RESUMO
Salmonella enterica serovar Typhimurium melibiose permease (MelBSt) is a prototype of the major facilitator superfamily (MFS) transporters, which play important roles in human health and diseases. MelBSt catalyzed the symport of galactosides with Na+, Li+, or H+ but prefers the coupling with Na+. Previously, we determined the structures of the inward- and outward-facing conformation of MelBSt and the molecular recognition for galactoside and Na+. However, the molecular mechanisms for H+- and Na+-coupled symport remain poorly understood. In this study, we solved two x-ray crystal structures of MelBSt, the cation-binding site mutants D59C at an unliganded apo-state and D55C at a ligand-bound state, and both structures display the outward-facing conformations virtually identical as published. We determined the energetic contributions of three major Na+-binding residues for the selection of Na+ and H+ by free energy simulations. Transport assays showed that the D55C mutant converted MelBSt to a solely H+-coupled symporter, and together with the free-energy perturbation calculation, Asp59 is affirmed to be the sole protonation site of MelBSt. Unexpectedly, the H+-coupled melibiose transport exhibited poor activities at greater bulky ΔpH and better activities at reversal ΔpH, supporting the novel theory of transmembrane-electrostatically localized protons and the associated membrane potential as the primary driving force for the H+-coupled symport mediated by MelBSt. This integrated study of crystal structure, bioenergetics, and free energy simulations, demonstrated the distinct roles of the major binding residues in the cation-binding pocket of MelBSt.
Assuntos
Sódio , Simportadores , Simportadores/química , Simportadores/metabolismo , Simportadores/genética , Sítios de Ligação , Cristalografia por Raios X , Sódio/metabolismo , Sódio/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Salmonella typhimurium/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/química , Melibiose/metabolismo , Melibiose/química , Cátions/metabolismo , Cátions/química , Conformação ProteicaRESUMO
The lysosomal degradation of macromolecules produces diverse small metabolites exported by specific transporters for reuse in biosynthetic pathways. Here we deorphanized the major facilitator superfamily domain containing 1 (MFSD1) protein, which forms a tight complex with the glycosylated lysosomal membrane protein (GLMP) in the lysosomal membrane. Untargeted metabolomics analysis of MFSD1-deficient mouse lysosomes revealed an increase in cationic dipeptides. Purified MFSD1 selectively bound diverse dipeptides, while electrophysiological, isotope tracer and fluorescence-based studies in Xenopus oocytes and proteoliposomes showed that MFSD1-GLMP acts as a uniporter for cationic, neutral and anionic dipeptides. Cryoelectron microscopy structure of the dipeptide-bound MFSD1-GLMP complex in outward-open conformation characterized the heterodimer interface and, in combination with molecular dynamics simulations, provided a structural basis for its selectivity towards diverse dipeptides. Together, our data identify MFSD1 as a general lysosomal dipeptide uniporter, providing an alternative route to recycle lysosomal proteolysis products when lysosomal amino acid exporters are overloaded.
