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At least 235 records · Page 13Linked to original sources
PubMed1999

The efficient export of NADP-containing glucose-fructose oxidoreductase to the periplasm of Zymomonas mobilis depends both on an intact twin-arginine motif in the signal peptide and on the generation of a structural export signal induced by cofactor binding.

The periplasmic, NADP-containing glucose-fructose oxidoreductase of the gram-negative bacterium Zymomonas mobilis belongs to a class of redox cofactor-dependent enzymes which are exported with the aid of a signal peptide containing a so-called twin-arginine motif. In this paper we show that the replacement of one or both arginine residues results in drastically reduced translocation of glucose-fructose oxidoreductase to the periplasm, showing that this motif is essential. Mutant proteins which, in contrast to wild-type glucose-fructose oxidoreductase, bind NADP in a looser and dissociable manner, were severely affected in the kinetics of plasma membrane translocation. These results strongly suggest that the translocation of glucose-fructose oxidoreductase into the periplasm uses a Sec-independent apparatus which recognizes, as an additional signal, a conformational change in the structure of the protein, most likely triggered by cofactor binding. Furthermore, these results suggest that glucose-fructose oxidoreductase is exported in a folded form. A glucose-fructose oxidoreductase:beta-galactosidase fusion protein is not lethal to Z. mobilis cells and leads to the accumulation of the cytosolic preform of wild-type glucose-fructose oxidoreductase expressed in trans but not of a typical Sec-substrate (OmpA), indicating that the glucose-fructose oxidoreductase translocation apparatus can be blocked without interfering with the export of essential proteins via the Sec pathway.

Arginine↗
PubMed2000

Proteomics of Synechocystis sp. strain PCC 6803. Identification of periplasmic proteins in cells grown at low and high salt concentrations.

Periplasmic proteins isolated by cold osmotic shock of Synechocystis sp. PCC 6803 cells were identified using 2D PAGE, MS and genome analysis. Most of the periplasmic proteins represent 'hypothetical proteins' with unknown function. A number of proteases of different specificity, and several enzymes involved in cell wall biosynthesis were also found. In salt-adapted cells, six proteins were greatly enhanced and three proteins were newly induced. Most of the salt-enhanced proteins are involved in the alteration of cell wall structure of salt-adapted cells. The precursors of all 57 periplasmic proteins identified have a signal peptide; 47 of them contain a typical Sec-dependent signal peptide, whereas 10 contain a putative twin-arginine signal peptide.

Amino Acid Sequence↗
PubMed1981

Role for membrane potential in the secretion of protein into the periplasm of Escherichia coli.

The leucine-specific binding protein of Escherichia coli is a periplasmic protein that is synthesized as a precursor and subsequently is processed during its secretion into the periplasmic space. The processing of both the leucine-specific binding protein and a plasmid-coded beta-lactamase is inhibited by phenethyl alcohol and by the proton ionophore, carbonylcyanide m-chlorophenylhydrazone (CCCP). The levels of CCCP that inhibit processing also produce significant decreases in the membrane potential. Valinomycin, a potassium ionophore, also inhibits processing of the leucine-specific binding protein in spheroplasts. Processing can be restored in CCCP-treated cells and in valinomycin-treated spheroplasts by dilution of the treated cells in fresh medium. These results suggest a role for membrane potential in the secretion of periplasmic proteins. A model is presented which suggests that membrane potential plays a primary role in the proper orientation of the precursor signal sequence within the membrane, thus promoting processing and secretion.

Bacterial Proteins↗
PubMed1998

The reductive enzyme thioredoxin 1 acts as an oxidant when it is exported to the Escherichia coli periplasm.

Thioredoxin 1 is a major thiol-disulfide oxidoreductase in the cytoplasm of Escherichia coli. One of its functions is presumed to be the reduction of the disulfide bond in the active site of the essential enzyme ribonucleotide reductase. Thioredoxin 1 is kept in a reduced state by thioredoxin reductase. In a thioredoxin reductase null mutant however, most of thioredoxin 1 is in the oxidized form; recent reports have suggested that this oxidized form might promote disulfide bond formation in vivo. In the Escherichia coli periplasm, the protein disulfide isomerase DsbC is maintained in the reduced and active state by the membrane protein DsbD. In a dsbD null mutant, DsbC accumulates in the oxidized form. This oxidized form is then able to promote disulfide bond formation. In both these cases, the inversion of the function of these thiol oxidoreductases appears to be due to an altered redox balance of the environment in which they find themselves. Here, we show that thioredoxin 1 attached to the alkaline phosphatase signal sequence can be exported into the E. coli periplasm. In this new environment for thioredoxin 1, we show that thioredoxin 1 can promote disulfide bond formation and, therefore, partially complement a dsbA strain defective for disulfide bond formation. Thus, we provide evidence that by changing the location of thioredoxin 1 from cytoplasm to periplasm, we change its function from a reductant to an oxidant. We conclude that the in vivo redox function of thioredoxin 1 depends on the redox environment in which it is localized.

