Complex formation between malate dehydrogenase and isocitrate dehydrogenase from Bacillus subtilis is regulated by tricarboxylic acid cycle metabolites.

In Bacillus subtilis, recent in vivo studies revealed that particular enzymes of the tricarboxylic acid cycle form complexes that allow an efficient transfer of metabolites. Remarkably, a complex of the malate dehydrogenase (Mdh) (EC 1.1.1.37) with isocitrate dehydrogenase (Icd) (EC 1.1.1.42) was identified, although both enzymes do not catalyze subsequent reactions. In the present study, the interactions between these enzymes were characterized in vitro by surface plasmon resonance in the absence and presence of their substrates and cofactors. These analyses revealed a weak but specific interaction between Mdh and Icd, which was specifically stimulated by a mixture of substrates and cofactors of Icd: isocitrate, NADP(+) and Mg(2+). Wild-type Icd converted these substrates too fast, preventing any valid quantitative analysis of the interaction with Mdh. Therefore, binding of the IcdS104P mutant to Mdh was quantified because the mutation reduced the enzymatic activity by 174-fold but did not affect the stimulatory effect of substrates and cofactors on Icd-Mdh complex formation. The analysis of the unstimulated Mdh-IcdS104P interaction revealed kinetic constants of k(a) = 2.0 ± 0.2 × 10(2) m(-1) ·s(-1) and k(d) = 1.0 ± 0.1 × 10(-3) ·s(-1) and a K(D) value of 5.0 ± 0.1 µm. Addition of isocitrate, NADP(+) and Mg(2+) stimulated the affinity of IcdS104P to Mdh by 33-fold (K(D) = 0.15 ± 0.01 µm, k(a) = 1.7 ± 0.7 × 10(3) m(-1) ·s(-1), k(d) = 2.6 ± 0.6 × 10(-4) ·s(-1)). Analyses of the enzymatic activities of wild-type Icd and Mdh showed that Icd activity doubles in the presence of Mdh, whereas Mdh activity was slightly reduced by Icd. In summary, these data indicate substrate control of complex formation in the tricarboxylic acid cycle metabolon assembly and maintenance of the α-ketoglutarate supply for amino acid anabolism in vivo.

A novel method to analyze nucleocytoplasmic transport in vivo by using short peptide tags.

Regulated nucleocytoplasmic transport is of vital importance for maintaining the physiology of the cell, and disturbed nucleocytoplasmic shuttling of certain proteins has been found in a variety of diseases including cancer. The most frequently used procedure to analyze those processes is to fuse the protein of interest to a fluorescent protein such as GFP (green fluorescent protein)--a technique that is prone to impair normal protein function and subcellular localization. We report a novel approach to monitor nucleocytoplasmic transport processes in vivo by combining short TetR inducing peptide tags (TIP) with a TetR-controlled reporter gene in a human cell line. The technology is exemplified by demonstrating nucleocytoplasmic shuttling of the glucocorticoid receptor and activity of two further TIP fusions to cancer-related proteins. The technology presented provides the basis for efficient screening systems to isolate compounds altering the nucleocytoplasmic distribution of a protein of interest.

Promoter strength driving TetR determines the regulatory properties of Tet-controlled expression systems.

Bacteria frequently rely on transcription repressors and activators to alter gene expression patterns in response to changes in the surrounding environment. Tet repressor (TetR) is a paradigm transcription factor that senses the environmental state by binding small molecule effectors, the tetracyclines. However, recently isolated peptides that act as inducers of TetR after having been fused to the C-terminus of a carrier protein, suggest that TetR can also regulate gene expression in a signal-transduction pathway. For this shift in regulatory mechanism to be successful, induction of TetR must be sensitive enough to respond to an inducing protein expressed at its endogenous level. To determine this regulatory parameter, a synthetic Tet-regulated system was introduced into the human pathogen Salmonella enterica serovar Typhimurium and tested for inducibility by a peptide. Reporter gene expression was detected if the peptide-containing carrier protein Thioredoxin 1 was strongly overproduced, but not if it was expressed at a level similar to the physiological level of Thioredoxin 1. This was attributed to high steady-state amounts of TetR which was expressed by the promoter of the chloramphenicol acetyl transferase gene (P(cat)). Reducing P(cat) strength either by directed or by random mutagenesis of its -10 element concomitantly reduced the intracellular amounts of TetR. Sensitive and quantitative induction of TetR by an inducing peptide, when it was fused to Thioredoxin 1 at its native locus in the genome, was only obtained with weak P(cat) promoter variants containing GC-rich -10 elements. A second important observation was that reducing the TetR steady-state level did not impair repression. This permits flexible adjustment of an inducible system's sensitivity simply by altering the expression level of the transcription factor. These two new layers of expression control will improve the quality and, thus, the applicability of the Tet and other regulatory systems.

