RESUMO
CorA, the major Mg(2+) uptake system in prokaryotes, is gated by intracellular Mg(2+) (KD â¼ 1-2 mM). X-ray crystallographic studies of CorA show similar conformations under Mg(2+)-bound and Mg(2+)-free conditions, but EPR spectroscopic studies reveal large Mg(2+)-driven quaternary conformational changes. Here, we determined cryo-EM structures of CorA in the Mg(2+)-bound closed conformation and in two open Mg(2+)-free states at resolutions of 3.8, 7.1, and 7.1 Å, respectively. In the absence of bound Mg(2+), four of the five subunits are displaced to variable extents (â¼ 10-25 Å) by hinge-like motions as large as â¼ 35° at the stalk helix. The transition between a single 5-fold symmetric closed state and an ensemble of low Mg(2+), open, asymmetric conformational states is, thus, the key structural signature of CorA gating. This mechanism is likely to apply to other structurally similar divalent ion channels.
Assuntos
Proteínas de Bactérias/ultraestrutura , Proteínas de Transporte de Cátions/ultraestrutura , Magnésio/metabolismo , Thermotoga maritima/química , Proteínas de Bactérias/química , Proteínas de Transporte de Cátions/química , Microscopia Crioeletrônica , Modelos Moleculares , Simulação de Dinâmica MolecularRESUMO
T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells1-3. Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRß). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Edição de Genes , Neoplasias , Medicina de Precisão , Receptores de Antígenos de Linfócitos T , Linfócitos T , Transgenes , Humanos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Biópsia , Terapia Baseada em Transplante de Células e Tecidos/efeitos adversos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Síndrome da Liberação de Citocina/complicações , Progressão da Doença , Encefalite/complicações , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Genes Codificadores da Cadeia alfa de Receptores de Linfócitos T , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Mutação , Neoplasias/complicações , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/terapia , Segurança do Paciente , Medicina de Precisão/efeitos adversos , Medicina de Precisão/métodos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transgenes/genética , Antígenos HLA/imunologia , Sistemas CRISPR-CasRESUMO
Neoantigens are peptides derived from non-synonymous mutations presented by human leukocyte antigens (HLAs), which are recognized by antitumour T cells1-14. The large HLA allele diversity and limiting clinical samples have restricted the study of the landscape of neoantigen-targeted T cell responses in patients over their treatment course. Here we applied recently developed technologies15-17 to capture neoantigen-specific T cells from blood and tumours from patients with metastatic melanoma with or without response to anti-programmed death receptor 1 (PD-1) immunotherapy. We generated personalized libraries of neoantigen-HLA capture reagents to single-cell isolate the T cells and clone their T cell receptors (neoTCRs). Multiple T cells with different neoTCR sequences (T cell clonotypes) recognized a limited number of mutations in samples from seven patients with long-lasting clinical responses. These neoTCR clonotypes were recurrently detected over time in the blood and tumour. Samples from four patients with no response to anti-PD-1 also demonstrated neoantigen-specific T cell responses in the blood and tumour to a restricted number of mutations with lower TCR polyclonality and were not recurrently detected in sequential samples. Reconstitution of the neoTCRs in donor T cells using non-viral CRISPR-Cas9 gene editing demonstrated specific recognition and cytotoxicity to patient-matched melanoma cell lines. Thus, effective anti-PD-1 immunotherapy is associated with the presence of polyclonal CD8+ T cells in the tumour and blood specific for a limited number of immunodominant mutations, which are recurrently recognized over time.
