RESUMO
Uptake of thiosulfate ions as an inorganic sulfur source from the environment is important for bacterial sulfur assimilation. Recently, a selective thiosulfate uptake pathway involving a membrane protein YeeE (TsuA) in Escherichia coli was characterized. YeeE-like proteins are conserved in some bacteria, archaea, and eukaryotes. However, the precise function of YeeE, along with its potential partner protein in the thiosulfate ion uptake pathway, remained unclear. Here, we assessed selective thiosulfate transport via Spirochaeta thermophila YeeE in vitro and characterized E. coli YeeD (TsuB) as an adjacent and essential protein for YeeE-mediated thiosulfate uptake in vivo. We further showed that S. thermophila YeeD possesses thiosulfate decomposition activity and that a conserved cysteine in YeeD was modified to several forms in the presence of thiosulfate. Finally, the crystal structures of S. thermophila YeeE-YeeD fusion proteins at 3.34-Å and 2.60-Å resolutions revealed their interactions. The association was evaluated by a binding assay using purified S. thermophila YeeE and YeeD. Based on these results, a model of the sophisticated uptake of thiosulfate ions by YeeE and YeeD is proposed.
Assuntos
Escherichia coli , Sulfurtransferases , Tiossulfatos , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico/genética , Cristalografia por Raios X , Cisteína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ligação Proteica , Sulfurtransferases/genética , Sulfurtransferases/metabolismo , Tiossulfatos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismoRESUMO
Membrane transport proteins require a gating mechanism that opens and closes the substrate transport pathway to carry out unidirectional transport. The "gating" involves large conformational changes and is achieved via multistep reactions. However, these elementary steps have not been clarified for most transporters due to the difficulty of detecting the individual steps. Here, we propose these steps for the gate opening of the bacterial Na+ pump rhodopsin, which outwardly pumps Na+ upon illumination. We herein solved an asymmetric dimer structure of Na+ pump rhodopsin from the bacterium Indibacter alkaliphilus. In one protomer, the Arg108 sidechain is oriented toward the protein center and appears to block a Na+ release pathway to the extracellular (EC) medium. In the other protomer, however, this sidechain swings to the EC side and then opens the release pathway. Assuming that the latter protomer mimics the Na+-releasing intermediate, we examined the mechanism for the swing motion of the Arg108 sidechain. On the EC surface of the first protomer, there is a characteristic cluster consisting of Glu10, Glu159, and Arg242 residues connecting three helices. In contrast, this cluster is disrupted in the second protomer. Our experimental results suggested that this disruption is a key process. The cluster disruption induces the outward movement of the Glu159-Arg242 pair and simultaneously rotates the seventh transmembrane helix. This rotation resultantly opens a space for the swing motion of the Arg108 sidechain. Thus, cluster disruption might occur during the photoreaction and then trigger sequential conformation changes leading to the gate-open state.
Assuntos
Rodopsina , Membrana Celular/metabolismo , Transporte de Íons , Íons/metabolismo , Subunidades Proteicas/metabolismo , Rodopsina/química , Rodopsina/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , AnimaisRESUMO
PpiD and YfgM are inner membrane proteins that are both composed of an N-terminal transmembrane segment and a C-terminal periplasmic domain. Escherichia coli YfgM and PpiD form a stable complex that interacts with the SecY/E/G (Sec) translocon, a channel that allows protein translocation across the cytoplasmic membrane. Although PpiD is known to function in protein translocation, the functional significance of PpiD-YfgM complex formation as well as the molecular mechanisms of PpiD-YfgM and PpiD/YfgM-Sec translocon interactions remain unclear. Here, we conducted genetic and biochemical studies using yfgM and ppiD mutants and demonstrated that a lack of YfgM caused partial PpiD degradation at its C-terminal region and hindered the membrane translocation of Vibrio protein export monitoring polypeptide (VemP), a Vibrio secretory protein, in both E. coli and Vibrio alginolyticus. While ppiD disruption also impaired VemP translocation, we found that the yfgM and ppiD double deletion exhibited no additive or synergistic effects. Together, these results strongly suggest that both PpiD and YfgM are required for efficient VemP translocation. Furthermore, our site-directed in vivo photocrosslinking analysis revealed that the tetratricopeptide repeat domain of YfgM and a conserved structural domain (NC domain) in PpiD interact with each other and that YfgM, like PpiD, directly interacts with the SecG translocon subunit. Crosslinking analysis also suggested that PpiD-YfgM complex formation is required for these proteins to interact with SecG. In summary, we propose that PpiD and YfgM form a functional unit that stimulates protein translocation by facilitating their proper interactions with the Sec translocon.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Canais de Translocação SEC/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Transporte Proteico , Periplasma/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Peptidilprolil Isomerase/químicaRESUMO
A transporter of the multidrug and toxic compound extrusion (MATE) family, Nicotiana tabacum MATE2 (NtMATE2), is located in the vacuole membrane of the tobacco plant root and is involved in the transportation of nicotine, a secondary or specialized metabolic compound in Solanaceae. Here, we report the crystal structures of NtMATE2 in its outward-facing forms. The overall structure has a bilobate V-shape with pseudo-symmetrical assembly of the N- and C-lobes. In one crystal structure, the C-lobe cavity of NtMATE2 interacts with an unidentified molecule that may partially mimic a substrate. In addition, NtMATE2-specific conformational transitions imply that an unprecedented movement of the transmembrane α-helix 7 is related to the release of the substrate into the vacuolar lumen.
