RESUMO
Metabolic adaptation is essential for cell survival during nutrient deprivation. We report that eukaryotic elongation factor 2 kinase (eEF2K), which is activated by AMP-kinase (AMPK), confers cell survival under acute nutrient depletion by blocking translation elongation. Tumor cells exploit this pathway to adapt to nutrient deprivation by reactivating the AMPK-eEF2K axis. Adaptation of transformed cells to nutrient withdrawal is severely compromised in cells lacking eEF2K. Moreover, eEF2K knockdown restored sensitivity to acute nutrient deprivation in highly resistant human tumor cell lines. In vivo, overexpression of eEF2K rendered murine tumors remarkably resistant to caloric restriction. Expression of eEF2K strongly correlated with overall survival in human medulloblastoma and glioblastoma multiforme. Finally, C. elegans strains deficient in efk-1, the eEF2K ortholog, were severely compromised in their response to nutrient depletion. Our data highlight a conserved role for eEF2K in protecting cells from nutrient deprivation and in conferring tumor cell adaptation to metabolic stress. PAPERCLIP:
Assuntos
Caenorhabditis elegans/metabolismo , Quinase do Fator 2 de Elongação/metabolismo , Neoplasias/fisiopatologia , Elongação Traducional da Cadeia Peptídica , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Neoplasias Encefálicas/fisiopatologia , Caenorhabditis elegans/genética , Sobrevivência Celular , Transformação Celular Neoplásica , Quinase do Fator 2 de Elongação/genética , Privação de Alimentos , Glioblastoma/fisiopatologia , Células HeLa , Humanos , Camundongos , Camundongos Nus , Células NIH 3T3 , Transplante de Neoplasias , Fator 2 de Elongação de Peptídeos/metabolismo , Transplante HeterólogoRESUMO
Ion channels play central roles in biology and human health by catalyzing the transmembrane flow of electrical charge. These proteins are ideal targets for genetic code expansion (GCE) methods because it is feasible to measure ion channel activity from miniscule amounts of protein and to analyze the resulting data via rigorous, established biophysical methods. In an ideal scenario, the encoding of synthetic, noncanonical amino acids via GCE allows the experimenter to ask questions inaccessible to traditional methods. For this reason, GCE has been successfully applied to a variety of ligand- and voltage-gated channels wherein extensive structural, functional, and pharmacological data exist. Here, we provide a comprehensive summary of GCE as applied to ion channels. We begin with an overview of the methods used to encode noncanonical amino acids in channels and then describe mechanistic studies wherein GCE was used for photochemistry (cross-linking; caged amino acids) and atomic mutagenesis (isosteric manipulation of charge and aromaticity; backbone mutation). Lastly, we cover recent advances in the encoding of fluorescent amino acids for the real-time study of protein conformational dynamics.
RESUMO
Genetic code expansion has pushed protein chemistry past the canonical 22 amino acids. The key enzymes that make this possible are engineered aminoacyl tRNA synthetases. However, as the number of genetically encoded amino acids has increased over the years, obvious limits in the type and size of novel side chains that can be accommodated by the synthetase enzyme become apparent. Here, we show that chemically acylating tRNAs allow for robust, site-specific incorporation of unnatural amino acids into proteins in zebrafish embryos, an important model organism for human health and development. We apply this approach to incorporate a unique photocaged histidine analogue for which synthetase engineering efforts have failed. Additionally, we demonstrate optical control over different enzymes in live embryos by installing photocaged histidine into their active sites.
Assuntos
Aminoacil-tRNA Sintetases , RNA de Transferência , Peixe-Zebra , Animais , Aminoácidos/química , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Código Genético , Histidina/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Docosahexaenoic acid (DHA), a polyunsaturated ω-3 fatty acid enriched in oily fish, contributes to better health by affecting multiple targets. Large-conductance Ca2+- and voltage-gated Slo1 BK channels are directly activated by nanomolar levels of DHA. We investigated DHA-channel interaction by manipulating both the fatty acid structure and the channel composition through the site-directed incorporation of unnatural amino acids. Electrophysiological measurements show that the para-group of a Tyr residue near the ion conduction pathway has a critical role. To robustly activate the channel, ionization must occur readily by a fatty acid for a good efficacy, and a long nonpolar acyl tail with a Z double bond present at the halfway position for a high affinity. The results suggest that DHA and the channel form an ion-dipole bond to promote opening and demonstrate the channel druggability. DHA, a marine-derived nutraceutical, represents a promising lead compound for rational drug design and discovery.
