RESUMO
ß-arrestins bind G protein-coupled receptors to terminate G protein signaling and to facilitate other downstream signaling pathways. Using single-molecule fluorescence resonance energy transfer imaging, we show that ß-arrestin is strongly autoinhibited in its basal state. Its engagement with a phosphopeptide mimicking phosphorylated receptor tail efficiently releases the ß-arrestin tail from its N domain to assume distinct conformations. Unexpectedly, we find that ß-arrestin binding to phosphorylated receptor, with a phosphorylation barcode identical to the isolated phosphopeptide, is highly inefficient and that agonist-promoted receptor activation is required for ß-arrestin activation, consistent with the release of a sequestered receptor C tail. These findings, together with focused cellular investigations, reveal that agonism and receptor C-tail release are specific determinants of the rate and efficiency of ß-arrestin activation by phosphorylated receptor. We infer that receptor phosphorylation patterns, in combination with receptor agonism, synergistically establish the strength and specificity with which diverse, downstream ß-arrestin-mediated events are directed.
Assuntos
Fosfopeptídeos , Receptores Acoplados a Proteínas G , Fosfopeptídeos/metabolismo , Fosforilação , Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestina 1/metabolismo , beta-Arrestinas/metabolismoRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that regulates salt and fluid homeostasis across epithelial membranes1. Alterations in CFTR cause cystic fibrosis, a fatal disease without a cure2,3. Electrophysiological properties of CFTR have been analysed for decades4-6. The structure of CFTR, determined in two globally distinct conformations, underscores its evolutionary relationship with other ATP-binding cassette transporters. However, direct correlations between the essential functions of CFTR and extant structures are lacking at present. Here we combine ensemble functional measurements, single-molecule fluorescence resonance energy transfer, electrophysiology and kinetic simulations to show that the two nucleotide-binding domains (NBDs) of human CFTR dimerize before channel opening. CFTR exhibits an allosteric gating mechanism in which conformational changes within the NBD-dimerized channel, governed by ATP hydrolysis, regulate chloride conductance. The potentiators ivacaftor and GLPG1837 enhance channel activity by increasing pore opening while NBDs are dimerized. Disease-causing substitutions proximal (G551D) or distal (L927P) to the ATPase site both reduce the efficiency of NBD dimerization. These findings collectively enable the framing of a gating mechanism that informs on the search for more efficacious clinical therapies.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Humanos , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Cloretos/metabolismo , Fibrose Cística/tratamento farmacológico , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Condutividade Elétrica , Eletrofisiologia , Transferência Ressonante de Energia de Fluorescência , Ativação do Canal Iônico , Multimerização Proteica/genéticaRESUMO
In all species, ribosomes synthesize proteins by faithfully decoding messenger RNA (mRNA) nucleotide sequences using aminoacyl-tRNA substrates. Current knowledge of the decoding mechanism derives principally from studies on bacterial systems1. Although key features are conserved across evolution2, eukaryotes achieve higher-fidelity mRNA decoding than bacteria3. In human, changes in decoding fidelity are linked to ageing and disease and represent a potential point of therapeutic intervention in both viral and cancer treatment4-6. Here we combine single-molecule imaging and cryogenic electron microscopy methods to examine the molecular basis of human ribosome fidelity to reveal that the decoding mechanism is both kinetically and structurally distinct from that of bacteria. Although decoding is globally analogous in both species, the reaction coordinate of aminoacyl-tRNA movement is altered on the human ribosome and the process is an order of magnitude slower. These distinctions arise from eukaryote-specific structural elements in the human ribosome and in the elongation factor eukaryotic elongation factor 1A (eEF1A) that together coordinate faithful tRNA incorporation at each mRNA codon. The distinct nature and timing of conformational changes within the ribosome and eEF1A rationalize how increased decoding fidelity is achieved and potentially regulated in eukaryotic species.
