RESUMO
NaCT (SLC13A5; mINDY), a sodium-coupled citrate transporter, is the mammalian ortholog of Drosophila INDY. Loss-of-function mutations in human NaCT cause severe complications with neonatal epilepsy and encephalopathy (EIEE25). Surprisingly, mice lacking this transporter do not have this detrimental brain phenotype. The marked differences in transport kinetics between mouse and human NaCTs provide at least a partial explanation for this conundrum, but a structural basis for the differences is lacking. Neither human nor mouse NaCT has been crystallized, and any information known on their structures is based entirely on what was inferred from the structure of VcINDY, a related transporter in bacteria. Here, we highlight the functional features of human and mouse NaCTs and provide a plausible molecular basis for the differences based on a full-length homology modeling approach. The transport characteristics of human NaCT markedly differ from those of VcINDY. Therefore, the modeling with VcINDY as the template is flawed, but this is the best available option at this time. With the newly deduced model, we determined the likely locations of the disease-causing mutations and propose a new classification for the mutations based on their location and potential impact on transport function. This new information should pave the way for future design and development of novel therapeutics to restore the lost function of the mutant transporters as a treatment strategy for patients with EIEE25.
Assuntos
Transportadores de Ácidos Dicarboxílicos/química , Transportadores de Ácidos Dicarboxílicos/metabolismo , Simportadores/química , Simportadores/metabolismo , Animais , Sítios de Ligação , Drosophila , Células HEK293 , Humanos , Camundongos , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
ATP binding cassette (ABC) transporters play critical roles in maintaining sterol balance in higher eukaryotes. The ABCG5/ABCG8 heterodimer (G5G8) mediates excretion of neutral sterols in liver and intestines. Mutations disrupting G5G8 cause sitosterolaemia, a disorder characterized by sterol accumulation and premature atherosclerosis. Here we use crystallization in lipid bilayers to determine the X-ray structure of human G5G8 in a nucleotide-free state at 3.9 Å resolution, generating the first atomic model of an ABC sterol transporter. The structure reveals a new transmembrane fold that is present in a large and functionally diverse superfamily of ABC transporters. The transmembrane domains are coupled to the nucleotide-binding sites by networks of interactions that differ between the active and inactive ATPases, reflecting the catalytic asymmetry of the transporter. The G5G8 structure provides a mechanistic framework for understanding sterol transport and the disruptive effects of mutations causing sitosterolaemia.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Lipoproteínas/química , Esteróis/metabolismo , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise , Cristalografia por Raios X , Humanos , Hipercolesterolemia/genética , Enteropatias/genética , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Erros Inatos do Metabolismo Lipídico/genética , Lipoproteínas/genética , Lipoproteínas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Nucleotídeos , Fitosteróis/efeitos adversos , Fitosteróis/genética , Dobramento de Proteína , Multimerização Proteica , Estrutura Terciária de ProteínaRESUMO
NaCT (SLC13A5) is a Na+-coupled transporter for citrate, which is expressed in the liver, brain, testes, and bone. It is the mammalian homolog of Drosophila INDY, a cation-independent transporter for citrate, whose partial loss extends lifespan in the organism. In humans, loss-of-function mutations in NaCT cause a disease with severe neurological dysfunction, characterized by neonatal epilepsy and delayed brain development. In contrast with humans, deletion of NaCT in mice results in a beneficial metabolic phenotype with protection against diet-induced obesity and metabolic syndrome; the brain dysfunction is not readily noticeable. The disease-causing mutations are located in different regions of human NaCT protein, suggesting that different mutations might have different mechanisms for the loss of function. The beneficial effects of NaCT loss in the liver versus the detrimental effects of NaCT loss in the brain provide an opportunity to design high-affinity inhibitors for the transporter that do not cross the blood-brain barrier so that only the beneficial effects could be harnessed. To realize these goals, we need a detailed knowledge of the 3D structure of human NaCT. The recent report by Sauer et al. in Nature describing the cryo-EM structure of human NaCT represents such a milestone, paving the way for a better understanding of the structure-function relationship for this interesting and clinically important transporter.