Assuntos
Ácidos Docosa-Hexaenoicos/química , Ácidos Graxos Ômega-3/química , Ácidos Graxos Insaturados/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Ácidos Docosa-Hexaenoicos/metabolismo , Ácidos Docosa-Hexaenoicos/uso terapêutico , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Insaturados/metabolismo , Ácidos Graxos Insaturados/uso terapêutico , Óleos de Peixe/química , Óleos de Peixe/metabolismo , Humanos , Ativação do Canal Iônico/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Canais de Potássio Ativados por Cálcio de Condutância Alta/química , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismoRESUMO
Oxidative stress plays a key role in late onset diseases including cancer and neurodegenerative diseases such as Huntington disease. Therefore, uncovering regulators of the antioxidant stress responses is important for understanding the course of these diseases. Indeed, the nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of the cellular antioxidative stress response, is deregulated in both cancer and neurodegeneration. Similar to NRF2, the tumor suppressor Homologous to the E6-AP Carboxyl Terminus (HECT) domain and Ankyrin repeat containing E3 ubiquitin-protein ligase 1 (HACE1) plays a protective role against stress-induced tumorigenesis in mice, but its roles in the antioxidative stress response or its involvement in neurodegeneration have not been investigated. To this end we examined Hace1 WT and KO mice and found that Hace1 KO animals exhibited increased oxidative stress in brain and that the antioxidative stress response was impaired. Moreover, HACE1 was found to be essential for optimal NRF2 activation in cells challenged with oxidative stress, as HACE1 depletion resulted in reduced NRF2 activity, stability, and protein synthesis, leading to lower tolerance against oxidative stress triggers. Strikingly, we found a reduction of HACE1 levels in the striatum of Huntington disease patients, implicating HACE1 in the pathology of Huntington disease. Moreover, ectopic expression of HACE1 in striatal neuronal progenitor cells provided protection against mutant Huntingtin-induced redox imbalance and hypersensitivity to oxidative stress, by augmenting NRF2 functions. These findings reveal that the tumor suppressor HACE1 plays a role in the NRF2 antioxidative stress response pathway and in neurodegeneration.
Assuntos
Doença de Huntington/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Western Blotting , Fracionamento Celular , Corpo Estriado/metabolismo , Primers do DNA/genética , Imunofluorescência , Células HEK293 , Humanos , Proteína Huntingtina , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
KEY MESSAGE: Human glucocerebrosidase with vacuolar anchoring domains was targeted to protein storage vacuoles (PSVs) of Arabidopsis seeds, but unexpectedly via the Golgi complex. PSV-targeting to effectively avoid problematic N-glycans is protein dependent. Plant-specific N-glycosylation patterns elaborated within the Golgi complex are a major limitation of using plants to produce biopharmaceuticals as the presence of ß1,2 xylose and/or α1,3 fucose residues on the recombinant glycoprotein can render the product immunogenic if administrated parenterally. A reporter protein fused to a vacuolar membrane targeting motif comprised of the BP-80 transmembrane domain (TMD), and the cytoplasmic tail (CT) of α-tonoplast intrinsic protein (α-TIP) is delivered to protein storage vacuoles (PSVs) of tobacco seeds by ER-derived transport vesicles that bypass the Golgi complex. This prompted us to investigate whether a pharmaceutical glycoprotein is targeted to PSVs using the same targeting sequences, thus avoiding the unwanted plant-Golgi-specific complex N-glycan modifications. The human lysosomal acid ß-glucosidase (glucocerebrosidase; GCase) (EC 3.2.1.45) fused to the BP-80 TMD and α-TIP CT was produced in Arabidopsis thaliana wild-type (Col-0) seeds. The chimeric GCase became localized in PSVs but transited through the Golgi complex, as indicated by biochemical analyses of the recombinant protein's N-glycans. Our findings suggest that use of this PSV-targeting strategy to avoid problematic N-glycan maturation on recombinant therapeutic proteins is not consistently effective, as it is likely protein- and/or species-specific.