Assuntos
Nicotiana/metabolismo , Nicotina/química , Proteínas de Transporte de Cátions Orgânicos/química , Proteínas de Plantas/química , Vacúolos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Transporte Biológico , Clonagem Molecular , Cristalografia por Raios X , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Modelos Moleculares , Nicotina/metabolismo , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Células Vegetais/química , Células Vegetais/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Nicotiana/genética , Vacúolos/químicaRESUMO
We have demonstrated that a bacterial membrane protein, YeeE, mediates thiosulfate uptake. Thiosulfate is used for cysteine synthesis in bacteria as an inorganic sulfur source in the global biological sulfur cycle. The crystal structure of YeeE at 2.5-Å resolution reveals an unprecedented hourglass-like architecture with thiosulfate in the positively charged outer concave side. YeeE is composed of loops and 13 helices including 9 transmembrane α helices, most of which show an intramolecular pseudo 222 symmetry. Four characteristic loops are buried toward the center of YeeE and form its central region surrounded by the nine helices. Additional electron density maps and successive molecular dynamics simulations imply that thiosulfate can remain temporally at several positions in the proposed pathway. We propose a plausible mechanism of thiosulfate uptake via three important conserved cysteine residues of the loops along the pathway.
RESUMO
Protein secretion mediated by SecYEG translocon and SecA ATPase is enhanced by membrane-embedded SecDF by using proton motive force. A previous structural study of SecDF indicated that it comprises 12 transmembrane helices that can conduct protons and three periplasmic domains, which form at least two characterized transition states, termed the F and I forms. We report the structures of full-length SecDF in I form at 2.6- to 2.8-Å resolution. The structures revealed that SecDF in I form can generate a tunnel that penetrates the transmembrane region and functions as a proton pathway regulated by a conserved Asp residue of the transmembrane region. In one crystal structure, periplasmic cavity interacts with a molecule, potentially polyethylene glycol, which may mimic a substrate peptide. This study provides structural insights into the Sec protein translocation that allows future analyses to develop a more detailed working model for SecDF.
Assuntos
Proteínas de Bactérias/química , Prótons , Canais de Translocação SEC/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Membrana Celular/química , Membrana Celular/metabolismo , Cristalografia por Raios X , Deinococcus/química , Glicolipídeos , Peptídeos/química , Peptídeos/metabolismo , Ligação ProteicaRESUMO
The Sec translocon facilitates transportation of newly synthesized polypeptides from the cytoplasm to the lumen/periplasm across the phospholipid membrane. Although the polypeptide-conducting machinery is formed by the SecYEG-SecA complex in bacteria, its transportation efficiency is markedly enhanced by SecDF. A previous study suggested that SecDF assumes at least two conformations differing by a 120° rotation in the spatial orientation of the P1 head subdomain to the rigid base, and that the conformational dynamics plays a critical role in polypeptide translocation. Here we addressed this hypothesis by analyzing the 3D structure of SecDF using electron tomography and single particle reconstruction. Reconstruction of wt SecDF showed two major conformations; one resembles the crystal structure of full-length SecDF (F-form structure), while the other is similar to the hypothetical structural variant based on the crystal structure of the isolated P1 domain (I-form structure). The transmembrane domain of the I-form structure has a scissor like cleft open to the periplasmic side. We also report the structure of a double cysteine mutant designed to constrain SecDF to the I-form. This reconstruction has a protrusion at the periplasmic end that nicely fits the orientation of P1 in the I-from. These results provide firm evidence for the occurrence of the I-form in solution and support the proposed F- to I-transition of wt SecDF during polypeptide translocation.
