Alternating Access Model Research Articles

Substrate-induced conformational dynamics of the dopamine transporter

The dopamine transporter is a member of the neurotransmitter:sodium symporters (NSSs), which are responsible for termination of neurotransmission through Na+-driven reuptake of neurotransmitter from the extracellular space. Experimental evidence elucidating the coordinated conformational rearrangements related to the transport mechanism has so far been limited. Here we probe the global Na+- and dopamine-induced conformational dynamics of the wild-type Drosophila melanogaster dopamine transporter using hydrogen-deuterium exchange mass spectrometry. We identify Na+- and dopamine-induced changes in specific regions of the transporter, suggesting their involvement in protein conformational transitions. Furthermore, we detect ligand-dependent slow cooperative fluctuations of helical stretches in several domains of the transporter, which could be a molecular mechanism that assists in the transporter function. Our results provide a framework for understanding the molecular mechanism underlying the function of NSSs by revealing detailed insight into the state-dependent conformational changes associated with the alternating access model of the dopamine transporter.

Substrate-Induced Conformational Dynamics of the Dopamine Transporter

The dopamine transporter is a member of the neurotransmitter:sodium symporters (NSSs), which are responsible for termination of neurotransmission through Na+-driven reuptake of neurotransmitter from the extracellular space. The coordinated conformational rearrangements related to the transport mechanism have so far been poorly understood. Here we have probed the global Na+- and dopamine-induced conformational dynamics of the wild-type Drosophila melanogaster dopamine transporter using hydrogen-deuterium exchange mass spectrometry. We identify Na+- and dopamine-induced changes in specific regions of the transporter, suggesting their involvement in protein conformational transitions. Furthermore, we detect novel ligand-dependent slow cooperative fluctuations of helical stretches in several domains of the transporter, which could be a novel molecular mechanism that assists in the transporter function. Our results provide a framework for understanding the molecular mechanism underlying the function of NSSs by revealing the first detailed insight into the state-dependent conformational changes associated with the alternating access model of a eukaryotic NSS.

Secondary Active Transporters.

Transport of solutes across biological membranes is essential for cellular life. This process is mediated by membrane transport proteins which move nutrients, waste products, certain drugs and ions into and out of cells. Secondary active transporters couple the transport of substrates against their concentration gradients with the transport of other solutes down their concentration gradients. The alternating access model of membrane transporters and the coupling mechanism of secondary active transporters are introduced in this book chapter. Structural studies have identified typical protein folds for transporters that we exemplify by the major facilitator superfamily (MFS) and LeuT folds. Finally, substrate binding and substrate translocation of the transporters LacY of the MFS and AdiC of the amino acid-polyamine-organocation (APC) superfamily are described.

The C terminus of the bacterial multidrug transporter EmrE couples drug binding to proton release

Ion-coupled transporters must regulate access of ions and substrates into and out of the binding site to actively transport substrates and minimize dissipative leak of ions. Within the single-site alternating access model, competitive substrate binding forms the foundation of ion-coupled antiport. Strict competition between substrates leads to stoichiometric antiport without slippage. However, recent NMR studies of the bacterial multidrug transporter EmrE have demonstrated that this multidrug transporter can simultaneously bind drug and proton, which will affect the transport stoichiometry and efficiency of coupled antiport. Here, we investigated the nature of substrate competition in EmrE using multiple methods to measure proton release upon the addition of saturating concentrations of drug as a function of pH. The resulting proton-release profile confirmed simultaneous binding of drug and proton, but suggested that a residue outside EmrE's Glu-14 binding site may release protons upon drug binding. Using NMR-monitored pH titrations, we trace this drug-induced deprotonation event to His-110, EmrE's C-terminal residue. Further NMR experiments disclosed that the C-terminal tail is strongly coupled to EmrE's drug-binding domain. Consideration of our results alongside those from previous studies of EmrE suggests that this conserved tail participates in secondary gating of EmrE-mediated proton/drug transport, occluding the binding pocket of fully protonated EmrE in the absence of drug to prevent dissipative proton transport.

