RESUMO
Alginate is a polymer containing two uronic acid epimers, ß-d-mannuronate (M) and α-l-guluronate (G), and is a major component of brown seaweed that is depolymerized by alginate lyases. These enzymes have diverse specificity, cleaving the chain with endo- or exotype activity and with differential selectivity for the sequence of M or G at the cleavage site. Dp0100 is a 201-kDa multimodular, broad-specificity endotype alginate lyase from the marine thermophile Defluviitalea phaphyphila, which uses brown algae as a carbon source, converting it to ethanol, and bioinformatics analysis suggested that its catalytic domain represents a new polysaccharide lyase family, PL39. The structure of the Dp0100 catalytic domain, determined at 2.07 Å resolution, revealed that it comprises three regions strongly resembling those of the exotype lyase families PL15 and PL17. The conservation of key catalytic histidine and tyrosine residues belonging to the latter suggests these enzymes share mechanistic similarities. A complex of Dp0100 with a pentasaccharide, M5, showed that the oligosaccharide is located in subsites -2, -1, +1, +2, and +3 in a long, deep canyon open at both ends, explaining the endotype activity of this lyase. This contrasted with the hindered binding sites of the exotype enzymes, which are blocked such that only one sugar moiety can be accommodated at the -1 position in the catalytic site. The biochemical and structural analyses of Dp0100, the first for this new class of endotype alginate lyases, have furthered our understanding of the structure-function and evolutionary relationships within this important class of enzymes.
Assuntos
Proteínas de Bactérias/química , Clostridiales/enzimologia , Polissacarídeo-Liases/química , Proteínas de Bactérias/genética , Clostridiales/genética , Cristalografia por Raios X , Polissacarídeo-Liases/genética , Domínios ProteicosRESUMO
Many human-dwelling bacteria acquire sialic acid for growth or surface display. We identified previously a sialic acid utilization operon in Tannerella forsythia that includes a novel outer membrane sialic acid-transport system (NanOU), where NanO (neuraminate outer membrane permease) is a putative TonB-dependent receptor and NanU (extracellular neuraminate uptake protein) is a predicted SusD family protein. Using heterologous complementation of nanOU genes into an Escherichia coli strain devoid of outer membrane sialic acid permeases, we show that the nanOU system from the gut bacterium Bacteroides fragilis is functional and demonstrate its dependence on TonB for function. We also show that nanU is required for maximal function of the transport system and that it is expressed in a sialic acid-responsive manner. We also show its cellular localization to the outer membrane using fractionation and immunofluorescence experiments. Ligand-binding studies revealed high-affinity binding of sialic acid to NanU (Kd ~400 nM) from two Bacteroidetes species as well as binding of a range of sialic acid analogues. Determination of the crystal structure of NanU revealed a monomeric SusD-like structure containing a novel motif characterized by an extended kinked helix that might determine sugar-binding specificity. The results of the present study characterize the first bacterial extracellular sialic acid-binding protein and define a sialic acid-specific PUL (polysaccharide utilization locus).
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Bacteroidetes/metabolismo , Proteínas de Membrana/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Bacteroides fragilis/genética , Bacteroides fragilis/metabolismo , Bacteroidetes/genética , Transporte Biológico , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Intestinos/microbiologia , Boca/microbiologia , Mutação , Ligação Proteica , Estrutura Secundária de ProteínaRESUMO
The clamp-loader complex plays a crucial role in DNA replication by loading the ß-clamp onto primed DNA to be used by the replicative polymerase. Relatively little is known about the stoichiometry, structure and assembly pathway of this complex, and how it interacts with the replicative helicase, in Gram-positive organisms. Analysis of full and partial complexes by mass spectrometry revealed that a hetero-pentameric τ3-δ-δ' Bacillus subtilis clamp-loader assembles via multiple pathways, which differ from those exhibited by the Gram-negative model Escherichia coli. Based on this information, a homology model of the B. subtilis τ3-δ-δ' complex was constructed, which revealed the spatial positioning of the full C-terminal τ domain. The structure of the δ subunit was determined by X-ray crystallography and shown to differ from that of E. coli in the nature of the amino acids comprising the τ and δ' binding regions. Most notably, the τ-δ interaction appears to be hydrophilic in nature compared with the hydrophobic interaction in E. coli. Finally, the interaction between τ3 and the replicative helicase DnaB was driven by ATP/Mg(2+) conformational changes in DnaB, and evidence is provided that hydrolysis of one ATP molecule by the DnaB hexamer is sufficient to stabilize its interaction with τ3.