Assuntos
Bactérias , Biossíntese de Proteínas , Humanos , Bactérias/genética , Bactérias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Aminoacil-RNA de Transferência/genética , Aminoacil-RNA de Transferência/metabolismo , Imagem Individual de Molécula , Microscopia Crioeletrônica , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Single-molecule fluorescence resonance energy transfer (smFRET) methods employed to quantify time-dependent compositional and conformational changes within biomolecules require elevated illumination intensities to recover robust photon emission streams from individual fluorophores. Here we show that outside the weak-excitation limit, and in regimes where fluorophores must undergo many rapid cycles of excitation and relaxation, non-fluorescing, excitation-induced triplet states with lifetimes orders of magnitude longer lived than photon-emitting singlet states degrade photon emission streams from both donor and acceptor fluorophores resulting in illumination-intensity-dependent changes in FRET efficiency. These changes are not commonly taken into consideration; therefore, robust strategies to suppress excited state accumulations are required to recover accurate and precise FRET efficiency, and thus distance, estimates. We propose both robust triplet state suppression and data correction strategies that enable the recovery of FRET efficiencies more closely approximating true values, thereby extending the spatial and temporal resolution of smFRET.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Fótons , Corantes Fluorescentes/química , Imagem Individual de Molécula/métodosRESUMO
Peptide-chain elongation during protein synthesis entails sequential aminoacyl-tRNA selection and translocation reactions that proceed rapidly (2-20 per second) and with a low error rate (around 10-3 to 10-5 at each step) over thousands of cycles1. The cadence and fidelity of ribosome transit through mRNA templates in discrete codon increments is a paradigm for movement in biological systems that must hold for diverse mRNA and tRNA substrates across domains of life. Here we use single-molecule fluorescence methods to guide the capture of structures of early translocation events on the bacterial ribosome. Our findings reveal that the bacterial GTPase elongation factor G specifically engages spontaneously achieved ribosome conformations while in an active, GTP-bound conformation to unlock and initiate peptidyl-tRNA translocation. These findings suggest that processes intrinsic to the pre-translocation ribosome complex can regulate the rate of protein synthesis, and that energy expenditure is used later in the translocation mechanism than previously proposed.
Assuntos
Fator G para Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas , Aminoacil-RNA de Transferência/genética , Ribossomos/metabolismo , Códon , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , RNA Mensageiro/genéticaRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that regulates electrolyte and fluid balance in epithelial tissues. While activation of CFTR is vital to treating cystic fibrosis, selective inhibition of CFTR is a potential therapeutic strategy for secretory diarrhea and autosomal dominant polycystic kidney disease. Although several CFTR inhibitors have been developed by high-throughput screening, their modes of action remain elusive. In this study, we determined the structure of CFTR in complex with the inhibitor CFTRinh-172 to an overall resolution of 2.7 Å by cryogenic electron microscopy. We observe that CFTRinh-172 binds inside the pore near transmembrane helix 8, a critical structural element that links adenosine triphosphate hydrolysis with channel gating. Binding of CFTRinh-172 stabilizes a conformation in which the chloride selectivity filter is collapsed, and the pore is blocked from the extracellular side of the membrane. Single-molecule fluorescence resonance energy transfer experiments indicate that CFTRinh-172 inhibits channel gating without compromising nucleotide-binding domain dimerization. Together, these data reconcile previous biophysical observations and provide a molecular basis for the activity of this widely used CFTR inhibitor.
Assuntos
Trifosfato de Adenosina , Regulador de Condutância Transmembrana em Fibrose Cística , Tiazolidinas , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Dimerização , BenzoatosRESUMO
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
In this issue of Molecular Cell, Pantel et al. (2018) identify and synthetically optimize a novel ribosome-targeting antimicrobial from a potentially rich new source of bioactive natural products.