Assuntos
Dipeptídeos , Lisossomos , Lisossomos/metabolismo , Animais , Dipeptídeos/metabolismo , Oócitos/metabolismo , Microscopia Crioeletrônica , Camundongos , Xenopus laevis , Humanos , Camundongos Knockout , Simulação de Dinâmica Molecular , Simportadores/metabolismo , Simportadores/genética , Simportadores/química , Feminino , Canais de Potencial de Receptor TransitórioRESUMO
The Organic Cation Transporter Novel 1 (OCTN1), also known as SLC22A4, is widely expressed in various human tissues, and involved in numerous physiological and pathological processes remains. It facilitates the transport of organic cations, zwitterions, with selectivity for positively charged solutes. Ergothioneine, an antioxidant compound, and acetylcholine (Ach) are among its substrates. Given the lack of experimentally solved structures of this protein, this study aimed at generating a reliable 3D model of OCTN1 to shed light on its substrate-binding preferences and the role of sodium in substrate recognition and transport. A chimeric model was built by grafting the large extracellular loop 1 (EL1) from an AlphaFold-generated model onto a homology model. Molecular dynamics simulations revealed domain-specific mobility, with EL1 exhibiting the highest impact on overall stability. Molecular docking simulations identified cytarabine and verapamil as highest affinity ligands, consistent with their known inhibitory effects on OCTN1. Furthermore, MM/GBSA analysis allowed the categorization of substrates into weak, good, and strong binders, with molecular weight strongly correlating with binding affinity to the recognition site. Key recognition residues, including Tyr211, Glu381, and Arg469, were identified through interaction analysis. Ach demonstrated a low interaction energy, supporting the hypothesis of its one-directional transport towards to outside of the membrane. Regarding the role of sodium, our model suggested the involvement of Glu381 in sodium binding. Molecular dynamics simulations of systems at increasing levels of Na+ concentrations revealed increased sodium occupancy around Glu381, supporting experimental data associating Na+ concentration to molecule transport. In conclusion, this study provides valuable insights into the 3D structure of OCTN1, its substrate-binding preferences, and the role of sodium in the recognition. These findings contribute to the understanding of OCTN1 involvement in various physiological and pathological processes and may have implications for drug development and disease management.
Assuntos
Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas de Transporte de Cátions Orgânicos , Humanos , Proteínas de Transporte de Cátions Orgânicos/química , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/genética , Simportadores/química , Simportadores/metabolismo , Sítios de Ligação , Ligação Proteica , Ergotioneína/química , Ergotioneína/metabolismo , Sódio/metabolismo , Sódio/química , Simulação por Computador , Acetilcolina/metabolismo , Acetilcolina/química , LigantesRESUMO
The human Solute Carrier (SLC) family member, monocarboxylate transporter 1 (MCT1), transports lactic and pyruvic acid across biological membranes to regulate cellular pH and metabolism. Proper trafficking of MCT1 from the endoplasmic reticulum to the plasma membrane hinges on its interactions with the membrane-bound chaperone protein, CD147. Here, using AlphaFold2 modeling and copurification, we show how a conserved signature motif located in the flexible N-terminus of MCT1 is a crucial region of interaction between MCT1 and the C-terminus of CD147. Mutations to this motif-namely, the thymic cancer linked G19C and the highly conserved W20A-destabilize the MCT1-CD147 complex and lead to a loss of proper membrane localization and cellular substrate flux. Notably, the monomeric stability of MCT1 remains unaffected in mutants, thus supporting the role of CD147 in mediating the trafficking of the heterocomplex. Using the auxiliary chaperone, GP70, we demonstrated that W20A-MCT1 can be trafficked to the plasma membrane, while G19C-MCT1 remains internalized. Overall, our findings underscore the critical role of the MCT1 transmembrane one signature motif for engaging CD147 and identify altered chaperone binding mechanisms between the CD147 and GP70 glycoprotein chaperones.
Assuntos
Motivos de Aminoácidos , Basigina , Transportadores de Ácidos Monocarboxílicos , Transporte Proteico , Simportadores , Basigina/metabolismo , Basigina/genética , Basigina/química , Transportadores de Ácidos Monocarboxílicos/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/química , Humanos , Simportadores/metabolismo , Simportadores/química , Simportadores/genética , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Mutação de Sentido IncorretoAssuntos
Simportadores , Humanos , Simportadores/metabolismo , Simportadores/química , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/química , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Transporte Biológico , Especificidade por SubstratoRESUMO
Human Na+ taurocholate co-transporting protein (hNTCP) is a key bile salt transporter to maintain enterohepatic circulation and is responsible for the recognition of hepatitis B and D viruses. Despite landmark cryoelectron microscopy studies revealing open-pore and inward-facing states of hNTCP stabilized by antibodies, the transport mechanism remains largely unknown. To address this knowledge gap, we used molecular dynamics and enhanced sampling metadynamics simulations to elucidate the intrinsic mechanism of hNTCP-mediated taurocholate acid (TCA) transport driven by Na+ binding. We uncovered three TCA-binding modes, including one that closely matched the limited cryoelectron microscopy density observed in the open-pore hNTCP. We also captured several key hNTCP conformations in the substrate transport cycle, particularly including an outward-facing, substrate-bound state. Furthermore, we provided thermodynamic evidence supporting that changes in the Na+-binding state drive the TCA transport by exploiting the amphiphilic nature of the substrate and modulating the protein environment, thereby enabling the TCA molecule to flip through. Understanding these mechanistic details of Na+-driven bile acid transport may aid in the development of hNTCP-targeted therapies for liver diseases.