Assuntos
Bacillus subtilis/química , Proteínas de Bactérias/química , DnaB Helicases/química , Subunidades Proteicas/química , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , DnaB Helicases/metabolismo , Geobacillus stearothermophilus/enzimologia , Magnésio/química , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas/metabolismo , Homologia Estrutural de ProteínaRESUMO
Viral and bacterial Holliday junction resolvases differ in specificity with the former typically being more promiscuous, acting on a variety of branched DNA substrates, while the latter exclusively targets Holliday junctions. We have determined the crystal structure of a RuvC resolvase from bacteriophage bIL67 to help identify features responsible for DNA branch discrimination. Comparisons between phage and bacterial RuvC structures revealed significant differences in the number and position of positively-charged residues in the outer sides of the junction binding cleft. Substitutions were generated in phage RuvC residues implicated in branch recognition and six were found to confer defects in Holliday junction and replication fork cleavage in vivo. Two mutants, R121A and R124A that flank the DNA binding site were purified and exhibited reduced in vitro binding to fork and linear duplex substrates relative to the wild-type, while retaining the ability to bind X junctions. Crucially, these two variants cleaved Holliday junctions with enhanced specificity and symmetry, a feature more akin to cellular RuvC resolvases. Thus, additional positive charges in the phage RuvC binding site apparently stabilize productive interactions with branched structures other than the canonical Holliday junction, a feature advantageous for viral DNA processing but deleterious for their cellular counterparts.
Assuntos
Bacteriófagos/enzimologia , DNA Cruciforme/metabolismo , Resolvases de Junção Holliday/genética , Resolvases de Junção Holliday/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Resolvases de Junção Holliday/química , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/isolamento & purificação , Proteínas Mutantes/metabolismo , Ligação Proteica , Conformação Proteica , Especificidade por SubstratoRESUMO
The ring-shaped cohesin complex is a key player in sister chromatid cohesion, DNA repair, and gene transcription. The loading of cohesin to chromosomes requires the loader Scc2 and is regulated by ATP. This process is hindered by Smc3 acetylation. However, the molecular mechanism underlying this inhibition remains mysterious. Here, using Saccharomyces cerevisiae as a model system, we identify a novel configuration of Scc2 with pre-engaged cohesin and reveal dynamic conformations of the cohesin/Scc2 complex in the loading reaction. We demonstrate that Smc3 acetylation blocks the association of Scc2 with pre-engaged cohesin by impairing the interaction of Scc2 with Smc3's head. Lastly, we show that ATP binding induces the cohesin/Scc2 complex to clamp DNA by promoting the interaction between Scc2 and Smc3 coiled coil. Our results illuminate a dynamic reconfiguration of the cohesin/Scc2 complex during loading and indicate how Smc3 acetylation and ATP regulate this process.