Assuntos
Antígenos de Neoplasias , Linfócitos T CD8-Positivos , Inibidores de Checkpoint Imunológico , Imunoterapia , Melanoma , Humanos , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/imunologia , Melanoma/patologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Antígenos HLA/imunologia , Metástase Neoplásica , Medicina de Precisão , Edição de Genes , Sistemas CRISPR-Cas , MutaçãoRESUMO
Magnesium (Mg(2+)) plays a central role in biology, regulating the activity of many enzymes and stabilizing the structure of key macromolecules. In bacteria, CorA is the primary source of Mg(2+) uptake and is self-regulated by intracellular Mg(2+). Using a gating mutant at the divalent ion binding site, we were able to characterize CorA selectivity and permeation properties to both monovalent and divalent cations under perfused two-electrode voltage clamp. The present data demonstrate that under physiological conditions, CorA is a multioccupancy Mg(2+)-selective channel, fully excluding monovalent cations, and Ca(2+), whereas in absence of Mg(2+), CorA is essentially nonselective, displaying only mild preference against other divalents (Ca(2+) > Mn(2+) > Co(2+) > Mg(2+) > Ni(2)(+)). Selectivity against monovalent cations takes place via Mg(2+) binding at a high-affinity site, formed by the Gly-Met-Asn signature sequence (Gly312 and Asn314) at the extracellular side of the pore. This mechanism is reminiscent of repulsion models proposed for Ca(2+) channel selectivity despite differences in sequence and overall structure.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Permeabilidade da Membrana Celular/fisiologia , Magnésio/metabolismo , Modelos Moleculares , Thermotoga maritima/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Biologia Computacional , Primers do DNA/genética , Vetores Genéticos , Dados de Sequência Molecular , Oócitos/metabolismo , Técnicas de Patch-Clamp , Alinhamento de Sequência , Eletricidade Estática , Thermotoga maritima/química , Thermotoga maritima/metabolismo , Xenopus laevisRESUMO
The coupled interplay between activation and inactivation gating is a functional hallmark of K(+) channels. This coupling has been experimentally demonstrated through ion interaction effects and cysteine accessibility, and is associated with a well defined boundary of energetically coupled residues. The structure of the K(+) channel KcsA in its fully open conformation, in addition to four other partial channel openings, richly illustrates the structural basis of activation-inactivation gating. Here, we identify the mechanistic principles by which movements on the inner bundle gate trigger conformational changes at the selectivity filter, leading to the non-conductive C-type inactivated state. Analysis of a series of KcsA open structures suggests that, as a consequence of the hinge-bending and rotation of the TM2 helix, the aromatic ring of Phe 103 tilts towards residues Thr 74 and Thr 75 in the pore-helix and towards Ile 100 in the neighbouring subunit. This allows the network of hydrogen bonds among residues Trp 67, Glu 71 and Asp 80 to destabilize the selectivity filter, allowing entry to its non-conductive conformation. Mutations at position 103 have a size-dependent effect on gating kinetics: small side-chain substitutions F103A and F103C severely impair inactivation kinetics, whereas larger side chains such as F103W have more subtle effects. This suggests that the allosteric coupling between the inner helical bundle and the selectivity filter might rely on straightforward mechanical deformation propagated through a network of steric contacts. Average interactions calculated from molecular dynamics simulations show favourable open-state interaction-energies between Phe 103 and the surrounding residues. We probed similar interactions in the Shaker K(+) channel where inactivation was impaired in the mutant I470A. We propose that side-chain rearrangements at position 103 mechanically couple activation and inactivation in KcsA and a variety of other K(+) channels.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ativação do Canal Iônico , Canais de Potássio/química , Canais de Potássio/metabolismo , Streptomyces lividans/química , Regulação Alostérica , Proteínas de Bactérias/genética , Cisteína/genética , Cisteína/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Simulação de Dinâmica Molecular , Fenilalanina/metabolismo , Canais de Potássio/genética , Conformação Proteica , Superfamília Shaker de Canais de Potássio/química , Superfamília Shaker de Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/metabolismo , Relação Estrutura-AtividadeRESUMO
CorA, the primary magnesium ion channel in prokaryotes and archaea, is a prototypical homopentameric ion channel that undergoes ion-dependent conformational transitions. CorA adopts five-fold symmetric non-conductive states in the presence of high concentrations of Mg 2+ , and highly asymmetric flexible states in its complete absence. However, the latter were of insufficient resolution to be thoroughly characterized. In order to gain additional insights into the relationship between asymmetry and channel activation, we exploited phage display selection strategies to generate conformation-specific synthetic antibodies (sABs) against CorA in the absence of Mg 2+ . Two sABs from these selections, C12 and C18, showed different degrees of Mg 2+ -sensitivity. Through structural, biochemical, and biophysical characterization, we found the sABs are both conformation-specific but probe different features of the channel under open-like conditions. C18 is highly specific to the Mg 2+ -depleted state of CorA and through negative-stain electron microscopy (ns-EM), we show sAB binding reflects the asymmetric arrangement of CorA protomers in Mg 2+ -depleted conditions. We used X-ray crystallography to determine a structure at 2.0 Å resolution of sAB C12 bound to the soluble N-terminal regulatory domain of CorA. The structure shows C12 is a competitive inhibitor of regulatory magnesium binding through its interaction with the divalent cation sensing site. We subsequently exploited this relationship to capture and visualize asymmetric CorA states in different [Mg 2+ ] using ns-EM. We additionally utilized these sABs to provide insights into the energy landscape that governs the ion-dependent conformational transitions of CorA.
