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Choline is an essential nutrient that the human body needs in vast quantities for cell membrane synthesis, epigenetic modification and neurotransmission. The brain has a particularly high demand for choline, but how it enters the brain remains unknown1-3. The major facilitator superfamily transporter FLVCR1 (also known as MFSD7B or SLC49A1) was recently determined to be a choline transporter but is not highly expressed at the blood-brain barrier, whereas the related protein FLVCR2 (also known as MFSD7C or SLC49A2) is expressed in endothelial cells at the blood-brain barrier4-7. Previous studies have shown that mutations in human Flvcr2 cause cerebral vascular abnormalities, hydrocephalus and embryonic lethality, but the physiological role of FLVCR2 is unknown4,5. Here we demonstrate both in vivo and in vitro that FLVCR2 is a BBB choline transporter and is responsible for the majority of choline uptake into the brain. We also determine the structures of choline-bound FLVCR2 in both inward-facing and outward-facing states using cryo-electron microscopy. These results reveal how the brain obtains choline and provide molecular-level insights into how FLVCR2 binds choline in an aromatic cage and mediates its uptake. Our work could provide a novel framework for the targeted delivery of therapeutic agents into the brain.
Assuntos
Encéfalo , Colina , Proteínas de Membrana Transportadoras , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Colina/metabolismo , Microscopia Crioeletrônica , Técnicas In Vitro , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/ultraestrutura , Modelos MolecularesRESUMO
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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The human GABAB receptor-a member of the class C family of G-protein-coupled receptors (GPCRs)-mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction1. A unique GPCR that is known to require heterodimerization for function2-6, the GABAB receptor has two subunits, GABAB1 and GABAB2, that are structurally homologous but perform distinct and complementary functions. GABAB1 recognizes orthosteric ligands7,8, while GABAB2 couples with G proteins9-14. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail15. Although the VFT heterodimer structure has been resolved16, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here we present a near full-length structure of the GABAB receptor, captured in an inactive state by cryo-electron microscopy. Our structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. We also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique 'intersubunit latch' within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity.
Assuntos
Microscopia Crioeletrônica , Receptores de GABA-B/química , Receptores de GABA-B/ultraestrutura , Cálcio/metabolismo , Etanolaminas/química , Etanolaminas/metabolismo , Humanos , Ligantes , Modelos Moleculares , Fosforilcolina/química , Fosforilcolina/metabolismo , Domínios Proteicos , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Receptores de GABA-B/metabolismo , Relação Estrutura-AtividadeRESUMO
Members of the nucleobase/ascorbic acid transporter (NAT) gene family are found in all kingdoms of life. In mammals, the concentrative uptake of ascorbic acid (vitamin C) by members of the NAT family is driven by the Na+ gradient, while the uptake of nucleobases in bacteria is powered by the H+ gradient. Here, we report the structure and function of PurTCp, a NAT family member from Colwellia psychrerythraea. The structure of PurTCp was determined to 2.80 Å resolution by X-ray crystallography. PurTCp forms a homodimer, and each protomer has 14 transmembrane segments folded into a transport domain (core domain) and a scaffold domain (gate domain). A purine base is present in the structure and defines the location of the substrate binding site. Functional studies reveal that PurTCp transports purines but not pyrimidines and that purine binding and transport is dependent on the pH. Mutation of a conserved aspartate residue close to the substrate binding site reveals the critical role of this residue in H+-dependent transport of purines. Comparison of the PurTCp structure with transporters of the same structural fold suggests that rigid-body motions of the substrate-binding domain are central for substrate translocation across the membrane.
Assuntos
Ácido Ascórbico , Purinas , Animais , Transporte Biológico , Purinas/metabolismo , Mutação , Sítios de Ligação , Ácido Ascórbico/metabolismo , Mamíferos/metabolismoRESUMO
MhsT of Bacillus halodurans is a transporter of hydrophobic amino acids and a homologue of the eukaryotic SLC6 family of Na+ -dependent symporters for amino acids, neurotransmitters, osmolytes, or creatine. The broad range of transported amino acids by MhsT prompted the investigation of the substrate recognition mechanism. Here, we report six new substrate-bound structures of MhsT, which, in conjunction with functional studies, reveal how the flexibility of a Gly-Met-Gly (GMG) motif in the unwound region of transmembrane segment 6 (TM6) is central for the recognition of substrates of different size by tailoring the binding site shape and volume. MhsT mutants, harboring substitutions within the unwound GMG loop and substrate binding pocket that mimick the binding sites of eukaryotic SLC6A18/B0AT3 and SLC6A19/B0AT1 transporters of neutral amino acids, exhibited impaired transport of aromatic amino acids that require a large binding site volume. Conservation of a general (G/A/C)ΦG motif among eukaryotic members of SLC6 family suggests a role for this loop in a common mechanism for substrate recognition and translocation by SLC6 transporters of broad substrate specificity.