Assuntos
Arabidopsis/metabolismo , Membrana Celular/metabolismo , Glucosilceramidase/química , Glucosilceramidase/metabolismo , Polissacarídeos/metabolismo , Proteínas Recombinantes/metabolismo , Vacúolos/metabolismo , Arabidopsis/genética , Enteropeptidase/metabolismo , Complexo de Golgi/metabolismo , Humanos , Plantas Geneticamente Modificadas , Estrutura Terciária de Proteína , Sementes/metabolismo , Sementes/ultraestrutura , Vacúolos/ultraestruturaRESUMO
Binding-activated optical sensors are powerful tools for imaging, diagnostics, and biomolecular sensing. However, biosensor discovery is slow and requires tedious steps in rational design, screening, and characterization. Here we report on a platform that streamlines biosensor discovery and unlocks directed nanosensor evolution through genetically encodable fluorogenic amino acids (FgAAs). Building on the classical knowledge-based semisynthetic approach, we engineer ~15 kDa nanosensors that recognize specific proteins, peptides, and small molecules with up to 100-fold fluorescence increases and subsecond kinetics, allowing real-time and wash-free target sensing and live-cell bioimaging. An optimized genetic code expansion chemistry with FgAAs further enables rapid (~3 h) ribosomal nanosensor discovery via the cell-free translation of hundreds of candidates in parallel and directed nanosensor evolution with improved variant-specific sensitivities (up to ~250-fold) for SARS-CoV-2 antigens. Altogether, this platform could accelerate the discovery of fluorogenic nanosensors and pave the way to modify proteins with other non-standard functionalities for diverse applications.
Assuntos
Aminoácidos , Técnicas Biossensoriais , Corantes Fluorescentes , SARS-CoV-2 , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Humanos , SARS-CoV-2/genética , COVID-19/virologia , Nanotecnologia/métodos , Peptídeos/metabolismo , Peptídeos/química , Peptídeos/genéticaRESUMO
Voltage-sensor domains couple membrane potential to conformational changes in voltage-gated ion channels and phosphatases. Highly coevolved acidic and aromatic side chains assist the transfer of cationic side chains across the transmembrane electric field during voltage sensing. We investigated the functional contribution of negative electrostatic potentials from these residues to channel gating and voltage sensing with unnatural amino acid mutagenesis, electrophysiology, voltage-clamp fluorometry and ab initio calculations. The data show that neutralization of two conserved acidic side chains in transmembrane segments S2 and S3, namely Glu293 and Asp316 in Shaker potassium channels, has little functional effect on conductance-voltage relationships, although Glu293 appears to catalyze S4 movement. Our results suggest that neither Glu293 nor Asp316 engages in electrostatic state-dependent charge-charge interactions with S4, likely because they occupy, and possibly help create, a water-filled vestibule.