Assuntos
Simulação de Dinâmica Molecular , Transportadores de Ânions Orgânicos Dependentes de Sódio , Sódio , Simportadores , Ácido Taurocólico , Simportadores/metabolismo , Simportadores/química , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/química , Humanos , Sódio/metabolismo , Ácido Taurocólico/metabolismo , Transporte Biológico , Ácidos e Sais Biliares/metabolismo , TermodinâmicaRESUMO
Congenital myasthenic syndrome (CMS) is a heterogeneous condition associated with 34 different genes, including SLC5A7, which encodes the high-affinity choline transporter 1 (CHT1). CHT1 is expressed in presynaptic neurons of the neuromuscular junction where it uses the inward sodium gradient to reuptake choline. Biallelic CHT1 mutations often lead to neonatal lethality, and less commonly to non-lethal motor weakness and developmental delays. Here, we report detailed biochemical characterization of two novel mutations in CHT1, p.I294T and p.D349N, which we identified in an 11-year-old patient with a history of neonatal respiratory distress, and subsequent hypotonia and global developmental delay. Heterologous expression of each CHT1 mutant in human embryonic kidney cells showed two different mechanisms of reduced protein function. The p.I294T CHT1 mutant transporter function was detectable, but its abundance and half-life were significantly reduced. In contrast, the p.D349N CHT1 mutant was abundantly expressed at the cell membrane, but transporter function was absent. The residual function of the p.I294T CHT1 mutant may explain the non-lethal form of CMS in this patient, and the divergent mechanisms of reduced CHT1 function that we identified may guide future functional studies of the CHT1 myasthenic syndrome. Based on these in vitro studies that provided a diagnosis, treatment with cholinesterase inhibitor together with physical and occupational therapy significantly improved the patient's strength and quality of life.
Assuntos
Proteínas Mutantes , Mutação , Síndromes Miastênicas Congênitas , Simportadores , Síndromes Miastênicas Congênitas/tratamento farmacológico , Síndromes Miastênicas Congênitas/genética , Síndromes Miastênicas Congênitas/metabolismo , Síndromes Miastênicas Congênitas/reabilitação , Humanos , Masculino , Criança , Células HEK293 , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Meia-Vida , Membrana Celular/metabolismo , Transporte Proteico , Estaurosporina/farmacologia , Brometo de Piridostigmina/uso terapêutico , Qualidade de Vida , Simportadores/química , Simportadores/genética , Simportadores/metabolismoRESUMO
Osmosensing transporter ProP forestalls cellular dehydration by detecting environments with high osmotic pressure and mediating the accumulation of organic osmolytes by bacterial cells. It is composed of 12 transmembrane helices with cytoplasmic N- and C-termini. In Escherichia coli, dimers form when the C-terminal domains of ProP molecules form homodimeric, antiparallel, α-helical coiled coils. No dominant negative effect was detected when inactive and active ProP molecules formed heterodimers in vivo. Purification of ProP in detergent dodecylmaltoside yielded monomers, which were functional after reconstitution in proteoliposomes. With other evidence, this suggests that ProP monomers function independently whether in the monomeric or dimeric state. Amino acid replacements that disrupted or reversed the coiled coil did not prevent in vivo dimerization of ProP detected with a bacterial two-hybrid system. Maleimide labeling detected no osmolality-dependent variation in the reactivities of cysteine residues introduced to transmembrane helix (TM) XII. In contrast, coarse-grained molecular dynamic simulations detected deformation of the lipid around TMs III and VI, on the lipid-exposed protein surface opposite to TM XII. This suggests that the dimer interface of ProP includes the surfaces of TMs III and VI, not of TM XII as previously suggested by crosslinking data. Homology modeling suggested that coiled-coil formation and dimerization via such an interface are not mutually exclusive. In previous work, alterations to the C-terminal coiled coil blocked co-localization of ProP with phospholipid cardiolipin at E. coli cell poles. Thus, dimerization may contribute to ProP targeting, adjust its lipid environment, and hence indirectly modify its osmotic stress response.