Assuntos
Simportadores , Animais , Transporte Biológico , Encéfalo/metabolismo , Ácido Cítrico , Microscopia Crioeletrônica , Transportadores de Ácidos Dicarboxílicos/genética , Humanos , Masculino , Camundongos , Mutação , Simportadores/genéticaRESUMO
The Na+-coupled citrate transporter (NaCT/SLC13A5/mINDY) in the liver delivers citrate from the blood into hepatocytes. As citrate is a key metabolite and regulator of multiple biochemical pathways, deletion of Slc13a5 in mice protects against diet-induced obesity, diabetes, and metabolic syndrome. Silencing the transporter suppresses hepatocellular carcinoma. Therefore, selective blockers of NaCT hold the potential to treat various diseases. Here we report on the characteristics of one such inhibitor, BI01383298. It is known that BI01383298 is a high-affinity inhibitor selective for human NaCT with no effect on mouse NaCT. Here we show that this compound is an irreversible and non-competitive inhibitor of human NaCT, thus describing the first irreversible inhibitor for this transporter. The mouse NaCT is not affected by this compound. The inhibition of human NaCT by BI01383298 is evident for the constitutively expressed transporter in HepG2 cells and for the ectopically expressed human NaCT in HEK293 cells. The IC50 is â¼100â nM, representing the highest potency among the NaCT inhibitors known to date. Exposure of HepG2 cells to this inhibitor results in decreased cell proliferation. We performed molecular modeling of the 3D-structures of human and mouse NaCTs using the crystal structure of a humanized variant of VcINDY as the template, and docking studies to identify the amino acid residues involved in the binding of citrate and BI01383298. These studies provide insight into the probable bases for the differential effects of the inhibitor on human NaCT versus mouse NaCT as well as for the marked species-specific difference in citrate affinity.
Assuntos
Inibidores Enzimáticos/farmacocinética , Simportadores/antagonistas & inibidores , Simportadores/metabolismo , Animais , Ácido Cítrico/metabolismo , Inibidores Enzimáticos/farmacologia , Células HEK293 , Células Hep G2 , Humanos , Concentração Inibidora 50 , Camundongos , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Especificidade da Espécie , Simportadores/químicaRESUMO
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.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Bloqueadores dos Canais de Cálcio/metabolismo , Bicamadas Lipídicas/química , Modelos Moleculares , Verapamil/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/química , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Trifosfato de Adenosina/química , Substituição de Aminoácidos , Animais , Sítios de Ligação , Transporte Biológico Ativo , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Bloqueadores dos Canais de Cálcio/química , Cisteína/química , Európio/química , Hidrólise , Camundongos , Mutação , Nanoestruturas/química , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Redobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Térbio/química , Verapamil/químicaRESUMO
The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is an ABC transporter containing two transmembrane domains forming a chloride ion channel, and two nucleotide binding domains (NBD1 and NBD2). CFTR has presented a formidable challenge to obtain monodisperse, biophysically stable protein. Here we report a comprehensive study comparing effects of single and multiple NBD1 mutations on stability of both the NBD1 domain alone and on purified full length human CFTR. Single mutations S492P, A534P, I539T acted additively, and when combined with M470V, S495P, and R555K cumulatively yielded an NBD1 with highly improved structural stability. Strategic combinations of these mutations strongly stabilized the domain to attain a calorimetric Tmâ¯>â¯70⯰C. Replica exchange molecular dynamics simulations on the most stable 6SS-NBD1 variant implicated fluctuations, electrostatic interactions and side chain packing as potential contributors to improved stability. Progressive stabilization of NBD1 directly correlated with enhanced structural stability of full-length CFTR protein. Thermal unfolding of the stabilized CFTR mutants, monitored by changes in intrinsic fluorescence, demonstrated that Tm could be shifted as high as 67.4⯰C in 6SS-CFTR, more than 20⯰C higher than wild-type. H1402S, an NBD2 mutation, conferred CFTR with additional thermal stability, possibly by stabilizing an NBD-dimerized conformation. CFTR variants with NBD1-stabilizing mutations were expressed at the cell surface in mammalian cells, exhibited ATPase and channel activity, and retained these functions to higher temperatures. The capability to produce enzymatically active CFTR with improved structural stability amenable to biophysical and structural studies will advance mechanistic investigations and future cystic fibrosis drug development.