High- and low-affinity cre boxes for CcpA binding in Bacillus subtilis revealed by genome-wide analysis.

BACKGROUND: In Bacillus subtilis and its relatives carbon catabolite control, a mechanism enabling to reach maximal efficiency of carbon and energy sources metabolism, is achieved by the global regulator CcpA (carbon catabolite protein A). CcpA in a complex with HPr-Ser-P (seryl-phosphorylated form of histidine-containing protein, HPr) binds to operator sites called catabolite responsive elements, cre. Depending on the cre box position relative to the promoter, the CcpA/HPr-Ser-P complex can either act as a positive or a negative regulator. The cre boxes are highly degenerate semi-palindromes with a lowly conserved consensus sequence. So far, studies aimed at revealing how CcpA can bind such diverse sites were focused on the analysis of single cre boxes. In this study, a genome-wide analysis of cre sites was performed in order to identify differences in cre sequence and position, which determine their binding affinity. RESULTS: The transcriptomes of B. subtilis cultures with three different CcpA expression levels were compared. The higher the amount of CcpA in the cells, the more operons possessing cre sites were differentially regulated. The cre boxes that mediated regulation at low CcpA levels were designated as strong (high affinity) and those which responded only to high amounts of CcpA, as weak (low affinity). Differences in the sequence and position in relation to the transcription start site between strong and weak cre boxes were revealed. CONCLUSIONS: Certain residues at specific positions in the cre box as well as, to a certain extent, a more palindromic nature of cre sequences and the location of cre in close vicinity to the transcription start site contribute to the strength of CcpA-dependent regulation. The main factors contributing to cre regulatory efficiencies, enabling subtle differential control of various subregulons of the CcpA regulon, are identified.

Protein expression can be monitored in yeast by peptide-mediated induction of TetR-controlled gene expression.

The rapidly increasing number of completed genome sequences urgently calls for convenient and efficient methods for analysis of gene function and expression. TetR-inducing peptides (TIP) can induce reporter gene expression controlled by Tet repressor (TetR) when fused to a protein of choice which makes them a highly valuable tool for monitoring expression in vivo. However, TIP functionality has only been demonstrated in bacteria so far. Here, we report that TIP is also functional in yeast. An mCherry-TIP fusion that locates to the nucleus induces TetR-controlled gfp+ expression in a dose-dependent manner. This opens up potential applications in proteome research in which the expression of proteins can be analyzed in vivo by fusing TIP to proteins of choice in conjunction with a Tet-controlled reporter system.

CcpA forms complexes with CodY and RpoA in Bacillus subtilis.