Assuntos
Proteínas de Escherichia coli , Simportadores , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Sequência de Aminoácidos , Dimerização , Simportadores/química , Proteínas de Membrana Transportadoras/metabolismo , Fosfolipídeos/metabolismoRESUMO
The sodium/iodide symporter (NIS) is the essential plasma membrane protein that mediates active iodide (I-) transport into the thyroid gland, the first step in the biosynthesis of the thyroid hormones-the master regulators of intermediary metabolism. NIS couples the inward translocation of I- against its electrochemical gradient to the inward transport of Na+ down its electrochemical gradient1,2. For nearly 50 years before its molecular identification3, NIS was the molecule at the centre of the single most effective internal radiation cancer therapy: radioiodide (131I-) treatment for thyroid cancer2. Mutations in NIS cause congenital hypothyroidism, which must be treated immediately after birth to prevent stunted growth and cognitive deficiency2. Here we report three structures of rat NIS, determined by single-particle cryo-electron microscopy: one with no substrates bound; one with two Na+ and one I- bound; and one with one Na+ and the oxyanion perrhenate bound. Structural analyses, functional characterization and computational studies show the substrate-binding sites and key residues for transport activity. Our results yield insights into how NIS selects, couples and translocates anions-thereby establishing a framework for understanding NIS function-and how it transports different substrates with different stoichiometries and releases substrates from its substrate-binding cavity into the cytosol.
Assuntos
Iodetos , Sódio , Simportadores , Animais , Ratos , Microscopia Crioeletrônica , Iodetos/metabolismo , Sódio/metabolismo , Simportadores/química , Simportadores/metabolismo , Simportadores/ultraestrutura , Sítios de Ligação , Especificidade por Substrato , Transporte de ÍonsRESUMO
Found in all domains of life, transporters belonging to the LeuT-fold class mediate the import and exchange of hydrophilic and charged compounds such as amino acids, metals, and sugar molecules. Nearly two decades of investigations on the eponymous bacterial transporter LeuT have yielded a library of high-resolution snapshots of its conformational cycle linked by solution-state experimental data obtained from multiple techniques. In parallel, its topology has been observed in symporters and antiporters characterized by a spectrum of substrate specificities and coupled to gradients of distinct ions. Here we review and compare mechanistic models of transport for LeuT, its well-studied homologs, as well as functionally distant members of the fold, emphasizing the commonalities and divergences in alternating access and the corresponding energy landscapes. Our integrated summary illustrates how fold conservation, a hallmark of the LeuT fold, coincides with divergent choreographies of alternating access that nevertheless capitalize on recurrent structural motifs. In addition, it highlights the knowledge gap that hinders the leveraging of the current body of research into detailed mechanisms of transport for this important class of membrane proteins.