Assuntos
Potenciais da Membrana , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Sequência de Aminoácidos , Animais , Ácido Aspártico/química , Ácido Aspártico/genética , Ácido Glutâmico/química , Ácido Glutâmico/genética , Ativação do Canal Iônico/fisiologia , Dados de Sequência Molecular , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Eletricidade Estática , XenopusRESUMO
Phosphoregulation is ubiquitous in biology. Defining the functional roles of individual phosphorylation sites within a multivalent system remains particularly challenging. We have therefore applied a chemical biology approach to light-control the state of single candidate phosphoserines in the canonical anion channel CFTR while simultaneously measuring channel activity. The data show striking non-equivalency among protein kinase A consensus sites, which vary from <10% to >1,000% changes in channel activity upon phosphorylation. Of note, slow phosphorylation of S813 suggests that this site is rate-limiting to the full activation of CFTR. Further, this approach reveals an unexpected coupling between the phosphorylation of S813 and a nearby site, S795. Overall, these data establish an experimental route to understanding roles of specific phosphoserines within complex phosphoregulatory domains. This strategy may be employed in the study of phosphoregulation of other eukaryotic proteins.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fosforilação , Ânions/metabolismoRESUMO
Adrenomedullary chromaffin cells respond to splanchnic (sympathetic) nerve stimulation by releasing stress hormones into the circulation. The signal for hormone secretion is encoded in the neurotransmitters - especially acetylcholine (ACh) and pituitary adenylate cyclase activating polypeptide (PACAP) - that are released into the splanchnic-chromaffin cell synapse. However, functional differences in the effects of ACh and PACAP on the chromaffin cell secretory response are not well defined. Here, selective agonists of PACAP receptors or nicotinic and muscarinic acetylcholine receptors were applied to chromaffin cells. The major differences in the effects of these agents were not on exocytosis, per se, but rather on the steps upstream of exocytosis. In almost every respect, the properties of individual fusion events triggered by PACAP and cholinergic agonists were similar. On the other hand, the properties of the Ca2+ transients evoked by PACAP differed in several ways from those evoked by muscarinic and nicotinic receptor stimulation. A defining feature of the PACAP-stimulated secretory pathway was its dependence on signaling through exchange protein directly activated by cAMP (Epac) and PLCε. However, the absence of PLCε did not disrupt Ca2+ transients evoked by cholinergic agonists. Accordingly, inhibition of Epac activity did not disrupt secretion triggered by acetylcholine or specific agonists of muscarinic and nicotinic receptors. Thus, PACAP and acetylcholine stimulate chromaffin cell secretion via separate and independent pathways. This feature of stimulus-secretion coupling may be important for sustaining hormone release from the adrenal medulla under conditions associated with the sympathetic stress response.
Assuntos
Células Cromafins , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Acetilcolina/metabolismo , Catecolaminas/metabolismo , Catecolaminas/farmacologia , Agonistas Colinérgicos/metabolismo , Agonistas Colinérgicos/farmacologia , Células Cromafins/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hormônios , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Animais , Camundongos , Receptores Colinérgicos/metabolismoRESUMO
The aromatic side-chains of phenylalanine, tyrosine, and tryptophan interact with their environments via both hydrophobic and electrostatic interactions. Determining the extent to which these contribute to protein function and stability is not possible with conventional mutagenesis. Serial fluorination of a given aromatic is a validated method in vitro and in silico to specifically alter electrostatic characteristics, but this approach is restricted to a select few experimental systems. Here, we report a group of pyrrolysine-based aminoacyl-tRNA synthetase/tRNA pairs (tRNA/RS pairs) that enable the site-specific encoding of a varied spectrum of fluorinated phenylalanine amino acids in E. coli and mammalian (HEK 293T) cells. By allowing the cross-kingdom expression of proteins bearing these unnatural amino acids at biochemical scale, these tools may potentially enable the study of biological mechanisms which utilize aromatic interactions in structural and cellular contexts.
Assuntos
Aminoacil-tRNA Sintetases , Fenilalanina , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Halogenação , Fenilalanina/metabolismo , RNA de Transferência/metabolismo , Humanos , Células HEK293RESUMO
Glycine receptors (GlyRs) are chloride channels that mediate fast inhibitory neurotransmission and are members of the pentameric ligand-gated ion channel (pLGIC) family. The interface between the ligand binding domain and the transmembrane domain of pLGICs has been proposed to be crucial for channel gating and is lined by a number of charged and aromatic side chains that are highly conserved among different pLGICs. However, little is known about specific interactions between these residues that are likely to be important for gating in α1 GlyRs. Here we use the introduction of cysteine pairs and the in vivo nonsense suppression method to incorporate unnatural amino acids to probe the electrostatic and hydrophobic contributions of five highly conserved side chains near the interface, Glu-53, Phe-145, Asp-148, Phe-187, and Arg-218. Our results suggest a salt bridge between Asp-148 in loop 7 and Arg-218 in the pre-M1 domain that is crucial for channel gating. We further propose that Phe-145 and Phe-187 play important roles in stabilizing this interaction by providing a hydrophobic environment. In contrast to the equivalent residues in loop 2 of other pLGICs, the negative charge at Glu-53 α1 GlyRs is not crucial for normal channel function. These findings help decipher the GlyR gating pathway and show that distinct residue interaction patterns exist in different pLGICs. Furthermore, a salt bridge between Asp-148 and Arg-218 would provide a possible mechanistic explanation for the pathophysiologically relevant hyperekplexia, or startle disease, mutant Arg-218 â Gln.