Carriers, exchangers, and cotransporters in the first 100 years of the Journal of General Physiology.

Transporters, pumps, and channels are proteins that catalyze the movement of solutes across membranes. The single-solute carriers, coupled exchangers, and coupled cotransporters that are collectively known as transporters are distinct from conductive ion channels, water channels, and ATP-hydrolyzing pumps. The main conceptual framework for studying transporter mechanisms is the alternating access model, which comprises substrate binding and release events on each side of the permeability barrier and translocation events involving conformational changes between inward-facing and outward-facing conformational states. In 1948, the Journal of General Physiology began to publish work that focused on the erythrocyte glucose transporter-the first transporter to be characterized kinetically-followed by articles on the rates, stoichiometries, asymmetries, voltage dependences, and regulation of coupled exchangers and cotransporters beginning in the 1960s. After the dawn of cDNA cloning and sequencing in the 1980s, heterologous expression systems and site-directed mutagenesis allowed identification of the functional roles of specific amino acid residues. In the past two decades, structures of transport proteins have made it possible to propose specific models for transporter function at the molecular level. Here, we review the contribution of JGP articles to our current understanding of solute transporter mechanisms. Whether the topic has been kinetics, energetics, regulation, mutagenesis, or structure-based modeling, a common feature of these articles has been a quantitative, mechanistic approach, leading to lasting insights into the functions of transporters.

Light-Driven Sodium-Pumping Rhodopsin: A New Concept of Active Transport.

Ion pumps perform active transport of ions by using energy. The active transport mechanism can be illustrated by the Panama Canal model, which considers two gates and a gain in energy. The Panama Canal model is consistent with the alternating access model that is used to describe active transport, in which the substrate ion is bound, energized, and released. It was generally accepted that energization occurs only for an ion-bound protein but not for an ion-unbound protein. Light-driven proton and chloride pumps, two of the best studied pumps, are represented by the Panama Canal model. In this case, light absorption takes place for the bound state of ions (proton and chloride ions) in the active center (protonated Schiff base of the retinal chromophore). In contrast, a recently discovered light-driven sodium pump, Krokinobacter eikastus rhodopsin 2 (KR2), is a unique active transporter that does not bind the transport substrate, the sodium ion, in its resting state. The molecular architecture and photoreaction cycle of the light-driven sodium pump are very similar to those of proton and chloride pumps, although sodium ions are actively transported without initial binding. Sodium uptake is a diffusive process, but the presence of two gates allows the unidirectional transport of sodium ions. In this sense, the light-driven sodium pump is also represented by a modified Panama Canal model. Current understanding of the light-driven sodium pump is reviewed.

Substrate-induced conformational changes in the nucleotide-binding domains of lipid bilayer–associated P-glycoprotein during ATP hydrolysis

P-glycoprotein (Pgp) is an efflux pump important in multidrug resistance of cancer cells and in determining drug pharmacokinetics. Pgp is a prototype ATP-binding cassette transporter with two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. Conformational changes at the NBDs (the Pgp engines) lead to changes across Pgp transmembrane domains that result in substrate translocation. According to current alternating access models (substrate-binding pocket accessible only to one side of the membrane at a time), binding of ATP promotes NBD dimerization, resulting in external accessibility of the drug-binding site (outward-facing, closed NBD conformation), and ATP hydrolysis leads to dissociation of the NBDs with the subsequent return of the accessibility of the binding site to the cytoplasmic side (inward-facing, open NBD conformation). However, previous work has not investigated these events under near-physiological conditions in a lipid bilayer and in the presence of transport substrate. Here, we used luminescence resonance energy transfer (LRET) to measure the distances between the two Pgp NBDs. Pgp was labeled with LRET probes, reconstituted in lipid nanodiscs, and the distance between the NBDs was measured at 37 °C. In the presence of verapamil, a substrate that activates ATP hydrolysis, the NBDs of Pgp reconstituted in nanodiscs were never far apart during the hydrolysis cycle, and we never observed the NBD-NBD distances of tens of Å that have previously been reported. However, we found two main conformations that coexist in a dynamic equilibrium under all conditions studied. Our observations highlight the importance of performing studies of efflux pumps under near-physiological conditions, in a lipid bilayer, at 37 °C, and during substrate-stimulated hydrolysis.