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Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Bacillus/metabolismo , Sítios de Ligação/fisiologia , Conformação Proteica , Especificidade por Substrato/fisiologiaRESUMO
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.
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Aminoácidos/metabolismo , Imagem Individual de Molécula , Simportadores/análise , Simportadores/metabolismo , Sítio Alostérico , Aminoácidos/análise , Aminoácidos/química , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Transporte Biológico , Sobrevivência Celular , Transferência Ressonante de Energia de Fluorescência , Interações Hidrofóbicas e Hidrofílicas , Cinética , Bicamadas Lipídicas/metabolismo , Conformação Proteica , Simportadores/químicaRESUMO
The emergence and spread of drug-resistant Plasmodium falciparum impedes global efforts to control and eliminate malaria. For decades, treatment of malaria has relied on chloroquine (CQ), a safe and affordable 4-aminoquinoline that was highly effective against intra-erythrocytic asexual blood-stage parasites, until resistance arose in Southeast Asia and South America and spread worldwide1. Clinical resistance to the chemically related current first-line combination drug piperaquine (PPQ) has now emerged regionally, reducing its efficacy2. Resistance to CQ and PPQ has been associated with distinct sets of point mutations in the P. falciparum CQ-resistance transporter PfCRT, a 49-kDa member of the drug/metabolite transporter superfamily that traverses the membrane of the acidic digestive vacuole of the parasite3-9. Here we present the structure, at 3.2 Å resolution, of the PfCRT isoform of CQ-resistant, PPQ-sensitive South American 7G8 parasites, using single-particle cryo-electron microscopy and antigen-binding fragment technology. Mutations that contribute to CQ and PPQ resistance localize primarily to moderately conserved sites on distinct helices that line a central negatively charged cavity, indicating that this cavity is the principal site of interaction with the positively charged CQ and PPQ. Binding and transport studies reveal that the 7G8 isoform binds both drugs with comparable affinities, and that these drugs are mutually competitive. The 7G8 isoform transports CQ in a membrane potential- and pH-dependent manner, consistent with an active efflux mechanism that drives CQ resistance5, but does not transport PPQ. Functional studies on the newly emerging PfCRT F145I and C350R mutations, associated with decreased PPQ susceptibility in Asia and South America, respectively6,9, reveal their ability to mediate PPQ transport in 7G8 variant proteins and to confer resistance in gene-edited parasites. Structural, functional and in silico analyses suggest that distinct mechanistic features mediate the resistance to CQ and PPQ in PfCRT variants. These data provide atomic-level insights into the molecular mechanism of this key mediator of antimalarial treatment failures.
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Microscopia Crioeletrônica , Resistência a Medicamentos/efeitos dos fármacos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/ultraestrutura , Plasmodium falciparum/química , Proteínas de Protozoários/química , Proteínas de Protozoários/ultraestrutura , Cloroquina/metabolismo , Cloroquina/farmacologia , Resistência a Medicamentos/genética , Concentração de Íons de Hidrogênio , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Mutação , Plasmodium falciparum/genética , Plasmodium falciparum/ultraestrutura , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Quinolinas/metabolismo , Quinolinas/farmacologiaRESUMO
Multidrug-resistant Plasmodium falciparum parasites have emerged in Cambodia and neighboring countries in Southeast Asia, compromising the efficacy of first-line antimalarial combinations. Dihydroartemisinin + piperaquine (PPQ) treatment failure rates have risen to as high as 50% in some areas in this region. For PPQ, resistance is driven primarily by a series of mutant alleles of the P. falciparum chloroquine resistance transporter (PfCRT). PPQ resistance was reported in China three decades earlier, but the molecular driver remained unknown. Herein, we identify a PPQ-resistant pfcrt allele (China C) from Yunnan Province, China, whose genotypic lineage is distinct from the PPQ-resistant pfcrt alleles currently observed in Cambodia. Combining gene editing and competitive growth assays, we report that PfCRT China C confers moderate PPQ resistance while re-sensitizing parasites to chloroquine (CQ) and incurring a fitness cost that manifests as a reduced rate of parasite growth. PPQ transport assays using purified PfCRT isoforms, combined with molecular dynamics simulations, highlight differences in drug transport kinetics and in this transporter's central cavity conformation between China C and the current Southeast Asian PPQ-resistant isoforms. We also report a novel computational model that incorporates empirically determined fitness landscapes at varying drug concentrations, combined with antimalarial susceptibility profiles, mutation rates, and drug pharmacokinetics. Our simulations with PPQ-resistant or -sensitive parasite lines predict that a three-day regimen of PPQ combined with CQ can effectively clear infections and prevent the evolution of PfCRT variants. This work suggests that including CQ in combination therapies could be effective in suppressing the evolution of PfCRT-mediated multidrug resistance in regions where PPQ has lost efficacy.
