transporter

Secondary active transporters use electrochemical gradient of ions to fuel the “uphill” translocation of the substrate following the alternating-access model. The coupling of ions to conformational dynamics of the protein remains one of the least characterized aspects of the transporter function. We employ extended molecular dynamics (MD) simulations to examine the Na+-binding effects on the structure and dynamics of a LeuT-fold, Na+-coupled secondary transporter (Mhp1) in its major conformational states, i.e., the outward-facing (OF) and inward-facing (IF) states, as well as on the OF ↔ IF state transition. Microsecond-long, unbiased MD simulations illustrate that Na+ stabilizes an OF conformation favorable for substrate association, by binding to a highly conserved site at the interface between the two helical bundles and restraining their relative position and motion. Furthermore, a special-protocol biased simulation for state transition suggests that Na+ binding hinders the OF ↔ IF transition. These synergistic Na+-binding effects allosterically couple the ion and substrate binding sites and modify the kinetics of state transition, collectively increasing the lifetime of an OF conformation with high substrate affinity, thereby facilitating substrate recruitment from a low-concentration environment. Based on the similarity between our findings for Mhp1 and experimental reports on LeuT, we propose that this model may represent a general Na+-coupling mechanism among LeuT-fold transporters.

Quantifying secondary transport at single-molecule resolution.

Secondary active transporters, which are vital for a multitude of physiological processes, use the energy of electrochemical ion gradients to power substrate transport across cell membranes1,2. Efforts to investigate their mechanisms of action have been hampered by their slow transport rates and the inherent limitations of ensemble methods. Here we quantify the activity of individual MhsT transporters, which are representative of the neurotransmitter:sodium symporter family of secondary transporters3, by imaging the transport of individual substrate molecules across lipid bilayers at both single- and multi-turnover resolution. We show that MhsT is active only when physiologically oriented and that the rate-limiting step of the transport cycle varies with the nature of the transported substrate. These findings are consistent with an extracellular allosteric substrate-binding site that modulates the rate-limiting aspects of the transport mechanism4,5, including the rate at which the transporter returns to an outward-facing state after the transported substrate is released.

In silico characterization of residues essential for substrate binding of human cystine transporter, xCT.

xCT is a sodium-independent amino acid antiporter that imports L-cystine and exports L-glutamate in a 1:1 ratio. It is a component of heterodimeric amino acid transporter system Xc- working at the cross-roads of maintaining neurological processes and regulating antioxidant defense. The transporter has 12 transmembrane domains with intracellular N- and C-termini, and like other transporter proteins can undergo various conformational changes while switching the ligand accessibilities from intracellular to extracellular site. In the present study, we generated two homology models of human xCT in two distinct conformations: inward-facing occluded state and outward-facing open state. Our results indicated the substrate translocation channel composed of transmembrane helices TMs 1, 3, 6, 8, and 10. We docked anionic L-cystine and L-glutamate within the cavities to assess the two distinct binding scenarios for xCT as antiporter. We also assessed the interactions between the ligands and transporter and observed that ligands bind to similar residues within the channel. Using MM-PBSA/MM-GBSA approach, we computed the binding energies of these ligands to different conformational states. Cystine and glutamate bind xCT with favorable binding energies, with more favorable binding observed in inward occluded state than in outward open state. We further computed the residue-wise decomposition of these binding energies and identified the residues as essential for substrate binding/permeation. Filtering the residues that form favorable energetic contributions to the ligand binding in both the states, our studies suggest T56, A60, R135, A138, V141, Y244, A247, F250, S330, L392, and R396 as critical residues for ligand binding as well as ligand transport for any conformational state adopted by xCT during its transport cycle. .Graphical Abstract.

Dynamics of ABC Transporter P-glycoprotein in Three Conformational States

We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to obtain a comprehensive view of transporter dynamics (85.8% sequence coverage) occurring throughout the multidrug efflux transporter P-glycoprotein (P-gp) in three distinct conformational states: predominantly inward-facing apo P-gp, pre-hydrolytic (E552Q/E1197Q) P-gp bound to Mg+2-ATP, and outward-facing P-gp bound to Mg+2-ADP-VO4−3. Nucleotide affinity was measured with bio-layer interferometry (BLI), which yielded kinetics data that fit a two Mg+2-ATP binding-site model. This model has one high affinity site (3.2 ± 0.3 µM) and one low affinity site (209 ± 25 µM). Comparison of deuterium incorporation profiles revealed asymmetry between the changes undergone at the critical interfaces where nucleotide binding domains (NBDs) contact intracellular helices (ICHs). In the pre-hydrolytic state, both interfaces between ICHs and NBDs decreased exchange to similar extents relative to inward-facing P-gp. In the outward-facing state, the ICH-NBD1 interface showed decreased exchange, while the ICH-NBD2 interface showed less of an effect. The extracellular loops (ECLs) showed reduced deuterium uptake in the pre-hydrolytic state, consistent with an occluded conformation. While in the outward-facing state, increased ECL exchange corresponding to EC domain opening was observed. These findings point toward asymmetry between both NBDs, and they suggest that pre-hydrolytic P-gp occupies an occluded conformation.

Exploring Conformational Transitions and Free-Energy Profiles of Proton-Coupled Oligopeptide Transporters.