Mitochondrial Pyruvate Carrier 1 and 2 Heterodimer, In Silico, Models of Plant and Human Complexes

The plant and human mitochondrial pyruvate carrier (MPC) had been studied in the 1970s-1990s providing many predictions on functional protein structure and mechanisms of substrate binding. Genes for human and plant MPC have been identified, but no crystal structure has yet been registered or deposited in a protein data bank. This report describes results for comparisons of structure for human and plant MPC1/2 heterodimer homology models. Key cysteine residues are identified for pyruvate and blocker binding and formation of thiohemiacetal or Michael addition bonds. Evidence is provided for an alternating access model in human, mouse ear-cress, castor and common beans, and corn.

Expression of a human NPT1/SLC17A1 missense variant which increases urate export

ABSTRACTHuman sodium-dependent phosphate cotransporter type 1 (NPT1/SLC17A1) is one of the urate transporters in the kidney. Our recent study revealed that a common missense variant, I269T (rs1165196), of NPT1 decreases the risk of renal underexcretion gout. Moreover, we demonstrated that human NPT1 is localized to the apical membrane of the renal proximal tubule, and that I269T is the gain-of-function variant which increases the NPT1-mediated urate export. However, the mechanism by which I269T variant increases the urate export remains to be clarified. Thus, we performed immunostaining and functional analysis of human NPT1 using the Xenopus oocyte expression system. For comparison of human NPT1 expression levels of oocyte membrane between 269I (wild type) and 269T (variant), immunostaining was performed with anti-human NPT1 antibodies. As a result, we showed that NPT1 I269T variant did not change the human NPT1 membrane expression levels, although NPT1 I269T variant increased the urate transport compared with NPT1 wild type. Combined with the previous report that I269T variant did not induce Km changes but increased the Vmax of urate transport in a proteoliposome system, our findings suggest that I269T variant increases NPT1-mediated urate export without increase of NPT1 expression levels on the membrane. Thus, I269T, a common missense variant of NPT1, might have faster conformation changes than NPT1 wild type in terms of the alternating-access model of transporters, and increases renal urate export in humans.

ATP-dependent substrate transport by the ABC transporter MsbA is proton-coupled.

ATP-binding cassette transporters mediate the transbilayer movement of a vast number of substrates in or out of cells in organisms ranging from bacteria to humans. Current alternating access models for ABC exporters including the multidrug and Lipid A transporter MsbA from Escherichia coli suggest a role for nucleotide as the fundamental source of free energy. These models involve cycling between conformations with inward- and outward-facing substrate-binding sites in response to engagement and hydrolysis of ATP at the nucleotide-binding domains. Here we report that MsbA also utilizes another major energy currency in the cell by coupling substrate transport to a transmembrane electrochemical proton gradient. The dependence of ATP-dependent transport on proton coupling, and the stimulation of MsbA-ATPase by the chemical proton gradient highlight the functional integration of both forms of metabolic energy. These findings introduce ion coupling as a new parameter in the mechanism of this homodimeric ABC transporter.

A current view of serotonin transporters.

Serotonin transporters (SERTs) are largely recognized for one aspect of their function—to transport serotonin back into the presynaptic terminal after its release. Another aspect of their function, however, may be to generate currents large enough to have physiological consequences. The standard model for electrogenic transport is the alternating access model, in which serotonin is transported with a fixed ratio of co-transported ions resulting in net charge per cycle. The alternating access model, however, cannot account for all the observed currents through SERT or other monoamine transporters. Furthermore, SERT agonists like ecstasy or antagonists like fluoxetine generate or suppress currents that the standard model cannot support. Here we survey evidence for a channel mode of transport in which transmitters and ions move through a pore. Available structures for dopamine and serotonin transporters, however, provide no evidence for a pore conformation, raising questions of whether the proposed channel mode actually exists or whether the structural data are perhaps missing a transient open state.