The Bacillus subtilis catabolite control protein A (CcpA) is a global transcriptional regulator that is controlled by interactions with the phosphoproteins histidine-containing protein (HPr)Ser46P and the catabolite responsive HPr (Crh)Ser46P and with low molecular weight effectors, depending on the availability of preferred carbon sources such as glucose. Distinct point mutations in CcpA abolish the regulation of some but not all target genes, suggesting additional interactions of CcpA. Therefore, in vivo crosslinking and MS were applied to identify CcpA complexes active in repression and activation. To compensate for an excess of promoters only repressed by CcpA, this experiment was accomplished with cells using multiple copies of the activated ackA promoter. Among the identified proteins HPr, RNA polymerase subunits and the global regulator transcriptional pleiotropic repressor (CodY) were observed. Bacterial two-hybrid assays combining each RNA polymerase subunit with CcpA localized CcpA binding at the α-subunit of the RNA polymerase (RpoA). In vivo crosslinking combined with immunoblot analyses revealed CcpA-RpoA complexes in cultures with or without glucose, whereas CcpA-HPr and CcpA-CodY complexes occurred only or predominantly in cultures with glucose. Surface plasmon resonance analyses confirmed the binding of CcpA to the N-terminal domain (αNTD) and C-terminal domain (αCTD) of RpoA, as well as to CodY. Furthermore, interactions of CodY with the αNTD and the αCTD were detected by surface plasmon resonance. The K(D) values of complexes of CcpA or CodY with the αNTD or the αCTD are in the range 5-8 µm. CcpA and CodY form a loose complex with a K(D) of 60 µm. These data were combined to propose a model for a transcription initiation complex at the ackA promoter.

Carbon source control of the phosphorylation state of the Bacillus subtilis carbon-flux regulator Crh in vivo.

Bacillus subtilis possesses carbon-flux regulating histidine protein (Crh), a paralog of the histidine protein (HPr) of the phosphotransferase system (PTS). Like HPr, Crh becomes (de)phosphorylated in vitro at residue Ser46 by the metabolite-controlled HPr kinase/phosphorylase HPrK/P. Depending on its phosphorylation state, Crh exerts regulatory functions in connection with carbohydrate metabolism. So far, knowledge on phosphorylation of Crh in vivo has been limited and derived from indirect evidence. Here, we studied the dynamics of Crh phosphorylation directly by non-denaturing gel electrophoresis followed by Western analysis. The results confirm that HPrK/P is the single kinase catalyzing phosphorylation of Crh in vivo. Accordingly, phosphorylation of Crh is triggered by the carbon source as observed previously for HPr, but with some differences. Phosphorylation of both proteins occurred during exponential growth and disappeared upon exhaustion of the carbon source. During exponential growth, ~80% of the Crh molecules were phosphorylated when cells utilized a preferred carbon source. The reverse distribution, i.e. around 20% of Crh molecules phosphorylated, was obtained upon utilization of less favorable substrates. This clear-cut classification of the substrates into two groups has not previously been observed for HPr(Ser)~P formation. The likely reason for this difference is the additional PTS-dependent phosphorylation of HPr at His15, which limits accumulation of HPr(Ser)~P.

An exclusive α/ß code directs allostery in TetR-peptide complexes.

The allosteric mechanism of one of the best characterized bacterial transcription regulators, tetracycline repressor (TetR), has recently been questioned. Tetracycline binding induces cooperative folding of TetR, as suggested by recent unfolding studies, rather than switching between two defined conformational states, namely a DNA-binding-competent conformation and a non-DNA-binding conformation. Upon ligand binding, a host of near-native multiconformational structures collapse into a single, highly stabilized protein conformation that is no longer able to bind DNA. Here, structure-function studies performed with four synthetic peptides that bind to TetR and mimic the function of low-molecular-weight effectors, such as tetracyclines, provide new means to discriminate between different allosteric models. Whereas two inducing peptides bind in an extended ß-like conformation, two anti-inducing peptides form an α-helix in the effector binding site of TetR. This exclusive bimodal interaction mode coincides with two distinct overall conformations of TetR, namely one that is identical with induced TetR and one that mirrors the DNA-bound state of TetR. Urea-induced unfolding studies show no increase in thermodynamic stability for any of the peptide complexes, although fluorescence measurements demonstrate peptide binding to TetR. This strongly suggests that, at least for these peptide effectors, a classical two-state allosteric model best describes TetR function.

Short peptides act as inducers, anti-inducers and corepressors of Tet repressor.