Alkaline Phosphatase↗
PubMed1996

Liganded and unliganded receptors interact with equal affinity with the membrane complex of periplasmic permeases, a subfamily of traffic ATPases.

The histidine-binding protein, HisJ, is the soluble receptor for the periplasmic histidine permease of Salmonella typhimurium. The receptor binds the substrate in the periplasm, interacts with the membrane-bound complex, transmits a transmembrane signal to hydrolyze ATP, and releases the ligand for translocation. HisJ, like other periplasmic receptors, has two lobes that are apart in the unliganded structure (open conformation) and drawn close together in the liganded structure (closed conformation), burying deeply the ligand. Such receptors are postulated to interact with the membrane-bound complex with high affinity in their liganded conformation, and, upon substrate translocation, to undergo a reduction in affinity and therefore be released. Here we show that in contrast to the current postulate, liganded and unliganded receptors have equal affinity for the membrane-bound complex. The affinity is measured both by chemical cross-linking and co-sedimentation procedures. An ATPase activity assay is also used to demonstrate the interaction of unliganded receptor with the membrane-bound complex. These findings support a new model for the transport mechanism, in which the soluble receptor functions independently of the commonly accepted high-low affinity switch.

ATP-Binding Cassette Transporters↗
PubMed1996

Properties of the periplasmic ModA molybdate-binding protein of Escherichia coli.

The modABCD operon, located at 17 min on the Escherichia coli chromosome, encodes the protein components of a high affinity molybdate uptake system. Sequence analysis of the modA gene (GenBank L34009) predicts that it encodes a periplasmic binding protein based on the presence of a leader-like sequence at its N terminus. To examine the properties of the ModA protein, the modA structural gene was overexpressed, and its product was purified. The ModA protein was localized to the periplasmic space of the cell, and it was released following a gentle osmotic shock. The N-terminal sequence of ModA confirmed that a leader region of 24 amino acids was removed upon export from the cell. The apparent size of ModA is 31.6 kDa as determined by gel sieve chromatography, whereas it is 22.5 kDa when examined by SDS-polyacrylamide gel electrophoresis. A ligand-dependent protein mobility shift assay was devised using a native polyacrylamide gel electrophoresis protocol to examine binding of molybdate and other anions to the ModA periplasmic protein. Whereas molybdate and tungstate were bound with high affinity (approximately 5 microM), sulfate, chromate, selenate, phosphate, and chlorate did not bind even when tested at 2 mM. A UV spectral assay revealed apparent Kd values of binding for molybdate and tungstate of 3 and 7 microM, respectively. Strains defective in the modA gene were unable to transport molybdate unless high levels of the anion were supplied in the medium. Therefore the modA gene product is essential for high affinity molybdate uptake by the cell. Tungstate interference of molybdate acquisition by the cell is apparently due in part to the high affinity of the ModA protein for this anion.

Amino Acid Sequence↗
PubMed2003

The structure of the periplasmic ligand-binding domain of the sensor kinase CitA reveals the first extracellular PAS domain.

The integral membrane sensor kinase CitA of Klebsiella pneumoniae is part of a two-component signal transduction system that regulates the transport and metabolism of citrate in response to its environmental concentration. Two-component systems are widely used by bacteria for such adaptive processes, but the stereochemistry of periplasmic ligand binding and the mechanism of signal transduction across the membrane remain poorly understood. The crystal structure of the CitAP periplasmic sensor domain in complex with citrate reveals a PAS fold, a versatile ligand-binding structural motif that has not previously been observed outside the cytoplasm or implicated in the transduction of conformational signals across the membrane. Citrate is bound in a pocket that is shared among many PAS domains but that shows structural variation according to the nature of the bound ligand. In CitAP, some of the citrate contact residues are located in the final strand of the central beta-sheet, which is connected to the C-terminal transmembrane helix. These secondary structure elements thus provide a potential conformational link between the periplasmic ligand binding site and the cytoplasmic signaling domains of the receptor.