Assuntos
Antibacterianos , Ribossomos , Produtos BiológicosRESUMO
Membrane transporters mediate cellular uptake of nutrients, signaling molecules, and drugs. Their overall mechanisms are often well understood, but the structural features setting their rates are mostly unknown. Earlier single-molecule fluorescence imaging of the archaeal model glutamate transporter homologue GltPh from Pyrococcus horikoshii suggested that the slow conformational transition from the outward- to the inward-facing state, when the bound substrate is translocated from the extracellular to the cytoplasmic side of the membrane, is rate limiting to transport. Here, we provide insight into the structure of the high-energy transition state of GltPh that limits the rate of the substrate translocation process. Using bioinformatics, we identified GltPh gain-of-function mutations in the flexible helical hairpin domain HP2 and applied linear free energy relationship analysis to infer that the transition state structurally resembles the inward-facing conformation. Based on these analyses, we propose an approach to search for allosteric modulators for transporters.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/metabolismo , Proteínas Arqueais/metabolismo , Transporte Biológico/fisiologia , Sistema X-AG de Transporte de Aminoácidos/genética , Archaea/genética , Archaea/metabolismo , Proteínas Arqueais/genética , Transporte Biológico/genética , Biologia Computacional/métodos , Mutação com Ganho de Função/genética , Modelos Moleculares , Pyrococcus horikoshii/genética , Pyrococcus horikoshii/metabolismo , Especificidade por Substrato/genéticaRESUMO
Secondary active transporters, which are vital for a multitude of physiological processes, use the energy of electrochemical ion gradients to power substrate transport across cell membranes1,2. Efforts to investigate their mechanisms of action have been hampered by their slow transport rates and the inherent limitations of ensemble methods. Here we quantify the activity of individual MhsT transporters, which are representative of the neurotransmitter:sodium symporter family of secondary transporters3, by imaging the transport of individual substrate molecules across lipid bilayers at both single- and multi-turnover resolution. We show that MhsT is active only when physiologically oriented and that the rate-limiting step of the transport cycle varies with the nature of the transported substrate. These findings are consistent with an extracellular allosteric substrate-binding site that modulates the rate-limiting aspects of the transport mechanism4,5, including the rate at which the transporter returns to an outward-facing state after the transported substrate is released.
Assuntos
Aminoácidos/metabolismo , Imagem Individual de Molécula , Simportadores/análise , Simportadores/metabolismo , Sítio Alostérico , Aminoácidos/análise , Aminoácidos/química , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Transporte Biológico , Sobrevivência Celular , Transferência Ressonante de Energia de Fluorescência , Interações Hidrofóbicas e Hidrofílicas , Cinética , Bicamadas Lipídicas/metabolismo , Conformação Proteica , Simportadores/químicaRESUMO
The HIV-1 envelope glycoprotein (Env) trimer mediates cell entry and is conformationally dynamic1-8. Imaging by single-molecule fluorescence resonance energy transfer (smFRET) has revealed that, on the surface of intact virions, mature pre-fusion Env transitions from a pre-triggered conformation (state 1) through a default intermediate conformation (state 2) to a conformation in which it is bound to three CD4 receptor molecules (state 3)8-10. It is currently unclear how these states relate to known structures. Breakthroughs in the structural characterization of the HIV-1 Env trimer have previously been achieved by generating soluble and proteolytically cleaved trimers of gp140 Env that are stabilized by a disulfide bond, an isoleucine-to-proline substitution at residue 559 and a truncation at residue 664 (SOSIP.664 trimers)5,11-18. Cryo-electron microscopy studies have been performed with C-terminally truncated Env of the HIV-1JR-FL strain in complex with the antibody PGT15119. Both approaches have revealed similar structures for Env. Although these structures have been presumed to represent the pre-triggered state 1 of HIV-1 Env, this hypothesis has never directly been tested. Here we use smFRET to compare the conformational states of Env trimers used for structural studies with native Env on intact virus. We find that the constructs upon which extant high-resolution structures are based predominantly occupy downstream conformations that represent states 2 and 3. Therefore, the structure of the pre-triggered state-1 conformation of viral Env that has been identified by smFRET and that is preferentially stabilized by many broadly neutralizing antibodies-and thus of interest for the design of immunogens-remains unknown.
Assuntos
Transferência Ressonante de Energia de Fluorescência , HIV-1/química , Imagem Individual de Molécula , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Animais , Anticorpos Neutralizantes/imunologia , Bovinos , Dissulfetos/química , Células HEK293 , HIV-1/genética , HIV-1/imunologia , Humanos , Modelos Moleculares , Mutação , Conformação Proteica , Multimerização Proteica , Estabilidade Proteica , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologiaRESUMO
Argyrins are a family of naturally produced octapeptides that display promising antimicrobial activity against Pseudomonas aeruginosa. Argyrin B (ArgB) has been shown to interact with an elongated form of the translation elongation factor G (EF-G), leading to the suggestion that argyrins inhibit protein synthesis by interfering with EF-G binding to the ribosome. Here, using a combination of cryo-electron microscopy (cryo-EM) and single-molecule fluorescence resonance energy transfer (smFRET), we demonstrate that rather than interfering with ribosome binding, ArgB rapidly and specifically binds EF-G on the ribosome to inhibit intermediate steps of the translocation mechanism. Our data support that ArgB inhibits conformational changes within EF-G after GTP hydrolysis required for translocation and factor dissociation, analogous to the mechanism of fusidic acid, a chemically distinct antibiotic that binds a different region of EF-G. These findings shed light on the mechanism of action of the argyrin-class antibiotics on protein synthesis as well as the nature and importance of rate-limiting, intramolecular conformational events within the EF-G-bound ribosome during late-steps of translocation.