How to move an amphipathic molecule across a lipid bilayer: different mechanisms for different ABC transporters?

Import of β-oxidation substrates into peroxisomes is mediated by ATP binding cassette (ABC) transporters belonging to subfamily D. In order to enter the β-oxidation pathway, fatty acids are activated by conversion to fatty acyl-CoA esters, a reaction which is catalysed by acyl-CoA synthetases (ACSs). Here, we present evidence for an unusual transport mechanism, in which fatty acyl-CoA substrates are accepted by ABC subclass D protein (ABCD) transporters, cleaved by the transporters during transit across the lipid bilayer to release CoA, and ultimately re-esterified in the peroxisome lumen by ACSs which interact with the transporter. We propose that this solves the biophysical problem of moving an amphipathic molecule across the peroxisomal membrane, since the intrinsic thioesterase activity of the transporter permits separate membrane translocation pathways for the hydrophobic fatty acid moiety and the polar CoA moiety. The cleavage/re-esterification mechanism also has the potential to control entry of disparate substrates into the β-oxidation pathway when coupled with distinct peroxisomal ACSs. A different solution to the movement of amphipathic molecules across a lipid bilayer is deployed by the bacterial lipid-linked oligosaccharide (LLO) flippase, PglK, in which the hydrophilic head group and the hydrophobic polyprenyl tail of the substrate are proposed to have distinct translocation pathways but are not chemically separated during transport. We discuss a speculative alternating access model for ABCD proteins based on the mammalian ABC transporter associated with antigen processing (TAP) and compare it to the novel mechanism suggested by the recent PglK crystal structures and biochemical data.

Shared Molecular Mechanisms of Membrane Transporters.

The determination of the crystal structures of small-molecule transporters has shed light on the conformational changes that take place during structural isomerization from outward- to inward-facing states. Rather than using a simple rocking movement of two bundles around a central substrate-binding site, it has become clear that even the most simplistic transporters utilize rearrangements of nonrigid bodies. In the most dramatic cases, one bundle is fixed while the other, structurally divergent, bundle carries the substrate some 18 Å across the membrane, which in this review is termed an elevator alternating-access mechanism. Here, we compare and contrast rocker-switch, rocking-bundle, and elevator alternating-access mechanisms to highlight shared features and novel refinements to the basic alternating-access model.

Elevating the alternating-access model.

50 years ago, Jardetzky proposed the alternating-access model, which has shaped the theoretical understanding of how substrates are carried across cell membranes. Two studies now demonstrate that transporters from distinct families undergo unexpectedly large elevator-like movements and also suggest that an 'elevate and twist' mechanism is a common means of achieving alternating access across the membrane.

Keeping Secondary Transporters under Control: Lessons from a Na+/Ca2+ Exchanger

Secondary-active transporters catalyze the translocation of substances across biological membranes, driven by the electrochemical gradient of one of the transported species, typically Na+ or H+. The mechanism of these transporters has been widely rationalized as a conformational cycle (alternating-access model) that exposes substrate binding sites within the protein to one or the other side of the membrane (or none), but not both simultaneously. The electrochemical gradient drives the cycle in one direction but is not required to interconvert between different conformational states (as opposed to voltage-gated ion-channels). However, active transport does require that the interconversion between outward and inward facing states occur only upon binding of specific substrates, and in uniquely defined stoichiometries. The physical basis of this mechanism of conformational control, however, remains to be established. Here, we address this central question for a prokaryotic homolog of the cardiac Na+/Ca2+ exchanger, NCX_Mj, which transports either three Na+ or one Ca2+ across the membrane. Specifically, we determine how Na+/Ca2+ recognition by the transporter outward-facing state reshapes its conformational free-energy landscape, which we examine via enhanced-sampling molecular-dynamics simulations and crystallographic titration experiments. Our results demonstrate that only upon binding of three Na+ or one Ca2+ can the protein adopt a state occluded to both sides of the membrane, which necessarily precedes the transition to the inward-facing conformation. Binding sites depletion and/or H+ binding, by contrast, eradicates the occluded state population, and induces the opening of hydrated access pathways connecting these sites to the surrounding solution. Interestingly, water in these pathways is markedly polarized, in part explaining the large energetic cost associated with occlusion. This study provides clear evidence that it is by inducing or precluding the formation of occluded, dehydrated states that substrate recognition controls the alternating-access transition in secondary transporters.