Amino Acid Sequence↗
PubMed2006

TorT, a member of a new periplasmic binding protein family, triggers induction of the Tor respiratory system upon trimethylamine N-oxide electron-acceptor binding in Escherichia coli.

In anaerobiosis, Escherichia coli can use trimethylamine N-oxide (TMAO) as a terminal electron acceptor. Reduction of TMAO in trimethylamine (TMA) is mainly performed by the respiratory TMAO reductase. This system is encoded by the torCAD operon, which is induced in the presence of TMAO. This regulation involves a two-component system comprising TorS, an unorthodox histidine kinase, and TorR, a response regulator. A third protein, TorT, sharing homologies with periplasmic binding proteins, plays a key role in this regulation because disruption of the torT gene abolishes tor expression. In this study we showed that TMAO protects TorT against degradation by the GluC endoproteinase and modifies its temperature-induced CD spectrum. We also isolated a TorT negative mutant that is no longer protected by TMAO from degradation by GluC. Isothermal titration calorimetry confirmed that TorT binds TMAO with a binding constant of 150 mum. Therefore, we conclude that TorT binds TMAO and that this binding promotes a conformational change of TorT. We also showed that TorT interacts with the periplasmic domain of TorS in both the presence and absence of TMAO but the TorT-TMAO complex induces a higher GluC protection of TorS than TorT alone. These results support the idea that TMAO binding to TorT induces a cascade of conformational changes from TorT to TorS, leading to TorS activation. We identified several homologues of the TorT protein that define a new family of periplasmic binding proteins. We thus propose that the members of this family bind TMAO or related compounds and that they are involved in signal transduction or even substrate transport.

Amino Acid Sequence↗
PubMed1998

The biophysics of the gram-negative periplasmic space.

When subject to an osmotic 'up-shock', water flows outward from bacterial cytoplasm of the bacterium. Lipid bilayers can shrink very little in area and therefore must wrinkle to accommodate the smaller volume. The usual consequence is that all the layers of the cell envelope must become wrinkled together because they adhere to each other and must now cover a smaller surface. Plasmolysis spaces are formed if the cytoplasmic membrane (CM) separates from the other components of the wall. However, because the CM bilayer is essentially an incompressible two-dimensional liquid, this constraint restricts the location and shape of plasmolysis spaces. With mild up-shocks they form at the pole and around constricting regions in the cell. Elsewhere their creation requires the formation of endocytotic or exocytotic vesicles. The formation of endocytotic vesicles occurs in animal and plant cells as well as in bacterial cells. With stronger up-shocks tubular structures (Bayer adhesion sites), or other special geometric shapes (e.g., Scheie structures) allow the bilayer to surround an irregular shaped cytoplast. Periosmotic agents, that is, those that extract water from the periplasm as well as the cytoplasm, are molecules such as poly-vinyl-pyrrolidone and alpha-cyclodextrin that are too large to pass through the porins in the outer membrane. They were found to significantly inhibit the formation of plasmolysis spaces. Presumably, they inhibit the plasmolysis process, which requires that extracellular fluid enter between the CM and the outer membrane (OM). In the extreme case, with the dehydrating action of both osmotic agents and periosmotic agents, periplasmic space formation tends to be prevented and a new kind of space develops within the cytoplasm. We have designated these as 'cytoplasmic voids'. These novel structures are not bounded by lipid bilayers, in contrast to the endocytotic vesicles. These new spaces appear to result from the negative turgor pressure generated by the application of the combination of osmotic and periosmotic agents causing bubble formation. Several ideas in the literature about the wall biology (periseptal annuli, leading edge, osmotic pressure in the periplasm) are presented and critiqued. The basic criticism of these is that much of the phenomena can be explained because of the physics of the phospholipid bilayers and osmotic forces and thus does not imply the existence of a special control mechanism to regulate growth and division.

Biophysical Phenomena↗
PubMed1998

Periplasmic chaperone recognition motif of subunits mediates quaternary interactions in the pilus.