Assuntos
Núcleo Celular , Saccharomyces cerevisiae , Acetilação , Trifosfato de Adenosina , Conformação Molecular , Saccharomyces cerevisiae/genética , CoesinasRESUMO
The PEB4 protein is an antigenic virulence factor implicated in host cell adhesion, invasion, and colonization in the food-borne pathogen Campylobacter jejuni. peb4 mutants have defects in outer membrane protein assembly and PEB4 is thought to act as a periplasmic chaperone. The crystallographic structure of PEB4 at 2.2-Å resolution reveals a dimer with distinct SurA-like chaperone and peptidyl-prolyl cis/trans isomerase (PPIase) domains encasing a large central cavity. Unlike SurA, the chaperone domain is formed by interlocking helices from each monomer, creating a domain-swapped architecture. PEB4 stimulated the rate of proline isomerization limited refolding of denatured RNase T(1) in a juglone-sensitive manner, consistent with parvulin-like PPIase domains. Refolding and aggregation of denatured rhodanese was significantly retarded in the presence of PEB4 or of an engineered variant specifically lacking the PPIase domain, suggesting the chaperone domain possesses a holdase activity. Using bioinformatics approaches, we identified two other SurA-like proteins (Cj1289 and Cj0694) in C. jejuni. The 2.3-Å structure of Cj1289 does not have the domain-swapped architecture of PEB4 and thus more resembles SurA. Purified Cj1289 also enhanced RNase T(1) refolding, although poorly compared with PEB4, but did not retard the refolding of denatured rhodanese. Structurally, Cj1289 is the most similar protein to SurA in C. jejuni, whereas PEB4 has most structural similarity to the Par27 protein of Bordetella pertussis. Our analysis predicts that Cj0694 is equivalent to the membrane-anchored chaperone PpiD. These results provide the first structural insights into the periplasmic assembly of outer membrane proteins in C. jejuni.
Assuntos
Proteínas de Bactérias/metabolismo , Campylobacter jejuni/metabolismo , Proteínas de Transporte/química , Proteínas de Escherichia coli/química , Chaperonas Moleculares/fisiologia , Peptidilprolil Isomerase/química , Fatores de Virulência/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Chaperoninas/química , Cristalografia por Raios X/métodos , Genoma Bacteriano , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética/métodos , Chaperonas Moleculares/genética , Plasmídeos/metabolismo , Conformação Proteica , Dobramento de Proteína , Propriedades de Superfície , Tiossulfato Sulfurtransferase/química , Fatores de Virulência/genética , Fatores de Virulência/fisiologiaRESUMO
bpsl0128, a gene encoding a putative response regulator from Burkholderia pseudomallei strain D286, has been cloned into a pETBLUE-1 vector system, overexpressed in Escherichia coli and purified. The full-length protein is degraded during purification to leave a fragment corresponding to the putative receiver domain, and crystals of this protein that diffracted to beyond 1.75â Å resolution have been grown by the hanging-drop vapour-diffusion technique using PEG 6000 as the precipitant. The crystals belonged to one of the enantiomorphic pair of space groups P3(1)21 and P3(2)21, with unit-cell parameters a = b = 65.69, c = 105.01â Å and either one or two molecules in the asymmetric unit.
Assuntos
Proteínas de Bactérias/química , Burkholderia pseudomallei/química , Proteínas de Membrana Transportadoras/química , Sequência de Aminoácidos , Cristalização , Cristalografia por Raios X , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The organic polymer lignin is a component of plant cell walls, which like (hemi)-cellulose is highly abundant in nature and relatively resistant to degradation. However, extracellular enzymes released by natural microbial consortia can cleave the ß-aryl ether linkages in lignin, releasing monoaromatic phenylpropanoids that can be further catabolised by diverse species of bacteria. Biodegradation of lignin is therefore important in global carbon cycling, and its natural abundance also makes it an attractive biotechnological feedstock for the industrial production of commodity chemicals. Whilst the pathways for degradation of lignin-derived aromatics have been extensively characterised, much less is understood about how they are recognised and taken up from the environment. The purple phototrophic bacterium Rhodopseudomonas palustris can grow on a range of phenylpropanoid monomers and is a model organism for studying their uptake and breakdown. R. palustris encodes a tripartite ATP-independent periplasmic (TRAP) transporter (TarPQM) linked to genes encoding phenylpropanoid-degrading enzymes. The periplasmic solute-binding protein component of this transporter, TarP, has previously been shown to bind aromatic substrates. Here, we determine the high-resolution crystal structure of TarP from R. palustris as well as the structures of homologous proteins from the salt marsh bacterium Sagittula stellata and the halophile Chromohalobacter salexigens, which also grow on lignin-derived aromatics. In combination with tryptophan fluorescence ligand-binding assays, our ligand-bound co-crystal structures reveal the molecular basis for high-affinity recognition of phenylpropanoids by these TRAP transporters, which have potential for improving uptake of these compounds for biotechnological transformations of lignin.