Assuntos
Receptores de Glicina/química , Sítio Alostérico , Sequência de Aminoácidos , Biofísica/métodos , Sequência Conservada , DNA Complementar/metabolismo , Eletrofisiologia/métodos , Glicina/química , Humanos , Ligantes , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Reflexo Anormal/genética , Reflexo de Sobressalto/genética , Sais/química , Homologia de Sequência de Aminoácidos , Eletricidade EstáticaRESUMO
There is a clear need for efficient methods to produce protein therapeutics requiring mannose-termination for therapeutic efficacy. Here we report on a unique system for production of active human lysosomal acid ß-glucosidase (glucocerebrosidase, GCase, EC 3.2.1.45) using seeds of the Arabidopsis thaliana complex-glycan-deficient (cgl) mutant, which are deficient in the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101). Gaucher disease is a prevalent lysosomal storage disease in which affected individuals inherit mutations in the gene (GBA1) encoding GCase. A gene cassette optimized for seed expression was used to generate the human enzyme in seeds of the cgl (C5) mutant, and the recombinant GCase was mainly accumulated in the apoplast. Importantly, the enzymatic properties including kinetic parameters, half-maximal inhibitory concentration of isofagomine and thermal stability of the cgl-derived GCase were comparable with those of imiglucerase, a commercially available recombinant human GCase used for enzyme replacement therapy in Gaucher patients. N-glycan structural analyses of recombinant cgl-GCase showed that the majority of the N-glycans (97%) were mannose terminated. Additional purification was required to remove â¼15% of the plant-derived recombinant GCase that possessed potentially immunogenic (xylose- and/or fucose-containing) N-glycans. Uptake of cgl-derived GCase by mouse macrophages was similar to that of imiglucerase. The cgl seed system requires no addition of foreign (non-native) amino acids to the mature recombinant GCase protein, and the dry transgenic seeds represent a stable repository of the therapeutic protein. Other strategies that may completely prevent plant-like complex N-glycans are discussed, including the use of a null cgl mutant.
Assuntos
Arabidopsis/genética , Glucosilceramidase/biossíntese , Plantas Geneticamente Modificadas , Sementes/genética , Animais , Arabidopsis/citologia , Configuração de Carboidratos , Sequência de Carboidratos , Células Cultivadas , Estabilidade Enzimática , Glucosilceramidase/isolamento & purificação , Glucosilceramidase/metabolismo , Glicosilação , Humanos , Cinética , Macrófagos/metabolismo , Manose , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Sementes/citologiaRESUMO
The essential transmembrane Na+ and K+ gradients in animal cells are established by the Na+/K+ pump, a P-type ATPase that exports three Na+ and imports two K+ per ATP hydrolyzed. The mechanism by which the Na+/K+ pump distinguishes between Na+ and K+ at the two membrane sides is poorly understood. Crystal structures identify two sites (sites I and II) that bind Na+ or K+ and a third (site III) specific for Na+. The side chain of a conserved tyrosine at site III of the catalytic α-subunit (Xenopus-α1 Y780) has been proposed to contribute to Na+ binding by cation-π interaction. We substituted Y780 with natural and unnatural amino acids, expressed the mutants in Xenopus oocytes and COS-1 cells, and used electrophysiology and biochemistry to evaluate their function. Substitutions disrupting H-bonds impaired Na+ interaction, while Y780Q strengthened it, likely by H-bond formation. Utilizing the non-sense suppression method previously used to incorporate unnatural derivatives in ion channels, we were able to analyze Na+/K+ pumps with fluorinated tyrosine or phenylalanine derivatives inserted at position 780 to diminish cation-π interaction strength. In line with the results of the analysis of mutants with natural amino acid substitutions, the results with the fluorinated derivatives indicate that Na+-π interaction with the phenol ring at position 780 contributes minimally, if at all, to the binding of Na+. All Y780 substitutions decreased K+ apparent affinity, highlighting that a state-dependent H-bond network is essential for the selectivity switch at sites I and II when the pump changes conformational state.
