RESUMO
Bacterial chemoreceptors regulate the cytosolic multidomain histidine kinase CheA through largely unknown mechanisms. Residue substitutions in the peptide linkers that connect the P4 kinase domain to the P3 dimerization and P5 regulatory domain affect CheA basal activity and activation. To understand the role that these linkers play in CheA activity, the P3-to-P4 linker (L3) and P4-to-P5 linker (L4) were extended and altered in variants of Thermotoga maritima (Tm) CheA. Flexible extensions of the L3 and L4 linkers in CheA-LV1 (linker variant 1) allowed for a well-folded kinase domain that retained wild-type (WT)-like binding affinities for nucleotide and normal interactions with the receptor-coupling protein CheW. However, CheA-LV1 autophosphorylation activity registered â¼50-fold lower compared to WT. Neither a WT nor LV1 dimer containing a single P4 domain could autophosphorylate the P1 substrate domain. Autophosphorylation activity was rescued in variants with extended L3 and L4 linkers that favor helical structure and heptad spacing. Autophosphorylation depended on linker spacing and flexibility and not on sequence. Pulse-dipolar electron-spin resonance (ESR) measurements with spin-labeled adenosine 5'-triphosphate (ATP) analogues indicated that CheA autophosphorylation activity inversely correlated with the proximity of the P4 domains within the dimers of the variants. Despite their separation in primary sequence and space, the L3 and L4 linkers also influence the mobility of the P1 substrate domains. In all, interactions of the P4 domains, as modulated by the L3 and L4 linkers, affect domain dynamics and autophosphorylation of CheA, thereby providing potential mechanisms for receptors to regulate the kinase.
Assuntos
Proteínas de Bactérias , Proteínas de Escherichia coli , Histidina Quinase/metabolismo , Proteínas Quimiotáticas Aceptoras de Metil/genética , Proteínas Quimiotáticas Aceptoras de Metil/química , Proteínas de Bactérias/química , Modelos Moleculares , Thermotoga maritima/metabolismo , Quimiotaxia , Proteínas de Escherichia coli/químicaRESUMO
Sulfur-insertion reactions are essential for the biosynthesis of several cellular metabolites, including enzyme cofactors. In Lactobacillus plantarum, a sulfur-containing nickel-pincer nucleotide (NPN) cofactor is used as a coenzyme of lactic acid racemase, LarA. During NPN biosynthesis in L. plantarum, sulfur is transferred to a nicotinic acid-derived substrate by LarE, which sacrifices the sulfur atom of its single cysteinyl side chain, forming a dehydroalanine residue. Most LarE homologs contain three conserved cysteine residues that are predicted to cluster at the active site; however, the function of this cysteine cluster is unclear. In this study, we characterized LarE from Thermotoga maritima (LarETm) and show that it uses these three conserved cysteine residues to bind a [4Fe-4S] cluster that is required for sulfur transfer. Notably, we found LarETm retains all side chain sulfur atoms, in contrast to LarELp. We also demonstrate that when provided with L-cysteine and cysteine desulfurase from Escherichia coli (IscSEc), LarETm functions catalytically with IscSEc transferring sulfane sulfur atoms to LarETm. Native mass spectrometry results are consistent with a model wherein the enzyme coordinates sulfide at the nonligated iron atom of the [4Fe-4S] cluster, forming a [4Fe-5S] species, and transferring the noncore sulfide to the activated substrate. This proposed mechanism is like that of TtuA that catalyzes sulfur transfer during 2-thiouridine synthesis. In conclusion, we found that LarE sulfur insertases associated with NPN biosynthesis function either by sacrificial sulfur transfer from the protein or by transfer of a noncore sulfide bound to a [4Fe-4S] cluster.