Proteins involved in peptide uptake and transport belong to the proton-coupled oligopeptide transporter (POT) family. Crystal structures of POT family members reveal a common fold consisting of two domains of six transmembrane α helices that come together to form a "V" shaped transporter with a central substrate binding site. Proton-coupled oligopeptide transporters operate through an alternate access mechanism, where the membrane transporter undergoes global conformational changes, alternating between inward-facing (IF), outward-facing (OF), and occluded (OC) states. Conformational transitions are promoted by proton and ligand binding; however, due to the absence of crystallographic models of the outward-open state, the role of H+ and ligands is still not fully understood. To provide a comprehensive picture of the POT conformational equilibrium, conventional and enhanced sampling molecular dynamics simulations of PepTst in the presence or absence of ligand and protonation were performed. Free-energy profiles of the conformational variability of PepTst were obtained from microseconds of adaptive biasing force (ABF) simulations. Our results reveal that both proton and ligand significantly change the conformational free-energy landscape. In the absence of ligand and protonation, only transitions involving IF and OC states are allowed. After protonation of the residue Glu300, the wider free-energy well for Glu300 protonated PepTst indicates a greater conformational variability relative to the apo system, and OF conformations became accessible. For the Glu300 protonated Holo-PepTst, the presence of a second free-energy minimum suggests that OF conformations are not only accessible, but also stable. The differences in the free-energy profiles demonstrate that transitions toward outward-facing conformation occur only after protonation, which is likely the first step in the mechanism of peptide transport. Our extensive ABF simulations provide a fully atomic description of all states of the transport process, offering a model for the alternating access mechanism and how protonation and ligand control the conformational changes.

Cryo-EM structures of the human glutamine transporter SLC1A5 (ASCT2) in the outward-facing conformation.

Alanine-serine-cysteine transporter 2 (ASCT2, SLC1A5) is the primary transporter of glutamine in cancer cells and regulates the mTORC1 signaling pathway. The SLC1A5 function involves finely tuned orchestration of two domain movements that include the substrate-binding transport domain and the scaffold domain. Here, we present cryo-EM structures of human SLC1A5 and its complex with the substrate, L-glutamine in an outward-facing conformation. These structures reveal insights into the conformation of the critical ECL2a loop which connects the two domains, thus allowing rigid body movement of the transport domain throughout the transport cycle. Furthermore, the structures provide new insights into substrate recognition, which involves conformational changes in the HP2 loop. A putative cholesterol binding site was observed near the domain interface in the outward-facing state. Comparison with the previously determined inward-facing structure of SCL1A5 provides a basis for a more integrated understanding of substrate recognition and transport mechanism in the SLC1 family.

A one-gate elevator mechanism for the human neutral amino acid transporter ASCT2

The human Alanine Serine Cysteine Transporter 2 (ASCT2) is a neutral amino acid exchanger that belongs to the solute carrier family 1 (SLC1A). SLC1A structures have revealed an elevator-type mechanism, in which the substrate is translocated across the cell membrane by a large displacement of the transport domain, whereas a small movement of hairpin 2 (HP2) gates the extracellular access to the substrate-binding site. However, it has remained unclear how substrate binding and release is gated on the cytoplasmic side. Here, we present an inward-open structure of the human ASCT2, revealing a hitherto elusive SLC1A conformation. Strikingly, the same structural element (HP2) serves as a gate in the inward-facing as in the outward-facing state. The structures reveal that SLC1A transporters work as one-gate elevators. Unassigned densities near the gate and surrounding the scaffold domain, may represent potential allosteric binding sites, which could guide the design of lipidic-inhibitors for anticancer therapy.

Inward-facing conformation of a multidrug resistance MATE family transporter

Multidrug and toxic compound extrusion (MATE) transporters mediate excretion of xenobiotics and toxic metabolites, thereby conferring multidrug resistance in bacterial pathogens and cancer cells. Structural information on the alternate conformational states and knowledge of the detailed mechanism of MATE transport are of great importance for drug development. However, the structures of MATE transporters are only known in V-shaped outward-facing conformations. Here, we present the crystal structure of a MATE transporter from Pyrococcus furiosus (PfMATE) in the long-sought-after inward-facing state, which was obtained after crystallization in the presence of native lipids. Transition from the outward-facing state to the inward-facing state involves rigid body movements of transmembrane helices (TMs) 2-6 and 8-12 to form an inverted V, facilitated by a loose binding of TM1 and TM7 to their respective bundles and their conformational flexibility. The inward-facing structure of PfMATE in combination with the outward-facing one supports an alternating access mechanism for the MATE family transporters.

The extracellular gate shapes the energy profile of an ABC exporter

ABC exporters harness the energy of ATP to pump substrates across membranes. Extracellular gate opening and closure are key steps of the transport cycle, but the underlying mechanism is poorly understood. Here, we generated a synthetic single domain antibody (sybody) that recognizes the heterodimeric ABC exporter TM287/288 exclusively in the presence of ATP, which was essential to solve a 3.2 Å crystal structure of the outward-facing transporter. The sybody binds to an extracellular wing and strongly inhibits ATPase activity by shifting the transporter’s conformational equilibrium towards the outward-facing state, as shown by double electron-electron resonance (DEER). Mutations that facilitate extracellular gate opening result in a comparable equilibrium shift and strongly reduce ATPase activity and drug transport. Using the sybody as conformational probe, we demonstrate that efficient extracellular gate closure is required to dissociate the NBD dimer after ATP hydrolysis to reset the transporter back to its inward-facing state.