Probing Conformational Changes of Secondary Active Transport Proteins

Secondary active transporters (SATs) require large protein conformational changes, which some believe follow the alternating access model. The structure of each characteristic conformational state is important for understanding the complete substrate transport process. However, the structure of certain state(s) can be very difficult to obtain by experiment, while certain enhanced simulation techniques may provide an approach to predict unknown structures. Self-guided Langevin dynamics (SGLD) (Chem. Phys. Lett., 381: 512-518) simulations allows proteins to overcome energy barriers and thus it can be used to search the structures of all the conformational states efficiently by enhancing the conformational sampling of low frequency motion. SGLD accelerates the conformational change while preserves the conformational distribution well. Therefore, this method is applied to study a SAT - lactose permease of E.coli. (LacY), which belongs to major facilitator superfamily (MFS). LacY structures of the cytoplasmic-open and occluded-like structure have been determined. To search the periplasmic-open LacY, SGLD simulations are performed with inward-facing LacY as initial structure, with different residues protonated, with and without ββ-(Galp)2, in the implicit membrane and in an explicit membrane in the NVT ensemble (available recently). The SGLD parameters adjusted are guiding factor and friction factor to control guiding force, and also local average time to set the boundary of low frequency motion to enhance. The preliminary broad SGLD parameters sweep are applied to find reasonable parameter set range for sampling protein structures. Then, a fine parameter sweep is applied to obtain the optimized structure. The effect of each parameter set, the protonation states, and presence of sugar on LacY residue distances and pore radii will be presented. These optimal SGLD parameter values may be applied to study conformational changes of other similar SAT proteins.

Conformational Changes of the Multidrug Transporter P-Glycoprotein in Solution and Lipid Discs

P-glycoprotein (Pgp) exports hundreds of chemically unrelated, hydrophobic compounds out of cells. Since Pgp can greatly affect bioavailability, pharmacokinetics and efficacy of therapeutic drugs, there is great interest in understanding the mechanism by which drugs are extruded. Pgp is a prototype ABC transporter with two nucleotide binding domains (NBDs) that bind and hydrolyze ATP, leading to extensive conformational changes across the transmembrane domains that result in substrate translocation across the cell membrane. According to current alternating access models, binding of ATP promotes NBDs dimerization (outward-facing conformation), and ATP-hydrolysis leads to their dissociation (inward-facing conformation). Despite decades of biochemical/biophysical studies, the mechanism by which the NBDs control these conformational changes is still controversial. We have used luminescence resonance energy transfer (LRET) to measure distances between the two NBDs during the ATP hydrolysis cycle. Pgp was labeled with the LRET probes, reconstituted in a lipid bilayer nanodisc, and the distance between the NBDs was measured at 37°C in nucleotide-free, ATP-bound, MgATP hydrolysis conditions. Pgp in nanodiscs showed increased basal ATPase activity and robust verapamil-stimulated ATP hydrolysis. Our LRET results suggest that the NBDs associate and dissociate in response to ATP binding and hydrolysis. Like the bacterial homolog MsbA, Pgp showed two main conformations (dimeric NBDs and dissociated NBDs) that coexist in equilibrium. However, the proportion of molecules in each conformation and the ATP-hydrolsysis driven changes are distinctly different suggesting that Pgp predominantly resides in the dissociated NBD state. The results are consistent with a recent snapshots analysis at ambient temperature by negative stain electron microscopy. The impact on substrate translocation mechanisms in Pgp vs. MsbA will be discussed. The LRET studies open new possibilities of studying conformational changes at physiological temperatures under native-like conditions.