Protein allostery plays a pivotal role in many regulatory processes. Prominent examples are cell-surface receptors, which allosterically transmit ligand-generated signals to their cytoplasmic domains, or bacterial transcription factors, which alternate between a free conformation and a DNA-bound conformation in response to binding an effector molecule. The bacterial transcription factor Tet repressor (TetR) belongs to the latter category and is regarded as highly adapted to tetracyclines (tc's) as effectors. However, peptides isolated in this study were able to trigger distinct allosteric behavior including induction, anti-induction and corepression. Binding of the peptides' C-terminal residues consistently occurs within the tc-binding pocket of TetR. However, an extensive analysis of TetR mutants revealed that inducing and anti-inducing peptides utilize different parts of the binding pocket to elicit their respective regulatory responses. This study demonstrates that even for transcription factors evolved for high effector specificity, alternative molecular structures can exert similar and even novel effects, provided that sufficient chemical diversity and molecular flexibility, as found in peptide libraries, is accompanied by an efficient in vivo selection system. The high number of bioactive peptides and their extensive sequence diversity suggests that switching from small-molecule-controlled transcription regulation to a signal transduction network might be rather easily accomplished. These findings will strongly affect protein-mediated regulation of gene expression.

Structures of carbon catabolite protein A-(HPr-Ser46-P) bound to diverse catabolite response element sites reveal the basis for high-affinity binding to degenerate DNA operators.

In Gram-positive bacteria, carbon catabolite protein A (CcpA) is the master regulator of carbon catabolite control, which ensures optimal energy usage under diverse conditions. Unlike other LacI-GalR proteins, CcpA is activated for DNA binding by first forming a complex with the phosphoprotein HPr-Ser46-P. Bacillus subtilis CcpA functions as both a transcription repressor and activator and binds to more than 50 operators called catabolite response elements (cres). These sites are highly degenerate with the consensus, WTGNNARCGNWWWCAW. How CcpA-(HPr-Ser46-P) binds such diverse sequences is unclear. To gain insight into this question, we solved the structures of the CcpA-(HPr-Ser46-P) complex bound to three different operators, the synthetic (syn) cre, ackA2 cre and gntR-down cre. Strikingly, the structures show that the CcpA-bound operators display different bend angles, ranging from 31° to 56°. These differences are accommodated by a flexible linkage between the CcpA helix-turn-helix-loop-helix motif and hinge helices, which allows independent docking of these DNA-binding modules. This flexibility coupled with an abundance of non-polar residues capable of non-specific nucleobase interactions permits CcpA-(HPr-Ser46-P) to bind diverse operators. Indeed, biochemical data show that CcpA-(HPr-Ser46-P) binds the three cre sites with similar affinities. Thus, the data reveal properties that license this protein to function as a global transcription regulator.

A single-chain triplebody with specificity for CD19 and CD33 mediates effective lysis of mixed lineage leukemia cells by dual targeting.

A single-chain triplebody (sctb) 33-ds16-ds19 comprising two distal single-chain Fv fragments (scFvs) specific for the lymphoid antigen CD19 and the myeloid antigen CD33 flanking a central scFv specific for CD16, which is the low affinity Fc-receptor (FcγRIII) present on natural killer cells and macrophages, was produced and its properties were investigated. CD33 and CD19 in combination are present on acute leukemiablasts with mixed lineage phenotype, but not on normal human hematopoietic cells. For comparison, two bispecific scFvs (bsscFvs), ds19-ds16 and 33-ds16, with monovalent binding to CD19 and CD33, respectively, were also studied. The sctb 33-ds16-ds19 specifically interacted with all 3 antigens. On the antigen double-positive cell line BV-173, the sctb bound with 2-fold greater avidity than bsscFv ds19-ds16 (KD = 21 vs. 42 nM) and with 1.4-fold greater avidity than bsscFv 33-ds16 (KD = 29 nM). All 3 fusion proteins had similar affinity for CD16 and sufficient thermic stability in human serum. In antibody-dependent cellular cytotoxicity (ADCC) reactions with human mononuclear cells as effectors, the sctb promoted lysis of BV-173 cells at 23-fold lower concentrations than bsscFv ds19-ds16 and at 1.4-fold lower concentrations than bsscFv 33-ds16. The sctb also mediated potent ADCC of the antigen double-positive mixed lineage leukemia cell line SEM, and the half-maximal concentration EC50 for BV-173 cells was 7 pM. Therefore, CD19 and CD33 are present on the surface of these leukemic cell lines such that they can be connected by a single sctb molecule, permitting the recruitment of NK cells via CD16 and tumor cell lysis.