Assuntos
Proteínas de Bactérias/genética , Biodegradação Ambiental , Lignina/genética , Proteínas de Ligação a RNA/genética , Rodopseudomonas/genética , Fatores de Transcrição/genética , Transporte Biológico/genética , Regulação Bacteriana da Expressão Gênica/genética , Ligantes , Lignina/química , Lignina/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Oxirredutases/genética , Periplasma/genética , Periplasma/microbiologia , Proteínas Periplásmicas de Ligação/genética , Proteobactérias/genética , Proteobactérias/crescimento & desenvolvimento , Rodopseudomonas/crescimento & desenvolvimentoRESUMO
During nutrient limitation, bacteria produce the alarmones (p)ppGpp as effectors of a stress signaling network termed the stringent response. RsgA, RbgA, Era, and HflX are four ribosome-associated GTPases (RA-GTPases) that bind to (p)ppGpp in Staphylococcus aureus. These enzymes are cofactors in ribosome assembly, where they cycle between the ON (GTP-bound) and OFF (GDP-bound) ribosome-associated states. Entry into the OFF state occurs upon hydrolysis of GTP, with GTPase activity increasing substantially upon ribosome association. When bound to (p)ppGpp, GTPase activity is inhibited, reducing 70S ribosome assembly and growth. Here, we determine how (p)ppGpp impacts RA-GTPase-ribosome interactions. We show that RA-GTPases preferentially bind to 5'-diphosphate-containing nucleotides GDP and ppGpp over GTP, which is likely exploited as a regulatory mechanism within the cell to shut down ribosome biogenesis during stress. Stopped-flow fluorescence and association assays reveal that when bound to (p)ppGpp, the association of RA-GTPases to ribosomal subunits is destabilized, both in vitro and within bacterial cells. Consistently, structural analysis of the ppGpp-bound RA-GTPase RsgA reveals an OFF-state conformation similar to the GDP-bound state, with the G2/switch I loop adopting a conformation incompatible with ribosome association. Altogether, we highlight (p)ppGpp-mediated inhibition of RA-GTPases as a major mechanism of stringent response-mediated ribosome assembly and growth control. IMPORTANCE The stringent response is a bacterial signaling network that utilizes the nucleotides pppGpp and ppGpp to reprogram cells in order to survive nutritional stresses. However, much about how these important nucleotides control cellular reprogramming is unknown. Our previous work revealed that (p)ppGpp can bind to and inhibit the enzymatic activity of four ribosome-associated GTPases (RA-GTPases), enzymes that facilitate maturation of the 50S and 30S ribosomal subunits. Here, we examine how this occurs mechanistically and demonstrate that this interaction prevents the accommodation of RA-GTPases on ribosomal subunits both in vitro and within bacterial cells, with the ppGpp-bound state structurally mimicking the inactive GDP-bound conformation of the enzyme. We additionally reveal that these GTPase enzymes have a greater affinity for OFF-state-inducing nucleotides, which is a mechanism likely to control ribosome assembly during growth. With this, we further our understanding of how ribosome function is controlled by (p)ppGpp, enabling bacterial survival during stress.
Assuntos
Proteínas de Bactérias/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Subunidades Ribossômicas/metabolismo , Staphylococcus aureus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , Guanosina Pentafosfato/química , Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/química , Guanosina Tetrafosfato/metabolismo , Modelos Moleculares , Ligação Proteica , Subunidades Ribossômicas/química , Subunidades Ribossômicas/genética , Staphylococcus aureus/química , Staphylococcus aureus/genéticaRESUMO
Tripartite members of the ClyA family of α-PFTs have recently been identified in a number of pathogenic Gram-negative bacteria, including the human pathogen Serratia marcescens. Structures of a Gram-negative A component and a tripartite α-PFT complete pore are unknown and a mechanism for pore formation is still uncertain. Here we characterise the tripartite SmhABC toxin from S. marcescens and propose a mechanism of pore assembly. We present the structure of soluble SmhA, as well as the soluble and pore forms of SmhB. We show that the ß-tongue soluble structure is well conserved in the family and propose two conserved latches between the head and tail domains that are broken on the soluble to pore conformational change. Using the structures of individual components, sequence analysis and docking predictions we illustrate how the A, B and C protomers would assemble on the membrane to produce a complete tripartite α-PFT pore.