Identification of Conformation Specific Binder for the NA+/Galactose Transporter

Na+/galactose transporters (SGLTs) are integral membrane proteins, which co-transport Na+ with sugars from the periplasmic space into the cytoplasm. According to the alternating access model for secondary active transporters, these proteins alternate between outward and inward-facing conformations during the transport cycle. The currently available structures from a bacterial homolog of SGLT from Vibrio parahaemolyticus, (vSGLT) are in the substrate-bound inward-occluded and the substrate-free inward-open conformations. Despite much effort, structures of the outward conformations remain elusive.Isolation of distinct conformations of transporter is a major obstacle for X-ray crystallography due to their conformational heterogeneity. Crystallization chaperones based on various protein scaffolds have emerged as a promising tool to increase the crystallization probability of a selected target protein. Sso7d is a highly stable binding protein derived from the hyperthermophilic archaeon Sulfolobus solfataricus. It has a versatile scaffold for generating binding protein for a wide spectrum of targets. Sso7d-derived proteins are far easier to produce in bacteria and due to their small size, they are capable of targeting areas that are not accessible to standards antibodies.To find binders for the SGLT transporter, we screened an Sso7d-based yeast display library using flow cytometry. After several screening steps, we isolated and purified clones that bind the SGLT protein. We are currently attempting to crystallize a complex of the binder with SGLT. Here we present the characterization of Sso7d-binder from the yeast display library using a two-step procedure involving magnetic and FACS-based screening. The micro-molar interaction between Sso7d-binder and vSGLT was determined by analytical gel filtration, SDS-PAGE, microscale thermophoresis and isothermal titration calorimetry. We anticipate this binder will assist in crystallization of the vSGLT protein. Future screening will utilize transport-impaired mutants to select for an outward-facing conformation, which will provide mechanistic insights of the transport mechanism.

Structure and Mechanism of an ATP-Driven Flippase of Lipid-Linked Oligosaccharides

The flipping of lipid-linked oligosaccharides (LLOs) that serve as glycan donors in N-linked protein glycosylation is slow and energetically unfavorable and therefore catalyzed by flippases. This is an essential step during protein N-glycosylation both at the endoplasmic reticulum and similarly at the membrane of bacteria like Campylobacter jejuni. Whereas the identity and potential energy source of the relevant flippase in eukaryotes remains under debate, LLO flipping in C. jejuni is catalyzed by the ABC transporter PglK. We propose a mechanism of PglK-catalyzed LLO flipping based on crystal structures in distinct states, a newly devised in vitro flipping assay, and in vivo studies. We conclude that although PglK can adopt inward- and outward-facing conformations in vitro, only outward-facing states are required for flipping. Whereas the pyrophosphate-oligosaccharide head group of LLO enters the translocation cavity and interacts with positively charged side chains, the lipidic polyprenyl tail binds and activates the transporter but remains exposed to the lipid bilayer during the reaction. Our results provide a framework for understanding lipid-linked oligosaccharide flipping. The proposed mechanism is distinct from the classical alternating-access model applied to other transporters.

Structure and mechanism of an active lipid-linked oligosaccharide flippase.

The flipping of membrane-embedded lipids containing large, polar head groups is slow and energetically unfavourable, and is therefore catalysed by flippases, the mechanisms of which are unknown. A prominent example of a flipping reaction is the translocation of lipid-linked oligosaccharides that serve as donors in N-linked protein glycosylation. In Campylobacter jejuni, this process is catalysed by the ABC transporter PglK. Here we present a mechanism of PglK-catalysed lipid-linked oligosaccharide flipping based on crystal structures in distinct states, a newly devised in vitro flipping assay, and in vivo studies. PglK can adopt inward- and outward-facing conformations in vitro, but only outward-facing states are required for flipping. While the pyrophosphate-oligosaccharide head group of lipid-linked oligosaccharides enters the translocation cavity and interacts with positively charged side chains, the lipidic polyprenyl tail binds and activates the transporter but remains exposed to the lipid bilayer during the reaction. The proposed mechanism is distinct from the classical alternating-access model applied to other transporters.