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Artemisininas/uso terapêutico , Cloroquina/uso terapêutico , Resistência a Múltiplos Medicamentos , Proteínas de Membrana Transportadoras/genética , Piperazinas/uso terapêutico , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Quinolinas/uso terapêutico , Alelos , Animais , Antimaláricos/uso terapêutico , Simulação por Computador , Humanos , Malária Falciparum/parasitologiaRESUMO
The neurotransmitter:sodium symporter (NSS) homolog LeuT from Aquifex aeolicus has proven to be a valuable model for studying the transport mechanism of the NSS family. Crystal structures have captured LeuT in key conformations visited during the transport cycle, allowing for the construction of a nearly complete model of transport, with much of the conformational dynamics studied by computational simulations. Here, we report crystal structures of LeuT representing new intermediate conformations between the outward-facing open and occluded states. These structures, combined with binding and accessibility studies, reveal details of conformational dynamics that can follow substrate binding at the central substrate binding site (S1) of LeuT in outward-facing states, suggesting a potential competition for direction between the outward-open and outward-occluded states at this stage during substrate transport. Our structures further support an intimate interplay between the protonation state of Glu290 and binding of Na1 that may ultimately regulate the outward-open-to-occluded transition.
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Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/química , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismo , Sódio/metabolismo , Aquifex/metabolismo , Cristalografia por Raios X , Leucina/metabolismo , Simulação de Dinâmica Molecular , Conformação Proteica , Simportadores/química , Simportadores/metabolismo , TermodinâmicaRESUMO
Neurotransmitter:sodium symporters (NSSs) in the SLC6 family terminate neurotransmission by coupling the thermodynamically favorable transport of ions to the thermodynamically unfavorable transport of neurotransmitter back into presynaptic neurons. Results from many structural, functional, and computational studies on LeuT, a bacterial NSS homolog, have provided critical insight into the mechanism of sodium-coupled transport, but the mechanism underlying substrate-specific transport rates is still not understood. We present a combination of molecular dynamics simulations, single-molecule fluorescence resonance energy transfer (smFRET) imaging, and measurements of Na+ binding and substrate transport that reveals an allosteric substrate specificity mechanism. In this mechanism, residues F259 and I359 in the substrate binding pocket couple the binding of substrate to Na+ release from the Na2 site by allosterically modulating the stability of a partially open, inward-facing state. We propose a model for transport selectivity in which residues F259 and I359 act as a volumetric sensor that inhibits the transport of bulky amino acids.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismo , Regulação Alostérica , Transporte Biológico , Glicina/metabolismo , Mutação/genética , Fenilalanina/metabolismo , Estabilidade Proteica , Rotação , Sódio/metabolismo , Especificidade por SubstratoRESUMO
Although long-studied in the central nervous system, there is increasing evidence that dopamine (DA) has important roles in the periphery including in metabolic regulation. Insulin-secreting pancreatic ß-cells express the machinery for DA synthesis and catabolism, as well as all five DA receptors. In these cells, DA functions as a negative regulator of glucose-stimulated insulin secretion (GSIS), which is mediated by DA D2-like receptors including D2 (D2R) and D3 (D3R) receptors. However, the fundamental mechanisms of DA synthesis, storage, release, and signaling in pancreatic ß-cells and their functional relevance in vivo remain poorly understood. Here, we assessed the roles of the DA precursor L-DOPA in ß-cell DA synthesis and release in conjunction with the signaling mechanisms underlying DA's inhibition of GSIS. Our results show that the uptake of L-DOPA is essential for establishing intracellular DA stores in ß-cells. Glucose stimulation significantly enhances L-DOPA uptake, leading to increased DA release and GSIS reduction in an autocrine/paracrine manner. Furthermore, D2R and D3R act in combination to mediate dopaminergic inhibition of GSIS. Transgenic knockout mice in which ß-cell D2R or D3R expression is eliminated exhibit diminished DA secretion during glucose stimulation, suggesting a new mechanism where D2-like receptors modify DA release to modulate GSIS. Lastly, ß-cell-selective D2R knockout mice exhibit marked postprandial hyperinsulinemia in vivo. These results reveal that peripheral D2R and D3R receptors play important roles in metabolism through their inhibitory effects on GSIS. This opens the possibility that blockade of peripheral D2-like receptors by drugs including antipsychotic medications may significantly contribute to the metabolic disturbances observed clinically.