The class of proteins collectively known as periplasmic immunoglobulin-like chaperones play an essential role in the assembly of a diverse set of adhesive organelles used by pathogenic strains of Gram-negative bacteria. Herein, we present a combination of genetic and structural data that sheds new light on chaperone-subunit and subunit-subunit interactions in the prototypical P pilus system, and provides new insights into how PapD controls pilus biogenesis. New crystallographic data of PapD with the C-terminal fragment of a subunit suggest a mechanism for how periplasmic chaperones mediate the extraction of pilus subunits from the inner membrane, a prerequisite step for subunit folding. In addition, the conserved N- and C-terminal regions of pilus subunits are shown to participate in the quaternary interactions of the mature pilus following their uncapping by the chaperone. By coupling the folding of subunit proteins to the capping of their nascent assembly surfaces, periplasmic chaperones are thereby able to protect pilus subunits from premature oligomerization until their delivery to the outer membrane assembly site.

Adhesins, Bacterial↗
PubMed1999

Six conserved cysteines of the membrane protein DsbD are required for the transfer of electrons from the cytoplasm to the periplasm of Escherichia coli.

The active-site cysteines of the Escherichia coli periplasmic protein disulfide bond isomerase (DsbC) are kept reduced by the cytoplasmic membrane protein, DsbD. DsbD, in turn, is reduced by cytoplasmic thioredoxin, indicating that DsbD transfers disulfidereducing potential from the cytoplasm to the periplasm. To understand the mechanism of this unusual mode of electron transfer, we have undertaken a genetic analysis of DsbD. In the process, we discovered that the previously suggested start site for the DsbD protein is incorrect. Our results permit the formulation of a model of DsbD membrane topology. Also, we show that six cysteines of DsbD conserved among DsbD homologs are essential for the reduction of DsbC, DsbG and for a reductive pathway leading to c-type cytochrome assembly in the periplasm. Our findings suggest a testable model for the DsbD-dependent transfer of electrons across the membrane, involving a cascade of disulfide bond reduction steps.

Alkaline Phosphatase↗
PubMed1996

Defect in export and synthesis of the periplasmic galactose receptor MglB in dnaK mutants of Escherichia coli, and decreased stability of the mglB mRNA.

The high-affinity galactose permease, which comprises the periplasmic galactose receptor MglB, the membrane translocator MglC and the membrane-associated ATPase MglA, displayed a reduced activity in a dnaK temperature-sensitive mutant of Escherichia coli. This reduced transport activity correlated with a reduction in the quantity of MglB. At 42 degrees C, an accumulation of pre-MglB in the dnaK temperature-sensitive mutant reflected a defect in MglB export. In addition, an accumulation of pre-MglB in secB, secA and secY mutants suggested that SecB and the Sec translocase are also involved in export of the periplasmic galactose receptor. At 30 degrees C, there was no accumulation of pre-MglB in the dnaK mutant, but there was still a decreased amount of MglB in the periplasm. The reduction in MglB expression was not the result of a decrease in its stability, nor was it the result of a general defect in translation or transcription, since the MglA protein (which is expressed from the same operon as MglB) was synthesized in normal amounts. Two mRNAs are implicated in the expression of the mgl genes, a polycistronic mglBAC mRNA, and a more stable and more abundant mglB mRNA, produced by 3'-5' degradation of the mglBAC mRNA (R. W. Hogg, C. Voelker & I. von Carlowitz, 1991, Mol Gen Genet 229, 453-459). The mglB mRNA is protected against exonucleases by a REP (Repetitive Extragenic Palindrome) sequence located at its 3' extremity, which is responsible for the higher expression of MglB compared to MglA and MglC. The decreased MglB expression in the dnaK mutant at 30 degrees C in the present work correlated with a reduced stability of the mglB mRNA, which may have resulted from a defective stabilization by the REP sequence, or from a defect in translation of the mglB gene.

Adenosine Triphosphatases↗
PubMed2000

Oxidase and periplasmic cytochrome assembly in Escherichia coli K-12: CydDC and CcmAB are not required for haem-membrane association.