Assuntos
Proteínas de Bactérias/ultraestrutura , Proteínas de Membrana/ultraestrutura , Proteínas de Membrana Transportadoras/ultraestrutura , Thermus thermophilus/genética , Proteínas de Bactérias/genética , Cristalografia por Raios X , Tomografia com Microscopia Eletrônica , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Mutação , Estrutura Terciária de Proteína , Transporte ProteicoRESUMO
Proton-dependent oligopeptide transporters (POTs) are major facilitator superfamily (MFS) proteins that mediate the uptake of peptides and peptide-like molecules, using the inwardly directed H(+) gradient across the membrane. The human POT family transporter peptide transporter 1 is present in the brush border membrane of the small intestine and is involved in the uptake of nutrient peptides and drug molecules such as ß-lactam antibiotics. Although previous studies have provided insight into the overall structure of the POT family transporters, the question of how transport is coupled to both peptide and H(+) binding remains unanswered. Here we report the high-resolution crystal structures of a bacterial POT family transporter, including its complex with a dipeptide analog, alafosfalin. These structures revealed the key mechanistic and functional roles for a conserved glutamate residue (Glu310) in the peptide binding site. Integrated structural, biochemical, and computational analyses suggested a mechanism for H(+)-coupled peptide symport in which protonated Glu310 first binds the carboxyl group of the peptide substrate. The deprotonation of Glu310 in the inward open state triggers the release of the bound peptide toward the intracellular space and salt bridge formation between Glu310 and Arg43 to induce the state transition to the occluded conformation.
Assuntos
Proteínas de Transporte/metabolismo , Peptídeos/metabolismo , Prótons , Alanina/análogos & derivados , Alanina/metabolismo , Proteínas de Transporte/química , Transporte de Íons , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação ProteicaRESUMO
Multidrug and toxic compound extrusion (MATE) family transporters are conserved in the three primary domains of life (Archaea, Bacteria and Eukarya), and export xenobiotics using an electrochemical gradient of H(+) or Na(+) across the membrane. MATE transporters confer multidrug resistance to bacterial pathogens and cancer cells, thus causing critical reductions in the therapeutic efficacies of antibiotics and anti-cancer drugs, respectively. Therefore, the development of MATE inhibitors has long been awaited in the field of clinical medicine. Here we present the crystal structures of the H(+)-driven MATE transporter from Pyrococcus furiosus in two distinct apo-form conformations, and in complexes with a derivative of the antibacterial drug norfloxacin and three in vitro selected thioether-macrocyclic peptides, at 2.1-3.0 Å resolutions. The structures, combined with functional analyses, show that the protonation of Asp 41 on the amino (N)-terminal lobe induces the bending of TM1, which in turn collapses the N-lobe cavity, thereby extruding the substrate drug to the extracellular space. Moreover, the macrocyclic peptides bind the central cleft in distinct manners, which correlate with their inhibitory activities. The strongest inhibitory peptide that occupies the N-lobe cavity may pave the way towards the development of efficient inhibitors against MATE transporters.
Assuntos
Antiporters/química , Antiporters/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Pyrococcus furiosus/química , Sequência de Aminoácidos , Apoproteínas/química , Apoproteínas/metabolismo , Ácido Aspártico/química , Cristalografia por Raios X , Análise Mutacional de DNA , Compostos Macrocíclicos/química , Compostos Macrocíclicos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Norfloxacino/química , Norfloxacino/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica , Prótons , Relação Estrutura-Atividade , Sulfetos/química , Sulfetos/metabolismoRESUMO
Protein translocation across the bacterial membrane, mediated by the secretory translocon SecYEG and the SecA ATPase, is enhanced by proton motive force and membrane-integrated SecDF, which associates with SecYEG. The role of SecDF has remained unclear, although it is proposed to function in later stages of translocation as well as in membrane protein biogenesis. Here, we determined the crystal structure of Thermus thermophilus SecDF at 3.3 Å resolution, revealing a pseudo-symmetrical, 12-helix transmembrane domain belonging to the RND superfamily and two major periplasmic domains, P1 and P4. Higher-resolution analysis of the periplasmic domains suggested that P1, which binds an unfolded protein, undergoes functionally important conformational changes. In vitro analyses identified an ATP-independent step of protein translocation that requires both SecDF and proton motive force. Electrophysiological analyses revealed that SecDF conducts protons in a manner dependent on pH and the presence of an unfolded protein, with conserved Asp and Arg residues at the transmembrane interface between SecD and SecF playing essential roles in the movements of protons and preproteins. Therefore, we propose that SecDF functions as a membrane-integrated chaperone, powered by proton motive force, to achieve ATP-independent protein translocation.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Thermus thermophilus/química , Trifosfato de Adenosina/metabolismo , Arginina/metabolismo , Asparagina/metabolismo , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Modelos Biológicos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Periplasma/química , Periplasma/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , Desdobramento de Proteína , Força Próton-Motriz , Eletricidade Estática , Relação Estrutura-Atividade , Thermus thermophilus/citologiaRESUMO