Assuntos
Antibacterianos , Fator G para Elongação de Peptídeos , Antibacterianos/metabolismo , Ácido Fusídico/farmacologia , Humanos , Oligopeptídeos , Fator G para Elongação de Peptídeos/metabolismo , Ribossomos/metabolismo , Translocação GenéticaRESUMO
Class C G protein-coupled receptors (GPCRs) are known to form stable homodimers or heterodimers critical for function, but the oligomeric status of class A and B receptors, which constitute >90% of all GPCRs, remains hotly debated. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful approach with the potential to reveal valuable insights into GPCR organization but has rarely been used in living cells to study protein systems. Here, we report generally applicable methods for using smFRET to detect and track transmembrane proteins diffusing within the plasma membrane of mammalian cells. We leverage this in-cell smFRET approach to show agonist-induced structural dynamics within individual metabotropic glutamate receptor dimers. We apply these methods to representative class A, B and C receptors, finding evidence for receptor monomers, density-dependent dimers and constitutive dimers, respectively.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Receptores Acoplados a Proteínas G/metabolismo , Dimerização , Conformação Proteica , Receptores Acoplados a Proteínas G/químicaRESUMO
Translocation of messenger RNA (mRNA) and transfer RNA (tRNA) substrates through the ribosome during protein synthesis, an exemplar of directional molecular movement in biology, entails a complex interplay of conformational, compositional, and chemical changes. The molecular determinants of early translocation steps have been investigated rigorously. However, the elements enabling the ribosome to complete translocation and reset for subsequent protein synthesis reactions remain poorly understood. Here, we have combined molecular simulations with single-molecule fluorescence resonance energy transfer imaging to gain insights into the rate-limiting events of the translocation mechanism. We find that diffusive motions of the ribosomal small subunit head domain to hyper-swivelled positions, governed by universally conserved rRNA, can maneuver the mRNA and tRNAs to their fully translocated positions. Subsequent engagement of peptidyl-tRNA and disengagement of deacyl-tRNA from mRNA, within their respective small subunit binding sites, facilitate the ribosome resetting mechanism after translocation has occurred to enable protein synthesis to resume.
Assuntos
Fator G para Elongação de Peptídeos , Ribossomos , Fator G para Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/química , RNA de Transferência/metabolismo , Ribossomos/metabolismoRESUMO
Transporters cycle through large structural changes to translocate molecules across biological membranes. The temporal relationships between these changes and function, and the molecular properties setting their rates, determine transport efficiency-yet remain mostly unknown. Using single-molecule fluorescence microscopy, we compare the timing of conformational transitions and substrate uptake in the elevator-type transporter GltPh We show that the elevator-like movements of the substrate-loaded transport domain across membranes and substrate release are kinetically heterogeneous, with rates varying by orders of magnitude between individual molecules. Mutations increasing the frequency of elevator transitions and reducing substrate affinity diminish transport rate heterogeneities and boost transport efficiency. Hydrogen deuterium exchange coupled to mass spectrometry reveals destabilization of secondary structure around the substrate-binding site, suggesting that increased local dynamics leads to faster rates of global conformational changes and confers gain-of-function properties that set transport rates.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/metabolismo , Proteínas Arqueais/metabolismo , Membrana Celular/metabolismo , Medição da Troca de Deutério , Sequência de Aminoácidos , Sistema X-AG de Transporte de Aminoácidos/genética , Proteínas Arqueais/genética , Transporte Biológico , Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Espectrometria de Massas , Mutação , Ligação Proteica , Imagem Individual de MoléculaRESUMO
G-protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiology and disease intervention, yet the molecular mechanisms responsible for ligand-dependent signalling responses remain poorly understood. In class A GPCRs, receptor activation and G-protein coupling entail outward movements of transmembrane helix 6 (TM6). Here, using single-molecule fluorescence resonance energy transfer imaging, we examine TM6 movements in the ß2 adrenergic receptor (ß2AR) upon exposure to orthosteric ligands with different efficacies, in the absence and presence of the Gs heterotrimer. We show that partial and full agonists differentially affect TM6 motions to regulate the rate at which GDP-bound ß2AR-Gs complexes are formed and the efficiency of nucleotide exchange leading to Gs activation. These data also reveal transient nucleotide-bound ß2AR-Gs species that are distinct from known structures, and provide single-molecule perspectives on the allosteric link between ligand- and nucleotide-binding pockets that shed new light on the G-protein activation mechanism.