Assuntos
Proteínas Ferro-Enxofre , Thermotoga maritima , Coenzimas/metabolismo , Cisteína/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Níquel/metabolismo , Nucleotídeos/metabolismo , Sulfetos/metabolismo , Enxofre/metabolismo , Thermotoga maritima/genética , Thermotoga maritima/metabolismoRESUMO
Membrane-spanning lipids are present in a wide variety of archaea, but they are rarely in bacteria. Nevertheless, the (hyper)thermophilic members of the order Thermotogales harbor tetraester, tetraether, and mixed ether/ester membrane-spanning lipids mostly composed of core lipids derived from diabolic acids, C30, C32, and C34 dicarboxylic acids with two adjacent mid-chain methyl substituents. Lipid analysis of Thermotoga maritima across growth phases revealed a decrease of the relative abundance of fatty acids together with an increase of diabolic acids with independence of growth temperature. We also identified isomers of C30 and C32 diabolic acids, i.e., dicarboxylic acids with only one methyl group at C-15. Their distribution suggests they are products of the condensation reaction but are preferably produced when the length of the acyl chains is not optimal. Compared with growth at the optimal temperature of 80°C, an increase of glycerol ether-derived lipids was observed at 55°C. Our analysis only detected diabolic acid-containing intact polar lipids with phosphoglycerol (PG) head groups. Considering these findings, we hypothesize a biosynthetic pathway for the synthesis of membrane-spanning lipids based on PG polar lipid formation, suggesting that the protein catalyzing this process is a membrane protein. We also identified, by genomic and protein domain analyses, a gene coding for a putative plasmalogen synthase homologue in T. maritima that is also present in other bacteria producing sn-1-alkyl ether lipids but not plasmalogens, suggesting it is involved in the conversion of the ester-to-ether bond in the diabolic acids bound in membrane-spanning lipids. IMPORTANCE Membrane-spanning lipids are unique compounds found in most archaeal membranes, but they are also present in specific bacterial groups like the Thermotogales. The synthesis and physiological role of membrane-spanning lipids in bacteria represent an evolutionary and biochemical open question that points to the differentiation of the membrane lipid composition. Understanding the formation of membrane-spanning lipids is crucial to solving this question and identifying the enzymatic and biochemical mechanism performing this procedure. In the present work, we found changes at the core lipid level, and we propose that the growth phase drives the biosynthesis of these lipids rather than temperature. Our results identified physiological conditions influencing the membrane-spanning lipid biosynthetic process, which can further clarify the pathway leading to the biosynthesis of these compounds.
Assuntos
Lipídeos de Membrana , Thermotoga maritima , Ácidos Dicarboxílicos , Éter , Éteres , Lipídeos de Membrana/metabolismo , Temperatura , Thermotoga maritima/genética , Thermotoga maritima/metabolismoRESUMO
Bacterial methionine biosynthesis can take place by either the trans-sulfurylation route or direct sulfurylation. The enzymes responsible for trans-sulfurylation have been characterized extensively because they occur in model organisms such as Escherichia coli. However, direct sulfurylation is actually the predominant route for methionine biosynthesis across the phylogenetic tree. In this pathway, most bacteria use an O-acetylhomoserine aminocarboxypropyltransferase (MetY) to catalyze the formation of homocysteine from O-acetylhomoserine and bisulfide. Despite the widespread distribution of MetY, this pyridoxal 5'-phosphate-dependent enzyme remains comparatively understudied. To address this knowledge gap, we have characterized the MetY from Thermotoga maritima (TmMetY). At its optimal temperature of 70 °C, TmMetY has a turnover number (apparent kcat = 900 s-1) that is 10- to 700-fold higher than the three other MetY enzymes for which data are available. We also present crystal structures of TmMetY in the internal aldimine form and, fortuitously, with a ß,γ-unsaturated ketimine reaction intermediate. This intermediate is identical to that found in the catalytic cycle of cystathionine γ-synthase (MetB), which is a homologous enzyme from the trans-sulfurylation pathway. By comparing the TmMetY and MetB structures, we have identified Arg270 as a critical determinant of specificity. It helps to wall off the active site of TmMetY, disfavoring the binding of the first MetB substrate, O-succinylhomoserine. It also ensures a strict specificity for bisulfide as the second substrate of MetY by occluding the larger MetB substrate, cysteine. Overall, this work illuminates the subtle structural mechanisms by which homologous pyridoxal 5'-phosphate-dependent enzymes can effect different catalytic, and therefore metabolic, outcomes.