Assuntos
ATPase Trocadora de Sódio-Potássio , Tirosina , Animais , Sítios de Ligação , Cátions/metabolismo , Potássio/metabolismo , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Chloride homeostasis is regulated in all cellular compartments. CLC-type channels selectively transport Cl- across biological membranes. It is proposed that side-chains of pore-lining residues determine Cl- selectivity in CLC-type channels, but their spatial orientation and contributions to selectivity are not conserved. This suggests a possible role for mainchain amides in selectivity. We use nonsense suppression to insert α-hydroxy acids at pore-lining positions in two CLC-type channels, CLC-0 and bCLC-k, thus exchanging peptide-bond amides with ester-bond oxygens which are incapable of hydrogen-bonding. Backbone substitutions functionally degrade inter-anion discrimination in a site-specific manner. The presence of a pore-occupying glutamate side chain modulates these effects. Molecular dynamics simulations show backbone amides determine ion energetics within the bCLC-k pore and how insertion of an α-hydroxy acid alters selectivity. We propose that backbone-ion interactions are determinants of Cl- specificity in CLC channels in a mechanism reminiscent of that described for K+ channels.
Assuntos
Amidas , Canais IônicosRESUMO
The human voltage-gated proton channel Hv1 is a drug target for cancer, ischemic stroke, and neuroinflammation. It resides on the plasma membrane and endocytic compartments of a variety of cell types, where it mediates outward proton movement and regulates the activity of NOX enzymes. Its voltage-sensing domain (VSD) contains a gated and proton-selective conduction pathway, which can be blocked by aromatic guanidine derivatives such as 2-guanidinobenzimidazole (2GBI). Mutation of Hv1 residue F150 to alanine (F150A) was previously found to increase 2GBI apparent binding affinity more than two orders of magnitude. Here, we explore the contribution of aromatic interactions between the inhibitor and the channel in the presence and absence of the F150A mutation, using a combination of electrophysiological recordings, classic mutagenesis, and site-specific incorporation of fluorinated phenylalanines via nonsense suppression methodology. Our data suggest that the increase in apparent binding affinity is due to a rearrangement of the binding site allowed by the smaller residue at position 150. We used this information to design new arginine mimics with improved affinity for the nonrearranged binding site of the wild-type channel. The new compounds, named "Hv1 Inhibitor Flexibles" (HIFs), consist of two "prongs," an aminoimidazole ring, and an aromatic group connected by extended flexible linkers. Some HIF compounds display inhibitory properties that are superior to those of 2GBI, thus providing a promising scaffold for further development of high-affinity Hv1 inhibitors.
Assuntos
Arginina , Canais Iônicos , Sítios de Ligação , Humanos , Canais Iônicos/metabolismo , Ligantes , PrótonsRESUMO
The CLC family comprises H+-coupled exchangers and Cl- channels, and mutations causing their dysfunction lead to genetic disorders. The CLC exchangers, unlike canonical 'ping-pong' antiporters, simultaneously bind and translocate substrates through partially congruent pathways. How ions of opposite charge bypass each other while moving through a shared pathway remains unknown. Here, we use MD simulations, biochemical and electrophysiological measurements to identify two conserved phenylalanine residues that form an aromatic pathway whose dynamic rearrangements enable H+ movement outside the Cl- pore. These residues are important for H+ transport and voltage-dependent gating in the CLC exchangers. The aromatic pathway residues are evolutionarily conserved in CLC channels where their electrostatic properties and conformational flexibility determine gating. We propose that Cl- and H+ move through physically distinct and evolutionarily conserved routes through the CLC channels and transporters and suggest a unifying mechanism that describes the gating mechanism of both CLC subtypes.