Assuntos
Antiporters , Proteínas de Bactérias , Leucina , Simportadores , Antiporters/química , Proteínas de Bactérias/química , Transporte Biológico , Leucina/metabolismo , Dobramento de Proteína , Simportadores/químicaRESUMO
Cation-chloride cotransporters (CCCs) catalyze electroneutral symport of Cl- with Na+ and/or K+ across membranes. CCCs are fundamental in cell volume homeostasis, transepithelia ion movement, maintenance of intracellular Cl- concentration, and neuronal excitability. Here, we present a cryoelectron microscopy structure of human K+-Cl- cotransporter (KCC)1 bound with the VU0463271 inhibitor in an outward-open state. In contrast to many other amino acid-polyamine-organocation transporter cousins, our first outward-open CCC structure reveals that opening the KCC1 extracellular ion permeation path does not involve hinge-bending motions of the transmembrane (TM) 1 and TM6 half-helices. Instead, rocking of TM3 and TM8, together with displacements of TM4, TM9, and a conserved intracellular loop 1 helix, underlie alternate opening and closing of extracellular and cytoplasmic vestibules. We show that KCC1 intriguingly exists in one of two distinct dimeric states via different intersubunit interfaces. Our studies provide a blueprint for understanding the mechanisms of CCCs and their inhibition by small molecule compounds.
Assuntos
Membro 4 da Família 12 de Carreador de Soluto , Simportadores , Cátions/metabolismo , Cloretos/metabolismo , Microscopia Crioeletrônica , Humanos , Transporte de Íons , Conformação Proteica em alfa-Hélice , Membro 4 da Família 12 de Carreador de Soluto/química , Simportadores/antagonistas & inibidores , Simportadores/química , Cotransportadores de K e Cl-RESUMO
Brain development and function require uptake of essential omega-3 fatty acids in the form of lysophosphatidylcholine via major-facilitator superfamily transporter MFSD2A, a potential pharmaceutical target to modulate blood-brain barrier (BBB) permeability. MFSD2A is also the receptor of endogenous retroviral envelope syncytin-2 (SYNC2) in human placenta, where it mediates cell-cell fusion and formation of the maternal-fetal interface. Here, we report a cryo-electron microscopy structure of the human MFSD2A-SYNC2 complex that reveals a large hydrophobic cavity in the transporter C-terminal domain to occlude long aliphatic chains. The transporter architecture suggests an alternating-access transport mechanism for lipid substrates in mammalian MFS transporters. SYNC2 establishes an extensive binding interface with MFSD2A, and a SYNC2-soluble fragment acts as a long-sought-after inhibitor of MFSD2A transport. Our work uncovers molecular mechanisms important to brain and placenta development and function, and SYNC2-mediated inhibition of MFSD2A transport suggests strategies to aid delivery of therapeutic macromolecules across the BBB.
Assuntos
Proteínas da Gravidez/química , Simportadores/química , Animais , Encéfalo/metabolismo , Microscopia Crioeletrônica , Feminino , Humanos , Lisofosfatidilcolinas/química , Lisofosfatidilcolinas/metabolismo , Mamíferos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Gravidez , Proteínas da Gravidez/metabolismo , Simportadores/metabolismoRESUMO
Chronic infection with hepatitis B virus (HBV) affects more than 290 million people worldwide, is a major cause of cirrhosis and hepatocellular carcinoma, and results in an estimated 820,000 deaths annually1,2. For HBV infection to be established, a molecular interaction is required between the large glycoproteins of the virus envelope (known as LHBs) and the host entry receptor sodium taurocholate co-transporting polypeptide (NTCP), a sodium-dependent bile acid transporter from the blood to hepatocytes3. However, the molecular basis for the virus-transporter interaction is poorly understood. Here we report the cryo-electron microscopy structures of human, bovine and rat NTCPs in the apo state, which reveal the presence of a tunnel across the membrane and a possible transport route for the substrate. Moreover, the cryo-electron microscopy structure of human NTCP in the presence of the myristoylated preS1 domain of LHBs, together with mutation and transport assays, suggest a binding mode in which preS1 and the substrate compete for the extracellular opening of the tunnel in NTCP. Our preS1 domain interaction analysis enables a mechanistic interpretation of naturally occurring HBV-insusceptible mutations in human NTCP. Together, our findings provide a structural framework for HBV recognition and a mechanistic understanding of sodium-dependent bile acid translocation by mammalian NTCPs.