RESUMO
CorA, the primary magnesium ion channel in prokaryotes and archaea, is a prototypical homopentameric ion channel that undergoes ion-dependent conformational transitions. CorA adopts five-fold symmetric non-conductive states in the presence of high concentrations of Mg2+, and highly asymmetric flexible states in its complete absence. However, the latter were of insufficient resolution to be thoroughly characterized. In order to gain additional insights into the relationship between asymmetry and channel activation, we exploited phage display selection strategies to generate conformation-specific synthetic antibodies (sABs) against CorA in the absence of Mg2+. Two sABs from these selections, C12 and C18, showed different degrees of Mg2+-sensitivity. Through structural, biochemical, and biophysical characterization, we found the sABs are both conformation-specific but probe different features of the channel under open-like conditions. C18 is highly specific to the Mg2+-depleted state of CorA and through negative-stain electron microscopy (ns-EM), we show sAB binding reflects the asymmetric arrangement of CorA protomers in Mg2+-depleted conditions. We used X-ray crystallography to determine a structure at 2.0 Å resolution of sAB C12 bound to the soluble N-terminal regulatory domain of CorA. The structure shows C12 is a competitive inhibitor of regulatory magnesium binding through its interaction with the divalent cation sensing site. We subsequently exploited this relationship to capture and visualize asymmetric CorA states in different [Mg2+] using ns-EM. We additionally utilized these sABs to provide insights into the energy landscape that governs the ion-dependent conformational transitions of CorA.
Assuntos
Anticorpos , Proteínas de Bactérias , Proteínas de Transporte de Cátions , Canais Iônicos , Magnésio , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Canais Iônicos/química , Canais Iônicos/imunologia , Magnésio/química , Magnésio/metabolismo , Conformação Proteica , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/imunologia , Anticorpos/químicaRESUMO
Distance determination from an echo intensity modulation obtained by pulsed double electron-electron resonance (DEER) experiment is a mathematically ill-posed problem. Tikhonov regularization yields distance distributions that can be difficult to interpret, especially in a system with multiple discrete distance distributions. Here, we show that by using geometric fit constraints in symmetric homo-oligomeric protein systems, we were able to increase the accuracy of a model-based fit solution based on a sum of Rice distributions. Our approach was validated on two different ion channels of known oligomeric states, KcsA (tetramer) and CorA (pentamer). Statistical analysis of the resulting fits was integrated within our method to help the experimenter evaluate the significance of a symmetry-constrained vs standard model distribution fit and to examine multidistance confidence regions. This approach was used to quantitatively evaluate the role of the C-terminal domain (CTD) on the flexibility and conformation of the activation gate of the K(+) channel KcsA. Our analysis reveals a significant increase in the dynamics of the inner bundle gate upon opening. Also, it explicitly demonstrates the degree to which the CTD restricts the motion of the lower gate at rest and during activation gating.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Ativação do Canal Iônico , Canais de Potássio/química , Canais de Potássio/metabolismo , Modelos Moleculares , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Reprodutibilidade dos Testes , Streptomyces lividansRESUMO
Outer membrane proteins (OMPs) in Gram-negative bacteria dictate permeability of metabolites, antibiotics, and toxins. Elucidating the structure-function relationships governing OMPs within native membrane environments remains challenging. We constructed a diverse library of >3000 monoclonal antibodies to assess the roles of extracellular loops (ECLs) in LptD, an essential OMP that inserts lipopolysaccharide into the outer membrane of Escherichia coli. Epitope binning and mapping experiments with LptD-loop-deletion mutants demonstrated that 7 of the 13 ECLs are targeted by antibodies. Only ECLs inaccessible to antibodies were required for the structure or function of LptD. Our results suggest that antibody-accessible loops evolved to protect key extracellular regions of LptD, but are themselves dispensable. Supporting this hypothesis, no α-LptD antibody interfered with essential functions of LptD. Our experimental workflow enables structure-function studies of OMPs in native cellular environments, provides unexpected insight into LptD, and presents a method to assess the therapeutic potential of antibody targeting.