The mechanism(s) that bacteria use to transport haem into and across the cytoplasmic membrane to complete the assembly of periplasmic cytochromes is unknown. The authors have tested directly the role(s) of two ATP-binding cassette (ABC) transporters - the cydDC and ccmAB gene products - in Escherichia coli by measuring haem uptake in everted (inside-out) membrane vesicles. If haem is exported to the periplasm in vivo, the same process should result in active accumulation in such everted vesicles. [14C]Haemin (chloride) with bovine serum albumin (BSA) as a carrier protein was accumulated in intact everted membrane vesicles by an energy-independent mechanism. The kinetics of this process were biphasic: rapid uptake/binding was followed by a slower uptake of haem, which was inhibited by a large excess of unlabelled haemin-BSA, but not by BSA. However, accumulated haemin was not chased out of the vesicles by unlabelled haemin-BSA, suggesting specific binding of haemin with the membrane or transport into the lumen of the vesicle. Neither ATP nor a protonmotive force (delta(p)) generated by lactate oxidation was required for haemin binding or subsequent transport, and carbonyl cyanide m-chlorophenylhydrazone (CCCP), sodium vanadate and monensin had no effect on haemin transport. The rate of haemin uptake following the initial rapid binding was proportional to the external haemin concentration, suggesting that the uptake process was driven by the haemin concentration gradient across the cell membrane. The kinetics of [14C]haemin uptake were similar in wild-type and cydD1 or delta(ccmA) mutants, suggesting that the activity of neither the CydDC nor CcmAB transporters is essential for haem export to the periplasm. Cytochrome d levels were unaffected by mutations in trxB (encoding thioredoxin reductase), trxA (thioredoxin), or grx (glutaredoxin), suggesting that the CydDC transporter does not export these components of reducing pathways for cytochrome assembly.

ATP-Binding Cassette Transporters↗
PubMed2002

Periplasmic maltose- and glucose-binding protein activities in cell-free extracts of Thermotoga maritima.

In this study, high-affinity maltose- and glucose-binding activities in cell-free extracts of Thermotoga maritima were detected; these activities were distinct and specific. At the gross level, the expression of binding-protein activities was repressed by growth of T. maritima in the presence of the cognate sugar. Growth of the organism in the presence of maltose reduced maltose-binding activity but not glucose-binding activity, while growth in the presence of glucose reduced glucose-binding activity but not maltose-binding activity. In competition assays, these binding activities showed distinct patterns of substrate specificity: whereas the maltose-binding activity showed specificity for alpha-linked glucosides, the glucose-binding activity showed a broader specificity. All maltose- and glucose-binding activity was found in the supernatant retrieved following centrifugation (100,000 g) of the cell-free extracts prepared by French-pressure-cell treatment; no activity was found in an octyl-glucoside-treated extract of the membrane fraction. The maltose-binding-protein activity was recovered from the periplasmic fraction by selective release of the periplasmic contents of T. maritima cells using a newly developed freeze-thaw procedure. Annotation of the complete genome sequence of T. maritima suggests that there may be at least two maltose-binding proteins, MalE1 and MalE2, encoded in the genome. The maltose-binding activity corresponded to a protein of 43 kDa, which was consistent in size with either of the putative proteins. These data demonstrate that the hyperthermophilic bacterium T. maritima possesses separate maltose- and glucose-binding-protein activities that are freely soluble in its periplasm, in contrast to the membrane-bound sugar-binding proteins found in archaeal hyperthermophiles.

Carrier Proteins↗
PubMed2002

The role of two periplasmic copper- and zinc-cofactored superoxide dismutases in the virulence of Salmonella choleraesuis.

Periplasmic copper- and zinc-cofactored superoxide dismutases ([Cu,Zn]-SODs, SodC) of several Gram-negative pathogens can protect against superoxide-radical-mediated host defences, and thus contribute to virulence. This role has been previously defined for one [Cu,Zn]-SOD in various Salmonella serovars. Following the recent discovery of a second periplasmic [Cu,Zn]-SOD in Salmonella, the effect of knockout mutations in one or both of the original sodC-1 and the new sodC-2 on the virulence of the porcine pathogen Salmonella choleraesuis is investigated here. In comparison to wild-type, while sodC mutants--whether single or double--showed no impairment in growth, they all showed equally enhanced sensitivity to superoxide and a dramatically increased sensitivity to the combination of superoxide and nitric oxide in vitro. This observation had its correlate in experimental infection both ex vivo and in vivo. Mutation of sodC significantly impaired survival of S. choleraesuis in interferon gamma-stimulated murine macrophages compared to wild-type organisms, and all S. choleraesuis sodC mutants persisted in significantly lower numbers than wild-type in BALB/c (Ity(s)) and C3H/HeN (Ity(r)) mice after experimental infection, but in no experimental system were sodC-1 sodC-2 double mutants more attenuated than either single mutant. These data suggest that both [Cu,Zn]-SODs are needed to protect bacterial periplasmic or membrane components. While SodC plays a role in S. choleraesuis virulence, the data presented here suggest that this is through overcoming a threshold effect, probably achieved by acquisition of sodC-1 on a bacteriophage. Loss of either sodC gene confers maximum vulnerability to superoxide on S. choleraesuis.