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Mutação , Nucleotídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Sítios de Ligação/genética , Células CHO , Cricetinae , Cricetulus , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/isolamento & purificação , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Estabilidade Enzimática/genética , Células HEK293 , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , Engenharia de Proteínas/métodos , Domínios e Motivos de Interação entre Proteínas/genética , Estabilidade Proteica , TemperaturaRESUMO
The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR, ABCC7) is a plasma membrane chloride ion channel in the ABC transporter superfamily. CFTR is a key target for cystic fibrosis drug development, and its structural elucidation would advance those efforts. However, the limited in vivo and in vitro stability of the protein, particularly its nucleotide binding domains, has made structural studies challenging. Here we demonstrate that phosphatidylserine uniquely stimulates and thermally stabilizes the ATP hydrolysis function of purified human CFTR. Among several lipids tested, the greatest stabilization was observed with brain phosphatidylserine, which shifted the Tm for ATPase activity from 22.7±0.8°C to 35.0±0.2°C in wild-type CFTR, and from 26.6±0.7°C to 42.1±0.2°C in a more stable mutant CFTR having deleted regulatory insertion and S492P/A534P/I539T mutations. When ATPase activity was measured at 37°C in the presence of brain phosphatidylserine, Vmax for wild-type CFTR was 240±60nmol/min/mg, a rate higher than previously reported and consistent with rates for other purified ABC transporters. The significant thermal stabilization of CFTR by phosphatidylserine may be advantageous in future structural and biophysical studies of CFTR.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fosfatidilserinas/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases , Trifosfato de Adenosina/metabolismo , Sítios de Ligação/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Hidrólise , Mutação/genética , Nucleotídeos/metabolismo , Ligação Proteica/fisiologiaRESUMO
Structural changes in mouse P-glycoprotein (Pgp) induced by thermal unfolding were studied by differential scanning calorimetry (DSC), circular dichroism and fluorescence spectroscopy to gain insight into the solution conformation(s) of this ABC transporter that may not be apparent from current crystal structures. DSC of reconstituted Pgp showed two thermal unfolding transitions in the absence of MgATP, suggesting that each transition involved the cooperative unfolding of two or more interacting structural domains. A low calorimetric unfolding enthalpy and minimal structural changes were observed, which are hallmarks of the thermal unfolding of α-helical membrane proteins, because generally only the extramembranous regions undergo significant unfolding. Nucleotide binding increased the unfolding temperature of both transitions to the same extent, suggesting that one nucleotide binding domain (NBD) unfolds with each transition. Combined with the results from the two isolated NBDs, we propose that each DSC transition represents the cooperative unfolding of one NBD and the two contacting intracellular loops. Further, the presence of two transitions in both apo and MgATP bound wild-type Pgp suggests the NBD-dimeric conformation is transient, and that Pgp resides predominantly in the crystallographically observed inward-facing conformation with NBDs separated, even under conditions supporting continuous MgATP hydrolysis. In contrast, DSC of the vanadate-trapped MgADP·Pgp complex and the MgATP-bound catalytically inactive mutant, E552A/E1197A, show an additional transition at much higher temperature, corresponding to the unfolding of the nucleotide-trapped NBD-dimeric outward-facing conformation. The collective results indicate a strong preference for an NBD dissociated, inward-facing conformation of Pgp.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Lipossomas Unilamelares/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Hidrólise , Cinética , Camundongos , Modelos Moleculares , Pichia/genética , Pichia/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Desdobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia Estrutural de Proteína , Especificidade por Substrato , Termodinâmica , Lipossomas Unilamelares/metabolismoRESUMO