Assuntos
Anticorpos Monoclonais/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Animais , Antibacterianos/farmacologia , Sítios de Ligação , Mapeamento de Epitopos , Epitopos/metabolismo , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Camundongos Endogâmicos BALB C , Estrutura Secundária de Proteína , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
Ghrelin, an appetite-stimulatory hormone secreted by the stomach, was discovered as a ligand for the growth hormone secretagogue receptor (GHSR). Through GHSR, ghrelin stimulates growth hormone (GH) secretion, a function that evolved to protect against starvation-induced hypoglycemia. Though the biology mediated by ghrelin has been described in great detail, regulation of ghrelin action is poorly understood. Here, we report the discovery of liver-expressed antimicrobial peptide 2 (LEAP2) as an endogenous antagonist of GHSR. LEAP2 is produced in the liver and small intestine, and its secretion is suppressed by fasting. LEAP2 fully inhibits GHSR activation by ghrelin and blocks the major effects of ghrelin in vivo, including food intake, GH release, and maintenance of viable glucose levels during chronic caloric restriction. In contrast, neutralizing antibodies that block endogenous LEAP2 function enhance ghrelin action in vivo. Our findings reveal a mechanism for fine-tuning ghrelin action in response to changing environmental conditions.
Assuntos
Hepcidinas/metabolismo , Receptores de Grelina/antagonistas & inibidores , Animais , Cirurgia Bariátrica , Restrição Calórica , Ingestão de Alimentos , Jejum , Feminino , Grelina/antagonistas & inibidores , Grelina/metabolismo , Hormônio do Crescimento/metabolismo , Humanos , Intestino Delgado/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Ligação Proteica , Ratos , Receptores de Grelina/metabolismoRESUMO
A major challenge in membrane biophysics is to define the mechanistic linkages between a protein's conformational transitions and its function. We describe a novel approach to stabilize transient functional states of membrane proteins in native-like lipid environments allowing for their structural and biochemical characterization. This is accomplished by combining the power of antibody Fab-based phage display selection with the benefits of embedding membrane protein targets in lipid-filled nanodiscs. In addition to providing a stabilizing lipid environment, nanodiscs afford significant technical advantages over detergent-based formats. This enables the production of a rich pool of high-performance Fab binders that can be used as crystallization chaperones, as fiducial markers for single-particle cryoelectron microscopy, and as probes of different conformational states. Moreover, nanodisc-generated Fabs can be used to identify detergents that best mimic native membrane environments for use in biophysical studies.
Assuntos
Técnicas de Visualização da Superfície Celular/métodos , Fragmentos de Imunoglobulinas/química , Proteínas de Membrana/química , Chaperonas Moleculares/química , Nanoestruturas/química , Detergentes/química , Bicamadas Lipídicas/química , Modelos Moleculares , Biblioteca de Peptídeos , Conformação ProteicaRESUMO
ATP-binding cassette (ABC) transporters have often been refractory to over-expression. Using the C41(DE3) E. coli as a host strain, membrane vesicles highly enriched (>50%) in YvcC, a previously uncharacterized ABC transporter from Bacillus subtilis homologous to P-glycoprotein multidrug transporters, were obtained. The functionality of YvcC was assessed by its high vanadate-sensitive ATPase activity and its ability to transport a fluorescent drug, the Hoechst 33342.
Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Bacillus subtilis/genética , Proteínas da Membrana Bacteriana Externa/biossíntese , Escherichia coli/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Benzimidazóis/metabolismo , Escherichia coli/genética , Corantes Fluorescentes/metabolismo , VanadatosRESUMO
CorA is the major transport system responsible for Mg(2+) uptake in bacteria and can functionally substitute for its homologue Mrs2p in the yeast inner mitochondrial membrane. Although several CorA crystal structures are available, the molecular mechanism of Mg(2+) uptake remains to be established. Here we use electron paramagnetic resonance spectroscopy, electrophysiology and molecular dynamic simulations to show that CorA is regulated by cytoplasmic Mg(2+) acting as a ligand and elucidate the basic conformational rearrangements responsible for Mg(2+)-dependent gating. Mg(2+) unbinding at the divalent cation sensor triggers a conformational change that leads to the inward motion of the stalk helix, which propagates to the pore-forming transmembrane helix TM1. Helical tilting and rotation in TM1 generates an iris-like motion that increases the diameter of the permeation pathway, triggering ion conduction. This work establishes the molecular basis of a Mg(2+)-driven negative feedback loop in CorA as the key physiological event controlling Mg(2+) uptake and homeostasis in prokaryotes.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Magnésio/metabolismo , Thermotoga maritima/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Transporte Biológico , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Citoplasma/metabolismo , Regulação Bacteriana da Expressão Gênica , Íons/metabolismo , Estrutura Secundária de Proteína , Thermotoga maritima/química , Thermotoga maritima/genética , Regulação para CimaRESUMO
The transmembrane conformation of Thermotoga maritima CorA, a magnesium transport system, has been studied in its Mg(2+)-bound form by site-directed spin labeling and electron paramagnetic resonance spectroscopy. Probe mobility together with accessibility data were used to evaluate the overall dynamics and relative arrangement of individual transmembrane segments TM1 and TM2. TM1 extends toward the cytoplasmic side creating a water-filled cavity, while TM2 is located in the periphery of the oligomer, contacting the lipid bilayer. A structural model for the conserved extracellular loop was generated based on EPR data and MD simulations, in which residue E316 is located toward the five-fold symmetry axis in position to electrostatically influence divalent ion translocation. Electrostatic analysis of our model suggest that, in agreement with the crystal structure, Mg(2+) -bound CorA is in a closed conformation. The present results suggest that long-range structural rearrangements are necessary to allow Mg(2+) translocation.
Assuntos
Proteínas de Transporte de Cátions/química , Bicamadas Lipídicas/química , Magnésio/química , Modelos Moleculares , Conformação Proteica , Thermotoga maritima/química , Ácido Edético/análogos & derivados , Ácido Edético/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Oxigênio/metabolismo , Marcadores de Spin , Eletricidade EstáticaRESUMO
The ATP-binding cassette is the most abundant family of transporters including many medically relevant members and gathers both importers and exporters involved in the transport of a wide variety of substrates. Although three high resolution three-dimensional structures have been obtained for a prototypic exporter, MsbA, two have been subjected to much criticism. Here, conformational changes of BmrA, a multidrug bacterial transporter structurally related to MsbA, have been studied. A three-dimensional model of BmrA, based on the "open" conformation of Escherichia coli MsbA, was probed by simultaneously introducing two cysteine residues, one in the first intracellular loop of the transmembrane domain and the other in the Q-loop of the nucleotide-binding domain (NBD). Intramolecular disulfide bonds could be created in the absence of any effectors, which prevented both drug transport and ATPase activity. Interestingly, addition of ATP/Mg plus vanadate strongly prevented this bond formation in a cysteine double mutant, whereas ATP/Mg alone was sufficient when the ATPase-inactive E504Q mutation was also introduced, in agreement with additional BmrA models where the ATP-binding sites are positioned at the NBD/NBD interface. Furthermore, cross-linking between the two cysteine residues could still be achieved in the presence of ATP/Mg plus vanadate when homobifunctional cross-linkers separated by more than 13 Angstrom were added. Altogether, these results give support to the existence, in the resting state, of a monomeric conformation of BmrA similar to that found within the open MsbA dimer and show that a large motion is required between intracellular loop 1 and the nucleotide-binding domain for the proper functioning of a multidrug ATP-binding cassette transporter.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Benzimidazóis/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Dobramento de Proteína , Transportadores de Cassetes de Ligação de ATP/química , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Transporte Biológico , Catálise , Reagentes de Ligações Cruzadas/metabolismo , Dimerização , Dissulfetos , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