RESUMO
C4-dicarboxylates play a central role in cellular physiology as key metabolic intermediates. Under aerobic conditions, they participate in the citric acid cycle, while in anaerobic bacteria, they are important in energy-conserving fermentation and respiration processes. Ten different families of secondary transporters have been described to participate in C4-dicarboxylate movement across biological membranes, but only one of these utilizes an extracytoplasmic solute binding protein to achieve high-affinity uptake. Here, we identify the MatBAC system from the photosynthetic bacterium Rhodopseudomonas palustris as the first member of the tripartite tricarboxylate transport family to be involved in C4-dicarboxylate transport. Tryptophan fluorescence spectroscopy showed that MatC, the periplasmic binding protein from this system, binds to l- and d-malate with Kd values of 27 and 21â¯nM, respectively, the highest reported affinity to date for these C4-dicarboxylates, and to succinate (Kd = 110â¯nM) and fumarate (Kd = 400â¯nM). The 2.1-Å crystal structure of MatC with bound malate shows a high level of substrate coordination, with participation of two water molecules that bridge hydrogen bonds between the ligand proximal carboxylic group and the main chain of two conserved loops in the protein structure. The substrate coordination in MatC correlates with the binding data and explains the protein's selectivity for different substrates and respective binding affinities. Our results reveal a new function in C4-dicarboxylate transport by members of the poorly characterized tripartite tricarboxylate transport family, which are widely distributed in bacterial genomes but for which details of structure-function relationships and transport mechanisms have been lacking.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Periplasma/metabolismo , Proteínas Periplásmicas de Ligação/metabolismo , Rodopseudomonas/metabolismo , Sequência de Aminoácidos , Transporte Biológico/fisiologia , Fumaratos/metabolismo , Malatos/metabolismo , Ácido Succínico/metabolismoRESUMO
The alpha helical CytolysinA family of pore forming toxins (α-PFT) contains single, two, and three component members. Structures of the single component Eschericia coli ClyA and the two component Yersinia enterolytica YaxAB show both undergo conformational changes from soluble to pore forms, and oligomerization to produce the active pore. Here we identify tripartite α-PFTs in pathogenic Gram negative bacteria, including Aeromonas hydrophila (AhlABC). We show that the AhlABC toxin requires all three components for maximal cell lysis. We present structures of pore components which describe a bi-fold hinge mechanism for soluble to pore transition in AhlB and a contrasting tetrameric assembly employed by soluble AhlC to hide their hydrophobic membrane associated residues. We propose a model of pore assembly where the AhlC tetramer dissociates, binds a single membrane leaflet, recruits AhlB promoting soluble to pore transition, prior to AhlA binding to form the active hydrophilic lined pore.
Assuntos
Aeromonas hydrophila/metabolismo , Toxinas Bacterianas/química , Proteínas Hemolisinas/química , Proteínas Citotóxicas Formadoras de Poros/química , Aeromonas hydrophila/química , Aeromonas hydrophila/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Cristalografia por Raios X , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismoRESUMO
A knowledge of the structures of acyl chain loaded species of the acyl carrier protein (ACP) as used in fatty acid biosynthesis and a range of other metabolic events, is essential for a full understanding of the molecular recognition at the heart of these processes. To date the only crystal structure of an acylated species of ACP is that of a butyryl derivative of Escherichia coli ACP. We have now determined the structures of a family of acylated E. coli ACPs of varying acyl chain length. The acyl moiety is attached via a thioester bond to a phosphopantetheine linker that is in turn bound to a serine residue in ACP. The growing acyl chain can be accommodated within a central cavity in the ACP for transport during the elongation stages of lipid synthesis through changes in the conformation of a four alpha-helix bundle. The results not only clarify the means by which a substrate of varying size and complexity is transported in the cell but also suggest a mechanism by which interacting enzymes can recognize the loaded ACP through recognition of surface features including the conformation of the phosphopantetheine linker.