Assuntos
Dopamina , Células Secretoras de Insulina , Animais , Dopamina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Camundongos , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/genética , Receptores de Dopamina D3/metabolismoRESUMO
Crystal structures of the neurotransmitter:sodium symporter MhsT revealed occluded inward-facing states with one substrate (Trp) bound in the primary substrate (S1) site and a collapsed extracellular vestibule, which in LeuT contains the second substrate (S2) site. In n-dodecyl-ß-d-maltoside, the detergent used to prepare MhsT for crystallization, the substrate-to-protein binding stoichiometry was determined by using scintillation proximity to be 1 Trp:MhsT. Here, using the same experimental approach, as well as equilibrium dialysis, we report that in n-decyl-ß-d-maltoside, or after reconstitution in lipid, MhsT, like LeuT, can simultaneously bind two Trp substrate molecules. Trp binding to the S2 site sterically blocks access to a substituted Cys at position 33 in the S2 site, as well as access to the deeper S1 site. Mutation of either the S1 or S2 site disrupts transport, consistent with previous studies in LeuT showing that substrate binding to the S2 site is an essential component of the transport mechanism.
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Proteínas de Bactérias/química , Lactococcus lactis/química , Simportadores/química , Cristalografia por Raios X , Humanos , Domínios ProteicosRESUMO
Bile acids are synthesized from cholesterol in hepatocytes and secreted through the biliary tract into the small intestine, where they aid in absorption of lipids and fat-soluble vitamins. Through a process known as enterohepatic recirculation, more than 90% of secreted bile acids are then retrieved from the intestine and returned to the liver for resecretion. In humans, there are two Na(+)-dependent bile acid transporters involved in enterohepatic recirculation, the Na(+)-taurocholate co-transporting polypeptide (NTCP; also known as SLC10A1) expressed in hepatocytes, and the apical sodium-dependent bile acid transporter (ASBT; also known as SLC10A2) expressed on enterocytes in the terminal ileum. In recent years, ASBT has attracted much interest as a potential drug target for treatment of hypercholesterolaemia, because inhibition of ASBT reduces reabsorption of bile acids, thus increasing bile acid synthesis and consequently cholesterol consumption. However, a lack of three-dimensional structures of bile acid transporters hampers our ability to understand the molecular mechanisms of substrate selectivity and transport, and to interpret the wealth of existing functional data. The crystal structure of an ASBT homologue from Neisseria meningitidis (ASBT(NM)) in detergent was reported recently, showing the protein in an inward-open conformation bound to two Na(+) and a taurocholic acid. However, the structural changes that bring bile acid and Na(+) across the membrane are difficult to infer from a single structure. To understand the structural changes associated with the coupled transport of Na(+) and bile acids, here we solved two structures of an ASBT homologue from Yersinia frederiksenii (ASBTYf) in a lipid environment, which reveal that a large rigid-body rotation of a substrate-binding domain gives the conserved 'crossover' region, where two discontinuous helices cross each other, alternating accessibility from either side of the cell membrane. This result has implications for the location and orientation of the bile acid during transport, as well as for the translocation pathway for Na(+).