Assuntos
Proteínas de Bactérias/metabolismo , Metionina/metabolismo , Thermotoga maritima/metabolismo , Proteínas de Bactérias/química , Vias Biossintéticas , Cristalografia por Raios X , Cinética , Modelos Moleculares , Thermotoga maritima/químicaRESUMO
Phosphoglucose isomerases (PGIs) belong to a class of enzymes that catalyze the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. PGIs are crucial in glycolysis and gluconeogenesis pathways and proposed as serving additional extracellular functions in eukaryotic organisms. The phosphoglucose isomerase function of TM1385, a previously uncharacterized protein from Thermotoga maritima, was hypothesized based on structural similarity to established PGI crystal structures and computational docking. Kinetic and colorimetric assays combined with 1H nuclear magnetic resonance (NMR) spectroscopy experimentally confirm that TM1385 is a phosphoglucose isomerase (TmPGI). Evidence of solvent exchange in 1H NMR spectra supports that TmPGI isomerization proceeds through a cis-enediol-based mechanism. To determine which amino acid residues are critical for TmPGI catalysis, putative active site residues were mutated with alanine and screened for activity. Results support that E281 is most important for TmPGI formation of the cis-enediol intermediate, and the presence of either H310 or K422 may be required for catalysis, similar to previous observations from homologous PGIs. However, only TmPGI E281A/Q415A and H310A/K422A double mutations abolished activity, suggesting that there are redundant catalytic residues, and Q415 may participate in sugar phosphate isomerization upon E281 mutation. Combined, we propose that TmPGI E281 participates directly in the cis-enediol intermediate step, and either H310 or K422 may facilitate sugar ring opening and closure.
Assuntos
Proteínas de Bactérias/metabolismo , Glucose-6-Fosfato Isomerase/metabolismo , Thermotoga maritima/metabolismo , Proteínas de Bactérias/química , Catálise , Domínio Catalítico , Glucose-6-Fosfato Isomerase/química , Isomerismo , Cinética , Espectroscopia de Prótons por Ressonância Magnética , Especificidade por SubstratoRESUMO
Domain swapping is a widespread oligomerization process that is observed in a large variety of protein families. In the large superfamily of substrate-binding proteins, non-monomeric members have rarely been reported. The arginine-binding protein from Thermotoga maritima (TmArgBP), a protein endowed with a number of unusual properties, presents a domain-swapped structure in its dimeric native state in which the two polypeptide chains mutually exchange their C-terminal helices. It has previously been shown that mutations in the region connecting the last two helices of the TmArgBP structure lead to the formation of a variety of oligomeric states (monomers, dimers, trimers and larger aggregates). With the aim of defining the structural determinants of domain swapping in TmArgBP, the monomeric form of the P235GK mutant has been structurally characterized. Analysis of this arginine-bound structure indicates that it consists of a closed monomer with its C-terminal helix folded against the rest of the protein, as typically observed for substrate-binding proteins. Notably, the two terminal helices are joined by a single nonhelical residue (Gly235). Collectively, the present findings indicate that extending the hinge region and conferring it with more conformational freedom makes the formation of a closed TmArgBP monomer possible. On the other hand, the short connection between the helices may explain the tendency of the protein to also adopt alternative oligomeric states (dimers, trimers and larger aggregates). The data reported here highlight the importance of evolutionary control to avoid the uncontrolled formation of heterogeneous and potentially harmful oligomeric species through domain swapping.