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Imagem Individual de Molécula , Agonistas de Receptores Adrenérgicos beta 2/química , Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Sítio Alostérico , Membrana Celular/metabolismo , Clembuterol/química , Clembuterol/metabolismo , Clembuterol/farmacologia , Ativação Enzimática/efeitos dos fármacos , Epinefrina/química , Epinefrina/metabolismo , Epinefrina/farmacologia , Transferência Ressonante de Energia de Fluorescência , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Guanosina Difosfato/metabolismo , Humanos , Cinética , Ligantes , Modelos Moleculares , Movimento/efeitos dos fármacos , Estabilidade Proteica , Receptores Adrenérgicos beta 2/químicaRESUMO
Phosphorylation-type (P-type) ATPases are ubiquitous primary transporters that pump cations across cell membranes through the formation and breakdown of a phosphoenzyme intermediate. Structural investigations suggest that the transport mechanism is defined by conformational changes in the cytoplasmic domains of the protein that are allosterically coupled to transmembrane helices so as to expose ion binding sites to alternate sides of the membrane. Here, we have used single-molecule fluorescence resonance energy transfer to directly observe conformational changes associated with the functional transitions in the Listeria monocytogenes Ca2+-ATPase (LMCA1), an orthologue of eukaryotic Ca2+-ATPases. We identify key intermediates with no known crystal structures and show that Ca2+ efflux by LMCA1 is rate-limited by phosphoenzyme formation. The transport process involves reversible steps and an irreversible step that follows release of ADP and extracellular release of Ca2+.
Assuntos
Trifosfato de Adenosina/metabolismo , ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/metabolismo , Transferência Ressonante de Energia de Fluorescência , Listeria monocytogenes/enzimologia , Imagem Individual de Molécula , Difosfato de Adenosina/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Cinética , Modelos Moleculares , Fosforilação , Conformação ProteicaRESUMO
The increase in multi-drug-resistant bacteria is limiting the effectiveness of currently approved antibiotics, leading to a renewed interest in antibiotics with distinct chemical scaffolds. We have solved the structures of the Thermus thermophilus 70S ribosome with A-, P-, and E-site tRNAs bound and in complex with either the aminocyclitol-containing antibiotic hygromycin A (HygA) or the nucleoside antibiotic A201A. Both antibiotics bind at the peptidyl transferase center and sterically occlude the CCA-end of the A-tRNA from entering the A site of the peptidyl transferase center. Single-molecule Förster resonance energy transfer (smFRET) experiments reveal that HygA and A201A specifically interfere with full accommodation of the A-tRNA, leading to the presence of tRNA accommodation intermediates and thereby inhibiting peptide bond formation. Thus, our results provide not only insight into the mechanism of action of HygA and A201A, but also into the fundamental process of tRNA accommodation during protein synthesis.
Assuntos
Aminoglicosídeos/química , Antibacterianos/química , Cinamatos/química , Higromicina B/análogos & derivados , RNA de Transferência/química , Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/química , Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Cinamatos/farmacologia , Cristalografia por Raios X , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Ligação de Hidrogênio , Higromicina B/química , Higromicina B/farmacologia , Modelos Moleculares , Conformação Proteica , Thermus thermophilusRESUMO
The regulation of protein synthesis contributes to gene expression in both normal physiology and disease, yet kinetic investigations of the human translation mechanism are currently lacking. Using single-molecule fluorescence imaging methods, we have quantified the nature and timing of structural processes in human ribosomes during single-turnover and processive translation reactions. These measurements reveal that functional complexes exhibit dynamic behaviors and thermodynamic stabilities distinct from those observed for bacterial systems. Structurally defined sub-states of pre- and post-translocation complexes were sensitive to specific inhibitors of the eukaryotic ribosome, demonstrating the utility of this platform to probe drug mechanism. The application of three-color single-molecule fluorescence resonance energy transfer (smFRET) methods further revealed a long-distance allosteric coupling between distal tRNA binding sites within ribosomes bearing three tRNAs, which contributed to the rate of processive translation.