Assuntos
Proteína de Transporte de Acila/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Sítios de Ligação , Cristalografia por Raios X , Ácidos Graxos/biossíntese , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformação ProteicaRESUMO
We have determined the structure of a catalytically inactive D70N variant of the Escherichia coli RusA resolvase bound to a duplex DNA substrate that reveals critical protein-DNA interactions and permits a much clearer understanding of the interaction of the enzyme with a Holliday junction (HJ). The RusA enzyme cleaves HJs, the fourway DNA branchpoints formed by homologous recombination, by introducing symmetrical cuts in the phosphodiester backbone in a Mg2+ dependent reaction. Although, RusA shows a high level of selectivity for DNA junctions, preferring to bind fourway junctions over other substrates in vitro, it has also been shown to have appreciable affinity for duplex DNA. However, RusA does not show DNA cleavage activity with duplex substrates. Our structure suggests the possible basis for structural selectivity as well as sources of the sequence specificity observed for DNA cleavage by RusA.
Assuntos
DNA Cruciforme/química , Proteínas de Escherichia coli/química , Resolvases de Junção Holliday/química , Modelos Moleculares , Substituição de Aminoácidos , Sítios de Ligação , DNA/química , DNA Cruciforme/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Resolvases de Junção Holliday/genética , Resolvases de Junção Holliday/metabolismo , Especificidade por SubstratoRESUMO
The Lrp/AsnC family of transcriptional regulatory proteins is found in both archaea and bacteria. Members of the family influence cellular metabolism in both a global (Lrp) and specific (AsnC) manner, often in response to exogenous amino acid effectors. In the present study we have determined both the first bacterial and the highest resolution structures for members of the family. Escherichia coli AsnC is a specific gene regulator whose activity is triggered by asparagine binding. Bacillus subtilis LrpC is a global regulator involved in chromosome condensation. Our AsnC-asparagine structure is the first for a regulator-effector complex and is revealed as an octameric disc. Key ligand recognition residues are identified together with a route for ligand access. The LrpC structure reveals a stable octamer supportive of a topological role in dynamic DNA packaging. The structures yield significant clues to the functionality of Lrp/AsnC-type regulators with respect to ligand binding and oligomerization states as well as to their role in specific and global DNA regulation.
Assuntos
Proteínas de Bactérias/química , Proteínas de Escherichia coli/química , Modelos Moleculares , Transativadores/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Asparagina/química , Asparagina/metabolismo , Bacillus subtilis , Proteínas de Bactérias/classificação , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/classificação , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Ligantes , Dados de Sequência Molecular , Alinhamento de Sequência , Transativadores/classificação , Transativadores/metabolismo , Fatores de Transcrição/classificação , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The tripartite tricarboxylate transporter (TTT) family is a poorly characterised group of prokaryotic secondary solute transport systems, which employ a periplasmic substrate-binding protein (SBP) for initial ligand recognition. The substrates of only a small number of TTT systems are known and very few SBP structures have been solved, so the mechanisms of SBP-ligand interactions in this family are not well understood. The SBP RPA4515 (AdpC) from Rhodopseudomonas palustris was found by differential scanning fluorescence and isothermal titration calorimetry to bind aliphatic dicarboxylates of a chain length of six to nine carbons, with KD values in the µm range. The highest affinity was found for the C6-dicarboxylate adipate (1,6-hexanedioate). Crystal structures of AdpC, either adipate or 2-oxoadipate bound, revealed a lack of positively charged amino acids in the binding pocket and showed that water molecules are involved in bridging hydrogen bonds to the substrate, a conserved feature in the TTT SBP family that is distinct from other types of SBP. In AdpC, both of the ligand carboxylate groups and a linear chain conformation are needed for coordination in the binding pocket. RT-PCR showed that adpC expression is upregulated by low environmental adipate concentrations, suggesting adipate is a physiologically relevant substrate but as adpC is not genetically linked to any TTT membrane transport genes, the role of AdpC may be in signalling rather than transport. Our data expand the known ligands for TTT systems and identify a novel high-affinity binding protein for adipate, an important industrial chemical intermediate and food additive. DATABASES: Protein structure co-ordinates are available in the PDB under the accession numbers 5OEI and 5OKU.