Assuntos
Arginina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Thermotoga maritima/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Cristalização , Mutação/genética , Ligação Proteica , Homologia Estrutural de ProteínaRESUMO
D-Allulose is a rare monosaccharide that exists in extremely small quantities in nature, and it is also hard to prepare at a large scale via chemical or enzyme synthetic route due to low conversion and downstream separation complexity. Using D-psicose epimerase and L-rhamnulose kinase, a method enabling high conversion of D-allulose from D-fructose without the need for a tedious isomer separation step was established recently. However, this method requires expensive ATP to facilitate the reaction. In the present study, an ATP regenerate system was developed coupling with polyphosphate kinase. In our optimized reaction with purified enzymes, the conversion rate of 99% D-fructose was achieved at the concentrations of 2 mM ATP, 5 mM polyphosphate, 20 mM D-fructose, and 20 mM Mg2+ when incubated at 50 °C and at pH 7.5. ATP usage can be reduced to 10% of the theoretical amount compared to that without the ATP regeneration system. A fed-batch mode was also studied to minimize the inhibitory effect of polyphosphate. The biosynthetic system reported here offers a potential and promising platform for the conversion of D-fructose into D-allulose at reduced ATP cost.
Assuntos
Trifosfato de Adenosina/metabolismo , Carboidratos Epimerases/metabolismo , Frutose/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Biotransformação , Carboidratos Epimerases/genética , Cátions Bivalentes , Clonagem Molecular , Ensaios Enzimáticos , Escherichia coli/genética , Escherichia coli/metabolismo , Frutose/biossíntese , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Magnésio/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Polifosfatos/metabolismo , Proteínas Recombinantes de Fusão/genética , Frações Subcelulares/química , Frações Subcelulares/metabolismo , Thermotoga maritima/genética , Thermotoga maritima/metabolismoRESUMO
Most organisms contain multiple soluble protein-based redox carriers such as members of the ferredoxin (Fd) family, that contain one or more iron-sulfur clusters. The potential redundancy of Fd proteins is poorly understood, particularly in connection to the ability of Fd proteins to deliver reducing equivalents to members of the "radical SAM," or S-adenosylmethionine radical enzyme (ARE) superfamily, where the activity of all known AREs requires that an essential iron-sulfur cluster bound by the enzyme be reduced to the catalytically relevant [Fe4 S4 ]1+ oxidation state. As it is still unclear whether a single Fd in a given organism is specific to individual redox partners, we have examined the five Fd proteins found within Thermotoga maritima via direct electrochemistry, to compare them in a side-by-side fashion for the first time. While a single [Fe4 S4 ]-cluster bearing Fd (TM0927) has a potential of -420 mV, the other four 2x[Fe4 S4 ]-bearing Fds (TM1175, TM1289, TM1533, and TM1815) have potentials that vary significantly, including cases where the two clusters of the same Fd are essentially coincident (e.g., TM1175) and those where the potentials are well separate (TM1815).
Assuntos
Ferredoxinas/química , Thermotoga maritima/química , Ferredoxinas/metabolismo , Oxirredução , Thermotoga maritima/metabolismoRESUMO
While two-component systems (TCSs), composed of a sensor histidine kinase (HK) and a response regulator, are the main signaling pathways in bacteria, global TCS activity remains poorly described. Here, we report the kinetic parameters of the HK autophosphorylation reaction using previously uncharacterized γ-phosphate-modified ATP analogues to further elucidate their utility as activity-based probes for global TCS analysis. Given the increased stability of thiophosphorylated histidine in comparison to that of the native phosphoryl modification, which is attributed to the decreased electrophilicity of this moiety, we anticipated that ATPγS may be turned over much more slowly by the HKs. Surprisingly, we found this not to be the case, with the turnover numbers decreasing <1 order of magnitude. Instead, we found that alkylation of the thiophosphate had a much more dramatic effect on turnover and, in one case, the binding affinity of this substrate analogue (BODIPY-FL-ATPγS).