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Yersinia/química , Ácidos e Sais Biliares/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , Reprodutibilidade dos Testes , Rotação , Sódio/metabolismo , Relação Estrutura-AtividadeRESUMO
Neurotransmitter:sodium symporters (NSSs) terminate neurotransmission by the reuptake of released neurotransmitters. This active accumulation of substrate against its concentration gradient is driven by the transmembrane Na+ gradient and requires that the transporter traverses several conformational states. LeuT, a prokaryotic NSS homolog, has been crystallized in outward-open, outward-occluded, and inward-open states. Two crystal structures of another prokaryotic NSS homolog, the multihydrophobic amino acid transporter (MhsT) from Bacillus halodurans, have been resolved in novel inward-occluded states, with the extracellular vestibule closed and the intracellular portion of transmembrane segment 5 (TM5i) in either an unwound or a helical conformation. We have investigated the potential involvement of TM5i in binding and unbinding of Na2, i.e. the Na+ bound in the Na2 site, by carrying out comparative molecular dynamics simulations of the models derived from the two MhsT structures. We find that the helical TM5i conformation is associated with a higher propensity for Na2 release, which leads to the repositioning of the N terminus and transition to an inward-open state. By using comparative interaction network analysis, we also identify allosteric pathways connecting TM5i and the Na2 binding site to the extracellular and intracellular regions. Based on our combined computational and mutagenesis studies of MhsT and LeuT, we propose that TM5i plays a key role in Na2 binding and release associated with the conformational transition toward the inward-open state, a role that is likely to be shared across the NSS family.
Assuntos
Bacillus/química , Proteínas de Bactérias/química , Simulação de Dinâmica Molecular , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/química , Sódio/química , Regulação Alostérica , Sistemas de Transporte de Aminoácidos , Bacillus/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismo , Domínios Proteicos , Sódio/metabolismoRESUMO
Neurotransmitter:sodium symporters (NSS) terminate neurotransmission through Na+-driven reuptake of cognate neurotransmitters. Crystallographically, whereas both substrates and inhibitors have been found to bind in the central binding (S1) site of NSS, inhibitors were found to bind to a second binding (S2) site in the extracellular vestibule (EV) of transporters for leucine (LeuT) and serotonin. On the basis of computational and experimental studies, we proposed that substrates bind to the S2 site of LeuT as well and that substrate binding to the S2 site is essential for Na+-coupled symport. Recent binding experiments show that substrate (l-Trp) binding in the S2 site of MhsT, another bacterial NSS, is also central to the allosteric transport mechanism. Here, we used extensive molecular dynamics simulations combined with Markov state model analysis to investigate the interaction of l-Trp with the EV of MhsT and identified potential binding poses of l-Trp as well as induced conformational changes in the EV. Our computational findings were validated by experimental mutagenesis studies and shed light on the ligand binding characteristics of the EV of NSS, which may facilitate development of allosteric ligands targeting NSS.
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Bacillus/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismo , Bacillus/química , Proteínas de Bactérias/química , Sítios de Ligação , Cadeias de Markov , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/química , Ligação Proteica , Conformação Proteica , Especificidade por SubstratoRESUMO
Neurotransmitter/Na(+) symporters (NSSs) terminate neuronal signalling by recapturing neurotransmitter released into the synapse in a co-transport (symport) mechanism driven by the Na(+) electrochemical gradient. NSSs for dopamine, noradrenaline and serotonin are targeted by the psychostimulants cocaine and amphetamine, as well as by antidepressants. The crystal structure of LeuT, a prokaryotic NSS homologue, revealed an occluded conformation in which a leucine (Leu) and two Na(+) are bound deep within the protein. This structure has been the basis for extensive structural and computational exploration of the functional mechanisms of proteins with a LeuT-like fold. Subsequently, an 'outward-open' conformation was determined in the presence of the inhibitor tryptophan, and the Na(+)-dependent formation of a dynamic outward-facing intermediate was identified using electron paramagnetic resonance spectroscopy. In addition, single-molecule fluorescence resonance energy transfer imaging has been used to reveal reversible transitions to an inward-open LeuT conformation, which involve the movement of transmembrane helix TM1a away from the transmembrane helical bundle. We investigated how substrate binding is coupled to structural transitions in LeuT during Na(+)-coupled transport. Here we report a process whereby substrate binding from the extracellular side of LeuT facilitates intracellular gate opening and substrate release at the intracellular face of the protein. In the presence of alanine, a substrate that is transported â¼10-fold faster than leucine, we observed alanine-induced dynamics in the intracellular gate region of LeuT that directly correlate with transport efficiency. Collectively, our data reveal functionally relevant and previously hidden aspects of the NSS transport mechanism that emphasize the functional importance of a second substrate (S2) binding site within the extracellular vestibule. Substrate binding in this S2 site appears to act cooperatively with the primary substrate (S1) binding site to control intracellular gating more than 30 Å away, in a manner that allows the Na(+) gradient to power the transport mechanism.