Assuntos
Microscopia Crioeletrônica , Vírus da Hepatite B , Transportadores de Ânions Orgânicos Dependentes de Sódio , Receptores Virais , Simportadores , Animais , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Apoproteínas/ultraestrutura , Bovinos , Vírus da Hepatite B/metabolismo , Hepatócitos/metabolismo , Humanos , Mutação , Transportadores de Ânions Orgânicos Dependentes de Sódio/química , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/ultraestrutura , Ratos , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/metabolismo , Receptores Virais/ultraestrutura , Sódio/metabolismo , Simportadores/química , Simportadores/genética , Simportadores/metabolismo , Simportadores/ultraestruturaRESUMO
Around 250 million people are infected with hepatitis B virus (HBV) worldwide1, and 15 million may also carry the satellite virus hepatitis D virus (HDV), which confers even greater risk of severe liver disease2. The HBV receptor has been identified as sodium taurocholate co-transporting polypeptide (NTCP), which interacts directly with the first 48 amino acid residues of the N-myristoylated N-terminal preS1 domain of the viral large protein3. Despite the pressing need for therapeutic agents to counter HBV, the structure of NTCP remains unsolved. This 349-residue protein is closely related to human apical sodium-dependent bile acid transporter (ASBT), another member of the solute carrier family SLC10. Crystal structures have been reported of similar bile acid transporters from bacteria4,5, and these models are believed to resemble closely both NTCP and ASBT. Here we have used cryo-electron microscopy to solve the structure of NTCP bound to an antibody, clearly showing that the transporter has no equivalent of the first transmembrane helix found in other SLC10 proteins, and that the N terminus is exposed on the extracellular face. Comparison of our structure with those of related proteins indicates a common mechanism of bile acid transport, but the NTCP structure displays an additional pocket formed by residues that are known to interact with preS1, presenting new opportunities for structure-based drug design.
Assuntos
Ácidos e Sais Biliares , Microscopia Crioeletrônica , Vírus da Hepatite B , Transportadores de Ânions Orgânicos Dependentes de Sódio , Receptores Virais , Simportadores , Anticorpos , Ácidos e Sais Biliares/metabolismo , Vírus da Hepatite B/metabolismo , Hepatócitos/metabolismo , Humanos , Transportadores de Ânions Orgânicos Dependentes de Sódio/química , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/ultraestrutura , Receptores Virais/química , Receptores Virais/metabolismo , Receptores Virais/ultraestrutura , Simportadores/química , Simportadores/metabolismo , Simportadores/ultraestruturaRESUMO
The liver takes up bile salts from blood to generate bile, enabling absorption of lipophilic nutrients and excretion of metabolites and drugs1. Human Na+-taurocholate co-transporting polypeptide (NTCP) is the main bile salt uptake system in liver. NTCP is also the cellular entry receptor of human hepatitis B and D viruses2,3 (HBV/HDV), and has emerged as an important target for antiviral drugs4. However, the molecular mechanisms underlying NTCP transport and viral receptor functions remain incompletely understood. Here we present cryo-electron microscopy structures of human NTCP in complexes with nanobodies, revealing key conformations of its transport cycle. NTCP undergoes a conformational transition opening a wide transmembrane pore that serves as the transport pathway for bile salts, and exposes key determinant residues for HBV/HDV binding to the outside of the cell. A nanobody that stabilizes pore closure and inward-facing states impairs recognition of the HBV/HDV receptor-binding domain preS1, demonstrating binding selectivity of the viruses for open-to-outside over inward-facing conformations of the NTCP transport cycle. These results provide molecular insights into NTCP 'gated-pore' transport and HBV/HDV receptor recognition mechanisms, and are expected to help with development of liver disease therapies targeting NTCP.