Members of the ATP-binding cassette (ABC) transporters share the same basic architecture, with a four-core domain made of two transmembrane plus two nucleotide-binding domains. However, a supramolecular organization has been detected in some ABC transporters, which might be relevant to physiological regulation of substrate transport. Here, the oligomerization status of a bacterial half-ABC multidrug transporter, BmrA, was investigated. Each BmrA monomer containing a single cysteine residue introduced close to either the Walker A or the ABC signature motifs was labeled using two probes, 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (fluorescence donor) or 4-dimethylaminophenylazophenyl-4'-maleimide (fluorescence acceptor). Reconstitution into proteoliposomes of BmrA monomers labeled separately with either the fluorescence donor or the fluorescence acceptor allowed measurement of time-resolved fluorescence resonance energy transfer between the two probes, showing that efficient reassociation of the singly labeled BmrA monomers occurred upon reconstitution. The efficiency of energy transfer studied as a function of increasing concentration of BmrA-labeled with the fluorescence acceptor argues for a dimeric association of BmrA instead of a tetrameric one. Furthermore, the efficiency of energy transfer allowed estimation of the distances between the two bound probes. Results suggest that, in the resting state, BmrA in a lipid bilayer environment preferentially adopts a closed conformation similar to that found in the BtuCD crystal structure and that the presence of different effectors does not substantially modify its global conformation.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , p-Dimetilaminoazobenzeno/análogos & derivados , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Dimerização , Proteínas de Membrana Transportadoras/genética , Modelos Químicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , Conformação Proteica , Proteolipídeos/química , Proteolipídeos/metabolismo , p-Dimetilaminoazobenzeno/metabolismoRESUMO
ATP-binding cassette (ABC) proteins constitute one of the widest families in all organisms, whose P-glycoprotein involved in resistance of cancer cells to chemotherapy is an archetype member. Although three-dimensional structures of several nucleotide-binding domains of ABC proteins are now available, the catalytic mechanism triggering the functioning of these proteins still remains elusive. In particular, it has been postulated that ATP hydrolysis proceeds via an acid-base mechanism catalyzed by the Glu residue adjacent to the Walker-B motif (Geourjon, C., Orelle, C., Steinfels, E., Blanchet, C., Deléage, G., Di Pietro, A., and Jault, J. M. (2001) Trends Biochem. Sci. 26, 539-544), but the involvement of such residue as the catalytic base in ABC transporters was recently questioned (Sauna, Z. E., Muller, M., Peng, X. H., and Ambudkar, S. V. (2002) Biochemistry, 41, 13989-14000). The equivalent glutamate residue (Glu504) of a half-ABC transporter involved in multidrug resistance in Bacillus subtilis, BmrA (formerly known as YvcC), was therefore mutated to Asp, Ala, Gln, Ser, and Cys residues. All these mutants were fully devoid of ATPase activity, yet they showed a high level of vanadate-independent trapping of 8-N3-alpha-32P-labeled nucleotide(s), following preincubation with 8-N3-[alpha-32P]ATP. However, and in contrast to the wild-type enzyme, the use of 8-N3-[gamma-32P]ATP unequivocally showed that all the mutants trapped exclusively the triphosphate form of the analogue, suggesting that they were not able to perform even a single hydrolytic turnover. These results demonstrate that Glu504 is the catalytic base for ATP hydrolysis in BmrA, and it is proposed that equivalent glutamate residues in other ABC transporters play the same role.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência Conservada , Ácido Glutâmico , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Bacillus subtilis/química , Proteínas de Bactérias/química , Catálise , Farmacorresistência Bacteriana , Hidrólise , Alinhamento de SequênciaRESUMO
The involvement of transporters in multidrug resistance of bacteria is an increasingly challenging problem, and most of the pumps identified so far use the protonmotive gradient as the energy source. A new member of the ATP-binding cassette (ABC) family, known in Bacillus subtilis as YvcC and homologous to each half of mammalian P-glycoprotein and to LmrA of Lactococcus lactis, has been studied here. The yvcC gene was constitutively expressed in B. subtilis throughout its growth, and a knockout mutant showed a lower rate of ethidium efflux than the wild-type strain. Overexpression of yvcC in Escherichia coli allowed the preparation of highly enriched inverted-membrane vesicles that exhibited high transport activities of three fluorescent drugs, namely, Hoechst 33342, doxorubicin, and 7-aminoactinomycin D. After solubilization with n-dodecyl beta-D-maltoside, the hexahistidine-tagged YvcC was purified by a one-step affinity chromatography, and its ability to bind many P-glycoprotein effectors was evidenced by fluorescence spectroscopy experiments. Collectively, these results showed that YvcC is a multidrug ABC transporter functionally active in wild-type B. subtilis, and YvcC was therefore renamed BmrA for Bacillus multidrug resistance ATP. Besides, reconstitution of YvcC into liposomes led to the highest, vanadate-sensitive, ATPase activity reported so far for an ABC transporter. Interestingly, such a high ATP hydrolysis proceeds with a positive cooperativity mechanism, a property only found so far with ABC importers.