Assuntos
Trifosfato de Adenosina/análogos & derivados , Proteínas de Bactérias/metabolismo , Histidina Quinase/metabolismo , Thermotoga maritima/enzimologia , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Histidina/química , Histidina/metabolismo , Histidina Quinase/química , Cinética , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Fosforilação , Thermotoga maritima/química , Thermotoga maritima/metabolismoRESUMO
CheY is a response regulator of bacterial chemotaxis that is activated by phosphorylation of a conserved aspartate residue. However, studies of CheY-phosphate have proven challenging due to rapid hydrolysis of the aspartyl-phosphate in vitro. To combat this issue, we have designed a stable analog suitable for structural and functional studies. Herein, we describe a method for the chemical modification of Thermotoga maritima CheY to produce a phospho-analog designated as phosphono-CheY. Our modification produces a stable analog in the constitutively active form that enables the study of signal transfer to the downstream target.
Assuntos
Proteínas Quimiotáticas Aceptoras de Metil/síntese química , Thermotoga maritima/metabolismo , Proteínas de Bactérias/síntese química , Proteínas de Bactérias/química , Quimiotaxia , Ativação Enzimática , Proteínas Quimiotáticas Aceptoras de Metil/química , Fosforilação , Estabilidade Proteica , Transdução de Sinais , Thermotoga maritima/químicaRESUMO
Proper cell division at the mid-site of Gram-negative bacteria reflects stringent regulation by the min system (MinC, MinD and MinE). Herein we report crystal structure of the C-terminal domain of MinC from Escherichia coli (EcMinCCTD). The MinCCTD beta helical domain is engaged in a tight homodimer, similar to Thermotoga maritima MinCCTD (TmMinCCTD). However, both EcMinCCTD and TmMinCCTD lack an α-helix (helix3) at their C-terminal tail, in comparison to Aquifex aerolicu MinCCTD (AaMinCCTD) which forms an extra interaction interface with MinD. To understand the role of this extra binding element in MinC/MinD interactions, we fused this helix (Aahelix3) to the C-terminus of EcMinC and examined its effect on cell morphology and cell growth. Our results revealed that Aahelix3 impaired normal cell division in vivo. Furthermore, results of a co-pelleting assay and binding free energy calculation suggested that Aahelix3 plays an essential role in AaMinCD complex formation, under the circumstance of lacking MinE in A. aerolicu. Combining these results with sequence analysis of MinC and MinD in different organisms, we propose an evolutionary relationship to rationalize different mechanisms in cell division positioning in various organisms.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/genética , Divisão Celular , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Proteínas de Membrana/genética , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/citologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Evolução Molecular , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Termodinâmica , Thermotoga maritima/citologia , Thermotoga maritima/genética , Thermotoga maritima/metabolismoRESUMO
Thermotoga maritima is a hyperthermophilic anaerobic bacterium that produces molecular hydrogen (H2) by fermentation. It catabolizes a broad range of carbohydrates through the action of diverse ABC transporters. However, in T. maritima and related species, highly similar genes with ambiguous annotation obscure a precise understanding of genome function. In T. maritima, three putative malK genes, all annotated as ATPase subunits, exhibited high identity to each other. To distinguish between these genes, malK disruption mutants were constructed by gene replacement, and the resulting mutant cell lines were characterized. Only a disruption of malK3 produced a defect in maltose catabolism. To verify that the mutant phenotype arose specifically from malK3 inactivation, the malK3 mutation was repaired by recombination, and maltose catabolism was restored. This study demonstrates the importance of a maltose ABC-type transporter and its relationship to sugar metabolism in T. maritimaIMPORTANCE The application and further development of a genetic system was used here to investigate gene paralogs in the hyperthermophile Thermotoga maritima The occurrence of three ABC transporter ATPase subunits all annotated as malK was evaluated using a combination of genetic and bioinformatic approaches. The results clarify the role of only one malK gene in maltose catabolism in a nonmodel organism noted for fermentative hydrogen production.