Assuntos
Antiporters/fisiologia , Canais de Cloreto/fisiologia , Cloretos/metabolismo , Ativação do Canal Iônico/fisiologia , Transporte de Íons/fisiologia , Antiporters/química , Canais de Cloreto/química , Proteínas de Escherichia coli/fisiologia , Simulação de Dinâmica Molecular , PrótonsRESUMO
Membrane proteins are universal signal decoders. The helical transmembrane segments of these proteins play central roles in sensory transduction, yet the mechanistic contributions of secondary structure remain unresolved. To investigate the role of main-chain hydrogen bonding on transmembrane function, we encoded amide-to-ester substitutions at sites throughout the S4 voltage-sensing segment of Shaker potassium channels, a region that undergoes rapid, voltage-driven movement during channel gating. Functional measurements of ester-harboring channels highlight a transitional region between α-helical and 310 segments where hydrogen bond removal is particularly disruptive to voltage-gating. Simulations of an active voltage sensor reveal that this region features a dynamic hydrogen bonding pattern and that its helical structure is reliant upon amide support. Overall, the data highlight the specialized role of main-chain chemistry in the mechanism of voltage-sensing; other catalytic transmembrane segments may enlist similar strategies in signal transduction mechanisms.
Assuntos
Simulação de Dinâmica Molecular , Canais de Potássio/química , Canais de Potássio/metabolismo , Ligação de Hidrogênio , Mutagênese/genética , Mutagênese/fisiologia , Canais de Potássio/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Estrutura Secundária de Proteína , Superfamília Shaker de Canais de Potássio/química , Superfamília Shaker de Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/metabolismoRESUMO
Chemical aminoacylation of orthogonal tRNA allows for the genetic encoding of a wide range of synthetic amino acids without the need to evolve specific aminoacyl-tRNA synthetases. This method, when paired with protein expression in the Xenopus laevis oocyte expression system, can extract atomic scale functional data from a protein structure to advance the study of membrane proteins. The utility of the method depends on the orthogonality of the tRNA species used to deliver the amino acid. Here, we report that the pyrrolysyl tRNA (pylT) from Methanosarcina barkeri fusaro is orthogonal and highly competent for genetic code expansion experiments in the Xenopus oocyte. The data show that pylT is amendable to chemical acylation in vitro; it is then used to rescue a cytoplasmic site within a voltage-gated sodium channel. Further, the high fidelity of the pylT is demonstrated via encoding of lysine within the selectivity filter of the sodium channel, where sodium ion recognition by the distal amine of this side-chain is essential. Thus, pylT is an appropriate tRNA species for delivery of amino acids via nonsense suppression in the Xenopus oocyte. It may prove useful in experimental contexts wherein reacylation of suppressor tRNAs have been observed.
Assuntos
Lisina/análogos & derivados , Oócitos/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Xenopus laevis/metabolismo , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Aminoacilação , Animais , Anticódon , Códon de Terminação , Código Genético , Humanos , Lisina/metabolismo , Methanosarcina barkeri/química , Técnicas de Patch-Clamp/métodos , Biossíntese de Proteínas , Ratos , Tetrahymena thermophila/química , Aminoacilação de RNA de Transferência , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
Voltage-dependent activation of voltage-gated cation channels results from the outward movement of arginine-bearing helices within proteinaceous voltage sensors. The voltage-sensing residues in potassium channels have been extensively characterized, but current functional approaches do not allow a distinction between the electrostatic and steric contributions of the arginine side chain. Here we use chemical misacylation and in vivo nonsense suppression to encode citrulline, a neutral and nearly isosteric analogue of arginine, into the voltage sensor of the Shaker potassium channel. We functionally characterize the engineered channels and compare them with those bearing conventional mutations at the same positions. We observe effects on both voltage sensitivity and gating kinetics, enabling dissection of the roles of residue structure versus positive charge in channel function. In some positions, substitution with citrulline causes mild effects on channel activation compared with natural mutations. In contrast, substitution of the fourth S4 arginine with citrulline causes substantial changes in the conductance-voltage relationship and the kinetics of the channel, which suggests that a positive charge is required at this position for efficient voltage sensor deactivation and channel closure. The encoding of citrulline is expected to enable enhanced precision for the study of arginine residues located in crowded transmembrane environments in other membrane proteins. In addition, the method may facilitate the study of citrullination in vivo.