The Sec translocon, a protein-conducting channel, consists of a heterotrimeric complex (SecYEG in bacteria and Sec61 alpha beta gamma in eukaryotes) that provides a pathway for secretary proteins to cross membranes, or for membrane proteins to integrate into the membrane. The Sec translocon alone is a passive channel, and association with channel partners, including the ribosome or SecA ATPase in bacteria, is needed for protein translocation. Three recently published crystal structures of SecY are considered to represent the closed (resting state), pre-open (transitional state determined with the bound Fab fragment mimicking SecA interaction), and SecA-bound forms. To elucidate mechanisms of transition between closed and pre-open forms, we performed all-atom molecular dynamics simulations for the pre-open form of Thermus thermophilus SecYE and the closed form of Methanococcus janaschii SecYE beta in explicit solvent and membranes. We found that the closed form of SecY is stable, while the pre-open form without the Fab fragment undergoes large conformational changes toward the closed form. The pre-open form of SecY with Fab remains unchanged, suggesting that the cytosolic interaction mimicking SecA binding stabilizes the pre-open form of SecY. Importantly, a lipid molecule at the lateral gate region appears to be required to maintain the pre-open form in the membrane. We propose that the conformational transition from closed to pre-open states of SecY upon association with SecA facilitates intercalation of phospholipids at the lateral gate, inducing initial entry of the positively charged signal peptide into the channel.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Mathanococcus , Conformação Proteica , Transporte Proteico , Canais de Translocação SEC , Proteínas SecA , Thermus thermophilusAssuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/fisiologia , Transporte Proteico/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Permeabilidade da Membrana Celular , Cristalografia por Raios X , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Complexos Multiproteicos , Conformação Proteica , Canais de Translocação SEC , Proteínas SecARESUMO
The mechanism of pre-protein export through the bacterial cytoplasmic membrane, in which the SecA ATPase plays a crucial role as an "energy supplier", is poorly understood. In particular, biochemical and structural studies provide contradictory data as to the oligomeric state of SecA when it is integrated into the active trans-membrane translocase. Here, we report the 2.8 A resolution crystal structure of the Thermus thermophilus SecA protein (TtSecA). Whereas the structure of the TtSecA monomer closely resembles that from other bacteria, the oligomeric state of TtSecA is strikingly distinct. In contrast to the antiparallel (head-to-tail) dimerization reported previously for the other bacterial systems, TtSecA forms parallel (head-to-head) dimers that are reminiscent of open scissors. The dimer interface is abundant in bulky Arg and Lys side-chains from both subunits, which stack on one another to form an unusual "basic zipper" that is highly conserved, as revealed by homology modeling and sequence analysis. The basic zipper is sealed on both ends by two pairs of the salt bridges formed between the basic side-chains from the zipper and two invariant acidic residues. The organization of the dimers, in which the two pre-protein binding domains are located proximal to each other at the tip of the "scissors", might allow a concerted mode of substrate recognition while the opening/closing of the scissors might facilitate translocation.
Assuntos
Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Proteínas de Membrana Transportadoras/química , Modelos Moleculares , Thermus thermophilus/enzimologia , Sequência de Aminoácidos , Sequência Conservada , Cristalografia por Raios X , Dimerização , Evolução Molecular , Dados de Sequência Molecular , Conformação Proteica , Subunidades Proteicas/química , Canais de Translocação SEC , Proteínas SecARESUMO
The Thermus thermophilus gene encoding the preprotein translocation ATPase SecA was cloned and expressed and the purified protein was crystallized by the hanging-drop vapour-diffusion technique in two different space groups P3(1(2))21 (a = b = 168.6, c = 149.8 A) and P6(1(5))22 (a = b = 130.9, c = 564.6 A). The crystals, improved by macroseeding, diffracted to beyond 2.8 and 3.5 A resolution for the trigonal and hexagonal crystal forms, respectively. Structure determination using the multiple isomorphous replacement method is in progress.