ATP-binding cassette proteins are ubiquitously present throughout all known genomes. Their basic functional unit possesses two transmembrane domains and two nucleotide-binding domains. The nucleotide-binding domains are responsible for ATP binding and hydrolysis, and their 3-dimensional structure is conserved across ATP-binding cassette proteins. Binding of ATP produces nucleotide-binding domain dimerization, a step necessary for hydrolysis. However, the possibility that nucleotide-binding domains bind and/or hydrolyze nucleotide triphosphates different from ATP has not been explored in detail. Here, we studied that possibility using M. jannaschii MJ0796, a prototypical ATP-binding cassette nucleotide-binding domain. We found that nucleotide-binding domain dimerization occurs as a result of binding to the natural nucleotide triphosphates ATP, GTP, CTP and UTP, and also to the analog ATP-γ-S. All the natural nucleotide triphosphates are hydrolyzed at similar rates, whereas ATP-γ-S is not hydrolyzed. We also found that the non-hydrolyzable ATP analog AMP-PNP, frequently assumed to produce the nucleotide-bound conformation, failed to elicit nucleotide-binding domain dimerization. Our results raise the possibility that not all the nucleotide binding sites of nucleotide-binding domains are occupied by ATP under physiological conditions, and that ATP is not always the nucleotide hydrolyzed to dissociate the nucleotide-binding domain dimers.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Sítios de Ligação , Hidrólise , Mutação , Domínios Proteicos , Multimerização ProteicaRESUMO
P-glycoprotein (P-gp) is one of the best-known mediators of drug efflux-based multidrug resistance in many cancers. This validated therapeutic target is a prototypic, plasma membrane resident ATP-Binding Cassette transporter that pumps xenobiotic compounds out of cells. The large, polyspecific drug-binding pocket of P-gp recognizes a variety of structurally unrelated compounds. The transport of these drugs across the membrane is coincident with changes in the size and shape of this pocket during the course of the transport cycle. Here, we present the crystal structures of three inward-facing conformations of mouse P-gp derived from two different crystal forms. One structure has a nanobody bound to the C-terminal side of the first nucleotide-binding domain. This nanobody strongly inhibits the ATP hydrolysis activity of mouse P-gp by hindering the formation of a dimeric complex between the ATP-binding domains, which is essential for nucleotide hydrolysis. Together, these inward-facing conformational snapshots of P-gp demonstrate a range of flexibility exhibited by this transporter, which is likely an essential feature for the binding and transport of large, diverse substrates. The nanobody-bound structure also reveals a unique epitope on P-gp.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Sistemas de Liberação de Medicamentos/métodos , Modelos Moleculares , Conformação Proteica , Animais , Mapeamento de Epitopos , Camundongos , Anticorpos de Domínio Único/químicaRESUMO
Structural knowledge of the cystic fibrosis transmembrane conductance regulator (CFTR) requires developing methods to purify and stabilize this aggregation-prone membrane protein above 1mg/ml. Starting with green fluorescent protein- and epitope-tagged human CFTR produced in mammalian cells known to properly fold and process CFTR, we devised a rapid tandem affinity purification scheme to minimize CFTR exposure to detergent in order to preserve its ATPase function. We compared a panel of detergents, including widely used detergents (maltosides, neopentyl glycols (MNG), C12E8, lysolipids, Chaps) and innovative detergents (branched alkylmaltosides, facial amphiphiles) for CFTR purification, function, monodispersity and stability. ATPase activity after reconstitution into proteoliposomes was 2-3 times higher when CFTR was purified using facial amphiphiles. ATPase activity was also demonstrated in purified CFTR samples without detergent removal using a novel lipid supplementation assay. By electron microscopy, negatively stained CFTR samples were monodisperse at low concentration, and size exclusion chromatography showed a predominance of monomer even after CFTR concentration above 1mg/ml. Rates of CFTR aggregation quantified in an electrophoretic mobility shift assay showed that detergents which best preserved reconstituted ATPase activity also supported the greatest stability, with CFTR monomer half-lives of 6-9days in MNG or Chaps, and 12-17days in facial amphiphile. Cryoelectron microscopy of concentrated CFTR in MNG or facial amphiphile confirmed mostly monomeric protein, producing low resolution reconstructions in conformity with similar proteins. These protocols can be used to generate samples of pure, functional, stable CFTR at concentrations amenable to biophysical characterization.