Animals↗
PubMed1999

Erwinia carotovora DsbA mutants: evidence for a periplasmic-stress signal transduction system affecting transcription of genes encoding secreted proteins.

The dsbA genes, which encode major periplasmic disulfide-bond-forming proteins, were isolated from Erwinia carotovora subsp. carotovora (Ecc) and Erwinia carotovora subsp. atroseptica (Eca), and the dsbC gene, encoding another periplasmic disulfide oxidoreductase was isolated from Ecc. All three genes were sequenced and mutants deficient in these genes were created by marker exchange mutagenesis. The Ecc mutants were severely affected in activity and secretion of pectate lyase, probably due to the absence of functional PelC, which is predicted to require disulfide bond formation to achieve its correct conformation prior to secretion across the outer membrane. Similarly, endopolygalacturonase, also predicted to possess disulfide bonds, displayed reduced activity. The major Ecc cellulase (CelV) does not contain cysteine residues and was still secreted in dsbA-deficient strains. This observation demonstrated unequivocally that the localization and activity of the individual components of the Out apparatus are independent of disulfide bond formation. Surprisingly, cellulase activity was shown to be increased approximately two- to threefold in the DsbA mutant. This phenomenon resulted from transcriptional up-regulation of celV gene expression. In contrast, transcription of both pelC and peh were down-regulated in dsbA-deficient strains when compared to the wild-type. Protease (Prt) activity and secretion were unaffected in the Ecc dsbA mutant. Prt activity was considerably reduced in the double dsbA dsbC mutant. However Prt was secreted normally in this strain. The Eca dsbA mutant was found to be non-motile, suggesting that disulfide bond formation is essential for motility in this strain. All of the dsb mutants showed reduced tissue maceration in planta. These results suggest that a feedback regulation system operates in Ecc. In this system, defects in periplasmic disulfide bond formation act as a signal which is relayed to the transcription machinery regulating gene expression in diverse ways.

Cellulase↗
PubMed2006

Expression, crystallization and preliminary X-ray analysis of the periplasmic stress sensory protein RseB from Escherichia coli.

Sensing external stress in the bacterial periplasm and signal transduction to the cytoplasm are important functions of the CpxAR, Bae and sigma(E) signalling pathways. In Escherichia coli, the sigma(E) pathway can be activated through degradation of the antisigma factor RseA by DegS and YaeL. The periplasmic protein RseB plays an important role in this pathway by exerting a direct or indirect negative effect on YaeL cleavage efficiency. RseB from E. coli, missing the periplasmic signal sequence (RseB(DeltaN)), was cloned, expressed, purified and crystallized. Crystals were obtained in two different forms belonging to space group P42(1)2 (form I) and C222(1) (form II) and diffracted to 2.8 and 2.4 A resolution, respectively. In crystal form I two copies of the protein were located in the asymmetric unit according to heavy-atom analysis, while crystal form II contained three copies.

Crystallization↗
PubMed2005

Nuclear magnetic resonance solution structure of the periplasmic signalling domain of the TonB-dependent outer membrane transporter FecA from Escherichia coli.

Gram-negative bacteria possess outer membrane receptors that utilize energy provided by the TonB system to take up iron. Several of these receptors participate in extracytoplasmic factor (ECF) signalling through an N-terminal signalling domain that interacts with a periplasmic transmembrane anti-sigma factor protein and a cytoplasmic sigma factor protein. The structures of the intact TonB-dependent outer membrane receptor FecA from Escherichia coli and FpvA from Pseudomonas aeruginosa have recently been solved by protein crystallography; however, no electron density was detected for their periplasmic signalling domains, suggesting that it was either unfolded or flexible with respect to the remainder of the protein. Here we describe the well-defined solution structure of this domain solved by multidimensional nuclear magnetic resonance (NMR) spectroscopy. The monomeric protein construct contains the 79-residue N-terminal domain as well as the next 17 residues that are part of the receptor's plug domain. These form two clearly distinct regions: a highly structured domain at the N-terminal end followed by an extended flexible tail at the C-terminal end, which includes the 'TonB-box' region, and connects it to the plug domain of the receptor. The structured region consists of two alpha-helices that are positioned side by side and are sandwiched in between two small beta-sheets. This is a novel protein fold which appears to be preserved in all the periplasmic signalling domains of bacterial TonB-dependent outer membrane receptors that are involved in ECF signalling, because the hydrophobic residues that make up the core of the protein domain are highly conserved.

Amino Acid Sequence↗