Assuntos
Ácidos e Sais Biliares , Microscopia Crioeletrônica , Fígado , Transportadores de Ânions Orgânicos Dependentes de Sódio , Sódio , Simportadores , Bile/metabolismo , Ácidos e Sais Biliares/metabolismo , Vírus da Hepatite B/metabolismo , Vírus Delta da Hepatite/metabolismo , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/química , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/ultraestrutura , Conformação Proteica , Receptores Virais/metabolismo , Anticorpos de Domínio Único , Sódio/metabolismo , Simportadores/química , Simportadores/metabolismo , Simportadores/ultraestrutura , Internalização do VírusRESUMO
Cation selectivity and coupling are important attributes of cation-coupled symporters. Salmonella typhimurium melibiose permease (MelBSt) catalyzes the co-transport of galactosides with cations (H+, Li+, or Na+). 3-D crystal structures of MelBSt have revealed the molecular recognition for sugar substrates, but the cation binding and coupling mechanisms have not been defined to atomic levels. In its human homolog MFSD2A, a lethal mutation was mapped at its Na+-binding pocket; however, none of the structures in this subfamily resolved its cation binding. In this study, molecular dynamics simulations reveal the binding interactions of Na+ and Li+ with MelBSt. Interestingly, Thr121, the lethal mutation position in MFSD2A, forms stable interaction with Na+ but is at a distance from Li+. Most mutations among 11 single-site Thr121 mutants of MelBSt exhibited little effects on the galactoside binding, but largely altered the cation selectivity with severe inhibitions on Na+ binding. Few mutants (Pro and Ala) completely lost the Na+ binding and Na+-coupled transport, but their Li+ or H+ modes of activity were largely retained. It can be concluded that Thr121 is necessary for Na+ binding, but not required for the binding of H+ or Li+, so a subset of the Na+-binding pocket is enough for Li+ binding. In addition, the protein stability for some mutants can be only retained in the presence of Li+, but not by Na+ due to the lack of affinity. This finding, together with other identified thermostable mutants, supports that the charge balance of the cation-binding site plays an important role in MelBSt protein stability.
Assuntos
Proteínas de Bactérias , Salmonella typhimurium , Simportadores , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cátions/metabolismo , Humanos , Lítio/metabolismo , Melibiose/metabolismo , Salmonella typhimurium/enzimologia , Salmonella typhimurium/genética , Sódio/metabolismo , Simportadores/química , Simportadores/genética , Simportadores/metabolismoRESUMO
The Novel Organic Cation Transporter, OCTN1, is the first member of the OCTN subfamily; it belongs to the wider Solute Carrier family SLC22, which counts many members including cation and anion organic transporters. The tertiary structure has not been resolved for any cation organic transporter. The functional role of OCNT1 is still not well assessed despite the many functional studies so far conducted. The lack of a definitive identification of OCTN1 function can be attributed to the different experimental systems and methodologies adopted for studying each of the proposed ligands. Apart from the contradictory data, the international scientific community agrees on a role of OCTN1 in protecting cells and tissues from oxidative and/or inflammatory damage. Moreover, the involvement of this transporter in drug interactions and delivery has been well clarified, even though the exact profile of the transported/interacting molecules is still somehow confusing. Therefore, OCTN1 continues to be a hot topic in terms of its functional role and structure. This review focuses on the most recent advances on OCTN1 in terms of functional aspects, physiological roles, substrate specificity, drug interactions, tissue expression, and relationships with pathology.