RESUMO
P-glycoprotein (P-gp) is a transporter of great clinical and pharmacological significance. Several structural studies of P-gp and its homologs have provided insights into its transport cycle, but questions remain regarding how P-gp recognizes diverse substrates and how substrate binding is coupled to ATP hydrolysis. Here, four new P-gp co-crystal structures with a series of rationally designed ligands are presented. It is observed that the binding of certain ligands, including an ATP-hydrolysis stimulator, produces a large conformational change in the fourth transmembrane helix, which is positioned to potentially transmit a signal to the nucleotide-binding domains. A new ligand-binding site on the surface of P-gp facing the inner leaflet of the membrane is also described, providing vital insights regarding the entry mechanism of hydrophobic drugs and lipids into P-gp. These results represent significant advances in the understanding of how P-gp and related transporters bind and export a plethora of metabolites, antibiotics and clinically approved and pipeline drugs.
Assuntos
Trifosfato de Adenosina/química , Subfamília B de Transportador de Cassetes de Ligação de ATP/química , Cristalografia por Raios X , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Multidrug ABC transporters can transport a wide range of drugs from the cell. Ongoing studies of the prototype mammalian multidrug resistance ATP-binding cassette transporter P-glycoprotein (ABCB1) have revealed many intriguing functional and biochemical features. However, a gap remains in our knowledge regarding the molecular basis of its broad specificity for structurally unrelated ligands. Recently, the first crystal structures of ligand-free and ligand-bound ABCB1 showed ligand binding in a cavity between its two membrane domains, and earlier observations on polyspecificity can now be interpreted in a structural context. Comparison of the new ABCB1 crystal structures with structures of bacterial homologs suggests a critical role for an axial rotation of transmembrane helices for high-affinity binding and low-affinity release of ligands during transmembrane transport.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/química , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Bactérias/química , Proteínas de Bactérias/química , Proteínas de Transporte/química , Cristalografia por Raios X , Proteínas Hemolisinas/química , Humanos , Especificidade por SubstratoRESUMO
P-glycoprotein (Pgp) is an important contributor to multidrug resistance of cancer. Pgp contains eleven native tryptophans (Trps) that are highly conserved among orthologs. We replaced each Trp by a conservative substitution to determine which Trps are important for function. Individual Trp mutants W44R, W208Y, W132Y, W704Y and W851Y, situated at the membrane surface, revealed significantly reduced Pgp induced drug resistance against one or more fungicides and/or reduced mating efficiencies in Saccharomyces cerevisiae. W158F and W799F, located in the intracellular coupling helices, abolished mating but retained resistance against most drugs. In contrast, W228F and W311Y, located within the membrane, W694L, at the cytoplasmic membrane interface, and W1104Y in NBD2 retained high levels of drug resistance and mating efficiencies similar to wild-type Pgp. Those were combined into pair (W228F/W311Y and W694L/W1104Y) and quadruple (W228F/W311Y/W694L/W1104Y) mutants that were fully active in yeast, and could be purified to homogeneity. Purified pair and quad mutants exhibited drug-stimulated ATPase activity with binding affinities very similar to wild-type Pgp. The combined mutations reduced Trp fluorescence by 35%, but drug induced fluorescence quenching was unchanged from wild-type Pgp suggesting that several membrane-bound Trps are sensitive to drug binding. Overall, we conclude that Trps at the membrane surface are critical for maintaining the integrity of the drug binding sites, while Trps in the coupling helices are important for proper interdomain communication. We also demonstrate that functional single Trp mutants can be combined to form a fully active Pgp that maintains drug polyspecificity, while significantly reducing intrinsic fluorescence.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Farmacorresistência Fúngica , Triptofano/química , Adenosina Trifosfatases/química , Antifúngicos/farmacologia , Sítios de Ligação , Membrana Celular/metabolismo , Cristalografia por Raios X/métodos , Ciclosporina/farmacologia , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Conformação Molecular , Mutação , Ligação Proteica , Saccharomyces cerevisiae/genética , Tacrolimo/farmacologia , Triptofano/genética , Valinomicina/farmacologia , Verapamil/farmacologiaRESUMO