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Ativação do Canal Iônico/efeitos dos fármacos , Modelos Moleculares , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismo , Humanos , Leucina/metabolismo , Lítio/metabolismo , Mutação , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/química , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , Ligação Proteica/genética , Estrutura Secundária de Proteína , Sódio/metabolismo , Sódio/farmacologiaRESUMO
Saccharides have a central role in the nutrition of all living organisms. Whereas several saccharide uptake systems are shared between the different phylogenetic kingdoms, the phosphoenolpyruvate-dependent phosphotransferase system exists almost exclusively in bacteria. This multi-component system includes an integral membrane protein EIIC that transports saccharides and assists in their phosphorylation. Here we present the crystal structure of an EIIC from Bacillus cereus that transports diacetylchitobiose. The EIIC is a homodimer, with an expansive interface formed between the amino-terminal halves of the two protomers. The carboxy-terminal half of each protomer has a large binding pocket that contains a diacetylchitobiose, which is occluded from both sides of the membrane with its site of phosphorylation near the conserved His250 and Glu334 residues. The structure shows the architecture of this important class of transporters, identifies the determinants of substrate binding and phosphorylation, and provides a framework for understanding the mechanism of sugar translocation.
Assuntos
Bacillus cereus/enzimologia , Proteínas de Membrana Transportadoras/química , Modelos Moleculares , Sítios de Ligação , Metabolismo dos Carboidratos , Cristalização , Fosforilação , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
The TrkH/TrkG/KtrB proteins mediate K(+) uptake in bacteria and probably evolved from simple K(+) channels by multiple gene duplications or fusions. Here we present the crystal structure of a TrkH from Vibrio parahaemolyticus. TrkH is a homodimer, and each protomer contains an ion permeation pathway. A selectivity filter, similar in architecture to those of K(+) channels but significantly shorter, is lined by backbone and side-chain oxygen atoms. Functional studies showed that TrkH is selective for permeation of K(+) and Rb(+) over smaller ions such as Na(+) or Li(+). Immediately intracellular to the selectivity filter are an intramembrane loop and an arginine residue, both highly conserved, which constrict the permeation pathway. Substituting the arginine with an alanine significantly increases the rate of K(+) flux. These results reveal the molecular basis of K(+) selectivity and suggest a novel gating mechanism for this large and important family of membrane transport proteins.
Assuntos
Canais de Potássio/química , Canais de Potássio/metabolismo , Vibrio parahaemolyticus/química , Transportadores de Cassetes de Ligação de ATP/química , Sequência de Aminoácidos , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Ativação do Canal Iônico , Transporte de Íons , Modelos Moleculares , Dados de Sequência Molecular , Potássio/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Recent work has shown that the choice of the type and concentration of detergent used for the solubilization of membrane proteins can strongly influence the results of functional experiments. In particular, the amino acid transporter LeuT can bind two substrate molecules in low concentrations of n-dodecyl ß-d-maltopyranoside (DDM), whereas high concentrations reduce the molar binding stoichiometry to 1:1. Subsequent molecular dynamics (MD) simulations of LeuT in DDM proteomicelles revealed that DDM can penetrate to the extracellular vestibule and make stable contacts in the functionally important secondary substrate binding site (S2), suggesting a potential competitive mechanism for the reduction in binding stoichiometry. Because annular lipids can be retained during solubilization, we performed MD simulations of LeuT proteomicelles at various stages of the solubilization process. We find that at low DDM concentrations, lipids are retained around the protein and penetration of detergent into the S2 site does not occur, whereas at high concentrations, lipids are displaced and the probability of DDM binding in the S2 site is increased. This behavior is dependent on the type of detergent, however, as we find in the simulations that the detergent lauryl maltose-neopentyl glycol, which is approximately twice the size of DDM and structurally more closely resembles lipids, does not penetrate the protein even at very high concentrations. We present functional studies that confirm the computational findings, emphasizing the need for careful consideration of experimental conditions, and for cautious interpretation of data in gathering mechanistic information about membrane proteins.