Assuntos
Biomarcadores , Suscetibilidade a Doenças , Interações Medicamentosas , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Simportadores/genética , Simportadores/metabolismo , Acetilação , Animais , Sítios de Ligação , Transporte Biológico , Ergotioneína/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Modelos Moleculares , Conformação Molecular , Especificidade de Órgãos , Proteínas de Transporte de Cátions Orgânicos/química , Ligação Proteica , Relação Estrutura-Atividade , Simportadores/químicaRESUMO
The crucial function of neurotransmitter:sodium symporters (NSS) in facilitating the reuptake of neurotransmitters into neuronal cells makes them attractive drug targets for treating multiple mental diseases. Due to the challenges in working with eukaryotic NSS proteins, LeuT, a prokaryotic amino acid transporter, has served as a model protein for studying structure-function relationships of NSS family proteins. With hydrogen-deuterium exchange mass spectrometry (HDX-MS), slow unfolding/refolding kinetics were identified in multiple regions of LeuT, suggesting that substrate translocation involves cooperative fluctuations of helical stretches. Earlier work has solely been performed at non-native temperatures (25 °C) for LeuT, which is evolutionarily adapted to function at high temperatures (85 - 95 °C). To address the effect of temperature on LeuT dynamics, we have performed HDX-MS experiments at elevated temperatures (45 °C and 60 °C). At these elevated temperatures, multiple regions in LeuT exhibited increased dynamics compared to 25 °C. Interestingly, coordinated slow unfolding/refolding of key regions could still be observed, though considerably faster. We have further investigated the conformational impact of binding the efficiently transported substrate alanine (Ala) relative to the much slower transported substrate leucine (Leu). Comparing the HDX of the Ala-bound versus Leu-bound state of LeuT, we observe distinct differences that could explain the faster transport rate (kcat) of Ala relative to Leu. Importantly, slow unfolding/refolding dynamics could still be observed in regions of Ala-bound LeuT . Overall, our work brings new insights into the conformational dynamics of LeuT and provides a better understanding of the transport mechanism of LeuT and possibly other transporters bearing the LeuT fold.
Assuntos
Conformação Molecular , Neurotransmissores , Simportadores/química , Temperatura , Cinética , Proteínas de Membrana , Simulação de Dinâmica Molecular , Preparações Farmacêuticas , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/química , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismo , Conformação Proteica , SódioRESUMO
Major facilitator superfamily_2 transporters are widely found from bacteria to mammals. The melibiose transporter MelB, which catalyzes melibiose symport with either Na+, Li+, or H+, is a prototype of the Na+-coupled MFS transporters, but its sugar recognition mechanism has been a long-unsolved puzzle. Two high-resolution X-ray crystal structures of a Salmonella typhimurium MelB mutant with a bound ligand, either nitrophenyl-α-D-galactoside or dodecyl-ß-D-melibioside, were refined to a resolution of 3.05 or 3.15 Å, respectively. In the substrate-binding site, the interaction of both galactosyl moieties on the two ligands with MelBSt are virturally same, so the sugar specificity determinant pocket can be recognized, and hence the molecular recognition mechanism for sugar binding in MelB has been deciphered. The conserved cation-binding pocket is also proposed, which directly connects to the sugar specificity pocket. These key structural findings have laid a solid foundation for our understanding of the cooperative binding and symport mechanisms in Na+-coupled MFS transporters, including eukaryotic transporters such as MFSD2A.
Assuntos
Proteínas de Bactérias/química , Cristalografia por Raios X , Melibiose/metabolismo , Salmonella typhimurium/metabolismo , Simportadores/químicaRESUMO
MFSD2A is a sodium-dependent lysophosphatidylcholine symporter that is responsible for the uptake of docosahexaenoic acid into the brain1,2, which is crucial for the development and performance of the brain3. Mutations that affect MFSD2A cause microcephaly syndromes4,5. The ability of MFSD2A to transport lipid is also a key mechanism that underlies its function as an inhibitor of transcytosis to regulate the blood-brain barrier6,7. Thus, MFSD2A represents an attractive target for modulating the permeability of the blood-brain barrier for drug delivery. Here we report the cryo-electron microscopy structure of mouse MFSD2A. Our structure defines the architecture of this important transporter, reveals its unique extracellular domain and uncovers its substrate-binding cavity. The structure-together with our functional studies and molecular dynamics simulations-identifies a conserved sodium-binding site, reveals a potential lipid entry pathway and helps to rationalize MFSD2A mutations that underlie microcephaly syndromes. These results shed light on the critical lipid transport function of MFSD2A and provide a framework to aid in the design of specific modulators for therapeutic purposes.