The sodium-coupled citrate transporter (NaCT, SLC13A5) mediates citrate uptake across the plasma membrane via an inward Na + gradient. Mutations in SLC13A5 cause early infantile epileptic encephalopathy type-25 (EIEE25, SLC13A5 Epilepsy) due to impaired citrate uptake in neurons. Despite clinical identification of disease-causing mutations, underlying mechanisms and cures remain elusive. We mechanistically classify the molecular phenotypes of six mutations. C50R, T142M, and T227M exhibit impaired citrate transport despite normal expression at the cell surface. G219R, S427L, and L488P are hampered by low protein expression, ER retention, and reduced transport. Mutants' mRNA levels resemble wildtype, suggesting post-translational defects. Class II mutations display immature core-glycosylation and shortened half-lives, indicating protein folding defects. These experiments provide a comprehensive understanding of the mutation's defects in SLC13A5 Epilepsy at the biochemical and molecular level and shed light into the trafficking pathway(s) of NaCT. The two classes of mutations will require fundamentally different treatment approaches to either restore transport function, or enable correction of protein folding defects. Summary: Loss-of-function mutations in the SLC13A5 causes SLC13A5-Epilepsy, a devastating disease characterized by neonatal epilepsy. Currently no cure is available. We clarify the molecular-level defects to guide future developments for phenotype-specific treatment of disease-causing mutations.
RESUMO
P-glycoprotein (Pgp) is a multidrug transporter that uses the energy from ATP binding and hydrolysis to export from cells a wide variety of hydrophobic compounds including anticancer drugs, and mediates the bioavailability and pharmacokinetics of many drugs. Lipids and cholesterol have been shown to modulate the substrate-stimulated ATPase activity of purified Pgp in detergent solution and the substrate transport activity after reconstitution into proteoliposomes. While lipid extracts from E. coli, liver or brain tissues generally support well Pgp's functionality, their ill-defined composition and high UV absorbance make them less suitable for optical biophysical assays. On the other hand, studies with defined synthetic lipids, usually the bilayer-forming phosphatidylcholine with or without cholesterol, are often plagued by low ATPase activity and low binding affinity of Pgp for drugs. Drawing from the lipid composition of mammalian plasma membranes, we here investigate how different head groups modulate the verapamil-stimulated ATPase activity of purified Pgp in detergent-lipid micelles and compare them with components of E. coli lipids. Our general approach was to assay modulation of verapamil-stimulation of ATPase activity by artificial lipid mixtures starting with the bilayer-forming palmitoyloyl-phosphatidylcholine (POPC) and -phosphatidylethanolamine (POPE). We show that POPC/POPE supplemented with sphingomyelin (SM), cardiolipin, or phosphatidic acid enhanced the verapamil-stimulated activity (Vmax) and decreased the concentration required for half-maximal activity (EC50). Cholesterol (Chol) and more so its soluble hemisuccinate derivative cholesteryl hemisuccinate substantially decreased EC50, perhaps by supporting the functional integrity of the drug binding sites. High concentrations of CHS (>15%) resulted in a significantly increased basal activity which could be due to binding of CHS to the drug binding site as transport substrate or as activator, maybe acting cooperatively with verapamil. Lastly, Pgp reconstituted into liposomes or nanodiscs displayed higher basal activity and sustained high levels of verapamil stimulated activity. The findings establish a stable source of artificial lipid mixtures containing either SM and cholesterol or CHS that restore Pgp functionality with activities and affinities similar to those in the natural plasma membrane environment and will pave the way for future functional and biophysical studies.