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Maltose/metabolismo , Thermotoga maritima/enzimologia , Adenosina Trifosfatases/genética , Anaerobiose , Proteínas de Bactérias/genética , Transporte Biológico , Temperatura Alta , Mutação , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Thermotoga maritima/genética , Thermotoga maritima/metabolismoRESUMO
Phosphorylation of CheY promotes association with the flagellar motor and ultimately controls the directional bias of the motor. However, biochemical studies of activated CheY-phosphate have been challenging due to the rapid hydrolysis of the aspartyl-phosphate in vitro. An inert analog of Tm CheY-phosphate, phosphono-CheY, was synthesized by chemical modification and purified by cation-exchange chromatography. Changes in HPLC retention times, chemical assays for phosphate and free thiol, and mass spectrometry experiments demonstrate modification of Cys54 with a phosphonomethyl group. Additionally, a crystal structure showed electron density for the phosphonomethyl group at Cys54, consistent with a modification at that position. Subsequent biochemical experiments confirmed that protein crystals were phosphono-CheY. Isothermal titration calorimetry and fluorescence polarization binding assays demonstrated that phosphono-CheY bound a peptide derived from FliM, a native partner of CheY-phosphate, with a dissociation constant of â¼29 µM, at least sixfold more tightly than unmodified CheY. Taken together these results suggest that Tm phosphono-CheY is a useful and unique analog of Tm CheY-phosphate.
Assuntos
Proteínas de Bactérias/química , Proteínas Quimiotáticas Aceptoras de Metil/química , Organofosfonatos/química , Peptídeos/química , Thermotoga maritima/química , Sequência de Aminoácidos , Ácido Aspártico/análogos & derivados , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Quimiotaxia/fisiologia , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Flagelos/química , Flagelos/metabolismo , Expressão Gênica , Cinética , Proteínas Quimiotáticas Aceptoras de Metil/genética , Proteínas Quimiotáticas Aceptoras de Metil/metabolismo , Modelos Moleculares , Organofosfonatos/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Thermotoga maritima/genética , Thermotoga maritima/metabolismoRESUMO
The Thermotoga maritima arginine-binding protein (TmArgBP) has been modified to create a reagentless fluorescent protein biosensor. Two design methods for biosensor construction are compared: 1) solvent accessibility of environmentally-sensitive probes and 2) fluorescence deactivation due to photo-induced electron transfer (PET). Nine single cysteine TmArgBP mutants were created and labeled with three different environmentally sensitive fluorescent probes. These mutants demonstrated limited changes in fluorescence emission upon the addition of arginine. In contrast, the PET-based biosensor provides significant enhancements over the traditional approach and provides a fluorescence quenching mechanism that was capable of providing quantitative detection of arginine. Site-directed mutagenesis of TmArgBP was used to create attachment points for the fluorescent probe (K145C) and for an internal aromatic residue (D18X) to serve as the PET quencher. Both tyrosine and tryptophan, but not phenylalanine, were able to quench the emission of the fluorescent probe by more than 80% upon the addition of arginine. The dissociation constant for arginine ranged from 0.87 to 1.5 µM across the different sensors. This PET-based strategy provides a simple and broadly applicable approach for the analytical detection of small molecules that may be applied to any protein that exhibits conformational switching in a ligand dependent manner.