Assuntos
Adipatos/metabolismo , Transportadores de Ácidos Dicarboxílicos/metabolismo , Proteínas Periplásmicas de Ligação/metabolismo , Rodopseudomonas/metabolismo , Adipatos/farmacologia , Sequência de Aminoácidos , Cristalografia por Raios X , DNA Bacteriano/genética , Transportadores de Ácidos Dicarboxílicos/genética , Ácidos Dicarboxílicos/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Cinética , Ligantes , Modelos Moleculares , Proteínas Periplásmicas de Ligação/química , Proteínas Periplásmicas de Ligação/genética , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas Recombinantes de Fusão/química , Rodopseudomonas/genética , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Acyl carrier protein (ACP) is an essential cofactor in biosynthesis of fatty acids and many other reactions that require acyl transfer steps. We have determined the first crystal structures of an acylated form of ACP from E. coli, that of butyryl-ACP. Our analysis of the molecular surface of ACP reveals a plastic hydrophobic cavity in the vicinity of the phosphopantethylated Ser36 residue that is expanded and occupied by the butyryl and beta-mercaptoethylamine moieties of the acylated 4'-phosphopantetheine group in one of our crystal forms. In the other form, the cavity is contracted, and we propose that the protein has adopted the conformation after delivery of substrate into the active site of a partner enzyme.
Assuntos
Proteína de Transporte de Acila/química , Proteínas de Escherichia coli/química , Proteína de Transporte de Acila/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
The crystal structure of C. elegans Ap(4)A hydrolase has been determined for the free enzyme and a binary complex at 2.0 A and 1.8 A, respectively. Ap(4)A hydrolase has a key role in regulating the intracellular Ap(4)A levels and hence potentially the cellular response to metabolic stress and/or differentiation and apoptosis via the Ap(3)A/Ap(4)A ratio. The structures reveal that the enzyme has the mixed alpha/beta fold of the Nudix family and also show how the enzyme binds and locates its substrate with respect to the catalytic machinery of the Nudix motif. These results suggest how the enzyme can catalyze the hydrolysis of a range of related dinucleoside tetraphosphate, but not triphosphate, compounds through precise orientation of key elements of the substrate.
Assuntos
Hidrolases Anidrido Ácido/química , Proteínas de Caenorhabditis elegans/química , Estrutura Terciária de Proteína , Hidrolases Anidrido Ácido/genética , Hidrolases Anidrido Ácido/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cristalografia por Raios X , Humanos , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
We have determined the structure of Kid toxin protein from E. coli plasmid R1 involved in stable plasmid inheritance by postsegregational killing of plasmid-less daughter cells. Kid forms a two-component system with its antagonist, Kis antitoxin. Our 1.4 A crystal structure of Kid reveals a 2-fold symmetric dimer that closely resembles the DNA gyrase-inhibitory toxin protein CcdB from E. coli F plasmid despite the lack of any notable sequence similarity. Analysis of nontoxic mutants of Kid suggests a target interaction interface associated with toxicity that is in marked contrast to that proposed for CcdB. A possible region for interaction of Kid with the antitoxin is proposed that overlaps with the target binding site and may explain the mode of antitoxin action.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/farmacologia , Escherichia coli/química , Plasmídeos , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Holliday junction resolution performed by a variety of structure-specific endonucleases is a key step in DNA recombination and repair. It is believed that all resolvases carry out their reaction chemistries in a similar fashion, utilizing a divalent cation to facilitate the hydrolysis of the phosphodiester backbone of the DNA, but their architecture varies. To date, with the exception of bacteriophage T4 endonuclease VII, each of the known resolvase enzyme structures has been categorized into one of two families: the integrases and the nucleases. We have now determined the structure of the Escherichia coli RusA Holliday junction resolvase, which reveals a fourth structural class for these enzymes. The structure suggests that dimer formation is essential for Mg(2+) cation binding and hence catalysis and that like the other resolvases, RusA distorts its Holliday junction target upon binding. Key residues identified by mutagenesis experiments are well positioned to interact with the DNA.