RESUMO
The transporter associated with antigen processing (TAP) plays a key role in adaptive immunity by translocating proteasomal degradation products from the cytosol into the endoplasmic reticulum lumen for subsequent loading onto major histocompatibility (MHC) class I molecules. For functional and structural analysis of this ATP-binding cassette complex, we established the overexpression of TAP in the methylotrophic yeast Pichia pastoris. Screening of optimal solubilization and purification conditions allowed the isolation of the heterodimeric transport complex, yielding 30 mg of TAP/liter of culture. Detailed analysis of TAP function in the membrane, solubilized, purified, and reconstituted states revealed a direct influence of the native lipid environment on activity. TAP-associated phospholipids, essential for function, were profiled by liquid chromatography Fourier transform mass spectrometry. The antigen translocation activity is stimulated by phosphatidylinositol and -ethanolamine, whereas cholesterol has a negative effect on TAP activity.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Lipídeos de Membrana/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/imunologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Humanos , Lipídeos de Membrana/genética , Lipídeos de Membrana/imunologia , Lipídeos de Membrana/metabolismo , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
Normal functions of cell-surface proteins are dependent on their proper trafficking from the site of synthesis to the cell surface. Transport proteins mediating solute transfer across the plasma membrane constitute an important group of cell-surface proteins. There are several diseases resulting from mutations in these proteins that interfere with their transport function or trafficking, depending on the impact of the mutations on protein folding and structure. Recent advances in successful treatment of some of these diseases with small molecules which correct the mutations-induced folding and structural changes underline the need for detailed structural and biophysical characterization of membrane proteins. This requires methods to express and purify these proteins using heterologous expression systems. Here, using the solute carrier (SLC) transporter NaCT (Na+-coupled citrate transporter) as an example, we describe experimental strategies for this approach. We chose this example because several mutations in NaCT, distributed throughout the protein, cause a severe neurologic disease known as early infantile epileptic encephalopathy-25 (EIEE-25). NaCT was modified with various peptide tags, including a RGS-His10, a Twin-Strep, the SUMOstar domain, and an enhanced green fluorescent protein (EGFP), each alone or in various combinations. When transiently expressed in HEK293 cells, recombinant NaCT proteins underwent complex glycosylation, compartmentalized with the plasma membrane, and exhibited citrate transport activity similar to the nontagged protein. Surface NaCT expression was enhanced by the presence of SUMOstar on the N-terminus. The dual-purpose peptide epitopes RGS-His10 and Twin-Strep facilitated detection of NaCT by immunohistochemistry and western blot and may serve useful tags for affinity purification. This approach sets the stage for future analyses of mutant NaCT proteins that may alter protein folding and trafficking. It also demonstrates the capability of a transient mammalian cell expression system to produce human NaCT of sufficient quality and quantity to augment future biophysical and structural studies and drug discovery efforts.
Assuntos
Simportadores , Animais , Transporte Biológico , Membrana Celular/metabolismo , Células HEK293 , Humanos , Mamíferos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Mutantes/metabolismo , Peptídeos/metabolismo , Simportadores/genéticaRESUMO
Most analytical electrophoreses of proteins are achieved by separation in polyacrylamide gels under conditions that ensure dissociation of proteins into individual polypeptide subunits and minimize aggregation. Most commonly, the anionic detergent sodium dodecyl sulfate (SDS) is used in combination with a reducing agent (ß-mercaptoethanol or dithiothreitol) and with heating to dissociate proteins before loading onto the gel. SDS binding denatures the polypeptides and imparts a negative charge that masks their intrinsic charge. The amount of SDS bound is generally sequence-independent and proportional to molecular weight; at saturation, approximately one SDS molecule is bound per two amino acids, or â¼1.4 g of SDS per gram of polypeptide. Therefore, the migration of SDS-polypeptide complexes in an electric field is proportional to the relative size of the polypeptide chain, and its molecular weight can be estimated by comparison to protein markers of known molecular weight. However, hydrophobicity, highly charged sequences, and certain posttranslational modifications such as glycosylation or phosphorylation may also influence migration. Thus, the apparent molecular weight of modified proteins does not always accurately reflect the mass of the polypeptide chain. This protocol describes preparation and running of SDS-PAGE gels, followed by staining to detect proteins using Coomassie Brilliant Blue. Finally, the stained SDS-PAGE gel may be scanned to an image or preserved by drying.
Assuntos
Peptídeos , Proteínas , Eletroforese em Gel de Poliacrilamida , Géis , Peso Molecular , Proteínas/química , Dodecilsulfato de SódioRESUMO
Many variations of the original Coomassie Brilliant Blue staining procedure are in use. This protocol describes some selected variations on the standard procedure that give comparable and consistent staining results for proteins in the 20- to 200-kDa range.