Assuntos
Arginina/análise , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Proteínas Periplásmicas de Ligação/metabolismo , Thermotoga maritima/metabolismo , Arginina/genética , Arginina/metabolismo , Proteínas de Bactérias , Sítios de Ligação , Fluorescência , Conformação Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Proteínas Periplásmicas de Ligação/química , Proteínas Periplásmicas de Ligação/genética , Ligação Proteica , Espectrometria de Fluorescência , Thermotoga maritima/genética , Thermotoga maritima/crescimento & desenvolvimento , Triptofano/química , Triptofano/genética , Triptofano/metabolismoRESUMO
TM287/288 is a heterodimeric ATP-binding cassette (ABC) transporter, which harnesses the energy of ATP binding and hydrolysis at the nucleotide-binding domains (NBDs) to transport a wide variety of molecules through the transmembrane domains (TMDs) by alternating inward- and outward-facing conformations. Here, we conducted multiple 100 ns molecular dynamics simulations of TM287/288 in different ATP- and substrate-bound states to elucidate the effects of ATP and substrate binding. As a result, the binding of two ATP molecules to the NBDs induced the formation of the consensus ATP-binding pocket (ABP2) or the NBD dimerization, whereas these processes did not occur in the presence of a single ATP molecule or when the protein was in its apo state. Moreover, binding of the substrate to the TMDs enhanced the formation of ABP2 through allosteric TMD-NBD communication. Furthermore, in the apo state, α-helical subdomains of the NBDs approached each other, acquiring a conformation with core half-pockets exposed to the solvent, appropriate for ATP binding. We propose a "core-exposed" model for this novel conformation found in the apo state of ABC transporters. These findings provide important insights into the structural dynamics of ABC transporters.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Multimerização Proteica , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Especificidade por Substrato , Thermotoga maritima/metabolismoRESUMO
Iron-sulfur (Fe-S) clusters are ubiquitously conserved and play essential cellular roles. The mechanism of Fe-S cluster biogenesis involves multiple proteins in a complex pathway. Cluster biosynthesis primarily occurs in the mitochondria, but key Fe-S proteins also exist in the cytosol. One such protein, glutaredoxin 3 (Grx3), is involved in iron regulation, sensing, and mediating [2Fe-2S] cluster delivery to cytosolic protein targets, but the cluster donor for cytosolic Grx3 has not been elucidated. Herein, we delineate the kinetic transfer of [2Fe-2S] clusters into Grx3 from potential cytosolic carrier/scaffold proteins, IscU and Nfu, to evaluate a possible model for Grx3 reconstitution in vivo.
Assuntos
Proteínas de Transporte/química , Ferro/química , Oxirredutases/química , Proteínas de Saccharomyces cerevisiae/química , Enxofre/química , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Citosol/química , Citosol/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Ferro/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Cinética , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Enxofre/metabolismo , Thermotoga maritima/química , Thermotoga maritima/metabolismoRESUMO
Oligopeptide-binding proteins (Opps) are part of the ATP-binding cassette system, playing a crucial role in nutrient uptake and sensing the external environment in bacteria, including hyperthermophiles. Opps serve as a binding platform for diverse peptides; however, how these peptides are recognized by Opps is still largely unknown and few crystal structures of Opps from hyperthermophiles have been determined. To facilitate such an understanding, the crystal structure of a putative Opp, OppA from Thermotoga maritima (TmOppA), was solved at 2.6-Å resolution in the open conformation. TmOppA is composed of three domains. The N-terminal domain consists of twelve strands, nine helices, and four 310 helices, and the C-terminal domain consists of five strands, ten helices, and one 310 helix. These two domains are connected by the linker domain, which consists of two strands, three helices, and three 310 helices. Based on structural comparisons of TmOppA with other OppAs and binding studies, we suggest that TmOppA might be a periplasmic Opp. The most distinct feature of TmOppA is the insertion of two helices, which are lacking in other OppAs. A cavity volume between the N-terminal and C-terminal domains is suggested to be responsible for binding peptides of various lengths.
Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte/química , Lipoproteínas/química , Periplasma/metabolismo , Thermotoga maritima/química , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Lipoproteínas/genética , Lipoproteínas/metabolismo , Ligação Proteica , Thermotoga maritima/genética , Thermotoga maritima/metabolismoRESUMO
ECF transporters are a family of active membrane transporters for essential micronutrients, such as vitamins and trace metals. Found exclusively in archaea and bacteria, these transporters are composed of four subunits: an integral membrane substrate-binding subunit (EcfS), a transmembrane coupling subunit (EcfT), and two ATP-binding cassette ATPases (EcfA and EcfA'). We have characterized the structural basis of substrate binding by the EcfS subunit for riboflavin from Thermotoga maritima, TmRibU. TmRibU binds riboflavin with high affinity, and the protein-substrate complex is exceptionally stable in solution. The crystal structure of riboflavin-bound TmRibU reveals an electronegative binding pocket at the extracellular surface in which the substrate is completely buried. Analysis of the intermolecular contacts indicates that nearly every available substrate hydrogen bond is satisfied. A conserved aromatic residue at the extracellular end of TM5, Tyr130, caps the binding site to generate a substrate-bound, occluded state, and non-conservative mutation of Tyr130 reduces the stability of this conformation. Using a novel fluorescence binding assay, we find that an aromatic residue at this position is essential for high-affinity substrate binding. Comparison with other S subunit structures suggests that TM5 and Loop5-6 contain a dynamic, conserved motif that plays a key role in gating substrate entry and release by S subunits of ECF transporters.
Assuntos
Sítios de Ligação/fisiologia , Proteínas de Membrana Transportadoras/metabolismo , Subunidades Proteicas/metabolismo , Riboflavina/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X/métodos , Modelos Moleculares , Conformação Proteica , Thermotoga maritima/metabolismo , Vitaminas/metabolismoRESUMO
The essential process of protein secretion is achieved by the ubiquitous Sec machinery. In prokaryotes, the drive for translocation comes from ATP hydrolysis by the cytosolic motor-protein SecA, in concert with the proton motive force (PMF). However, the mechanism through which ATP hydrolysis by SecA is coupled to directional movement through SecYEG is unclear. Here, we combine all-atom molecular dynamics (MD) simulations with single molecule FRET and biochemical assays. We show that ATP binding by SecA causes opening of the SecY-channel at long range, while substrates at the SecY-channel entrance feed back to regulate nucleotide exchange by SecA. This two-way communication suggests a new, unifying 'Brownian ratchet' mechanism, whereby ATP binding and hydrolysis bias the direction of polypeptide diffusion. The model represents a solution to the problem of transporting inherently variable substrates such as polypeptides, and may underlie mechanisms of other motors that translocate proteins and nucleic acids.
Assuntos
Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Escherichia coli/metabolismo , Methanocaldococcus/metabolismo , Canais de Translocação SEC/química , Thermotoga maritima/metabolismo , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Escherichia coli/genética , Expressão Gênica , Cinética , Methanocaldococcus/genética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canais de Translocação SEC/genética , Canais de Translocação SEC/metabolismo , Proteínas SecA , Especificidade por Substrato , Termodinâmica , Thermotoga maritima/genéticaRESUMO
SecYEG protein of bacteria or Sec61αßγ of eukaryotes is a universally conserved heterotrimeric protein channel complex that accommodates the partitioning of membrane proteins into the lipid bilayer as well as the secretion of proteins to the trans side of the plasma or endoplasmic reticular membrane, respectively. SecYEG function is facilitated by cytosolic partners, mainly a nascent chain-ribosome complex or the SecA ATPase motor protein. Extensive efforts utilizing both biochemical and biophysical approaches have been made to determine whether SecYEG functions as a monomer or a dimer, but such approaches have often generated conflicting results. Here we have employed site-specific in vivo photo-cross-linking or cysteine cross-linking, along with co-immunoprecipitation or SecA footprinting techniques to readdress this issue. Our findings show that the SecY dimer to monomer ratio is relatively constant regardless of whether translocons are actively engaged with protein substrate or not. Under the former conditions the SecY dimer can be captured associated with a translocon-jammed substrate, indicative of SecY dimer function. Furthermore, SecA ATPase can be cross-linked to two copies of SecY when the complex contains a translocation intermediate. Collectively, our results suggest that SecYEG dimers are functional units of the translocon.