Tetracycline sensing using novel doxycycline derivatives immobilized on different surface plasmon resonance biosensor surfaces.

BACKGROUND: This article aims to explore novel doxycycline derivatives for analyzing low concentrations of tetracyclines in biological matrices and food in competitive assays. RESULTS: Surface plasmon resonance (SPR) was employed in an indirect competitive format using a bacterial tetracycline-dependent regulatory protein as receptor. Three doxycycline derivatives were synthesized and covalently bound to the surface of four different sensor chips. Parameters that influence the immobilization of the doxycycline derivatives and subsequent binding of the receptor protein were studied. CONCLUSION: The novel doxycycline derivatives were successfully used as competitors in an indirect SPR assay.

Anhydrotetracycline-peptide conjugates as representatives for ligand-based transactivating systems.

Bioconjugates of anhydrotetracycline and minimal activation sequences (VP1, VP2) derived from the Herpes simplex virus protein VP16 were synthesized. Different ligation strategies were applied and the resulting molecules tested in HeLa cells expressing the reverse transactivator rtTA-S3 for activity. The data clearly demonstrate that the atc-peptide conjugates are able to penetrate the cell membrane. Furthermore, binding to and induction of rtTA-S3 were detected. Structure-activity relationships indicated that the biological activity of the atc-peptide strongly depends on the specific linker used. The N-terminally linked oxime derivative 10 proved excellent activity when the increase of luciferace activity indicated a transcriptional activation substantially exceeding the inducing properties of anhydrotetracycline.

Adjusting transgene expression levels in lymphocytes with a set of inducible promoters.

BACKGROUND: Inducible gene expression systems are powerful research tools and could be of clinical value in the future, with lymphocytes being likely prime application targets. However, currently available regulatable promoters exhibit variation in their efficiency in a cell line-dependent-manner and are notorious for basal leakiness or poor inducibility. Data concerning the regulatory properties of different inducible promoters are scarce for lymphocytes. In the present study, we report a comprehensive analysis of how various inducible promoters perform and how their combination with a transsilencer and a reverse transactivator can result in optimally controlled gene expression in T-cells. METHODS: The performance of the tetracycline-regulated (Tet)-inducible promoters Tet-responsive element (TRE), mouse mammary tumor virus (MMTV)/TRE, TREtight and second generation TRE (SG/TRE) was compared in several B-cell lines and in Jurkat T-cells using transient transfections in combination with Tet-On. To monitor transgene expression in a Jurkat cell line containing a transsilencer and a reverse transactivator, expression cassettes encoding enhanced green fluorescent protein, CD123 or a constitutively active, cytotoxic caspase-3 were flanked with insulators and stably integrated. The performance of TREtight and SG/TRE was furthermore analysed in transiently transfected primary CD4(+) human T-cells. RESULTS: The promoters exhibit greatly diverging characteristics. MMTV/TRE permits moderate, TRE and TREtight permits intermediate and SG/TRE permits very high expression levels. TRE and SG/TRE are leaky, whereas MMTV/TRE and TREtight provide stringent expression control. Tetracycline derivatives add flexibility to transgene expression by introducing intermediate expression levels. CONCLUSIONS: The different expression profiles of the promoters increase the flexibility to adjust transgene expression levels. The promoters provide an additional option to optimize system performance for many applications.

Click-chemistry-derived tetracycline-amino acid conjugates exhibiting exceptional potency and exclusive recognition of the reverse tet repressor.

A click-chemistry-based synthesis of biologically active doxycycline-amino acid conjugates is described. Starting from 9-aminodoxycycline derivatives and complementary functionalized amino acids, ligation was accomplished by copper(I)-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC). The final products were tested in a variety of TetR and revTetR systems, and the C-terminally linked phenylalanine conjugate 12 c exhibited high selectivity for revTetR over TetR. Besides the unique property of the specific effector 12 c to effectively differentiate TetR and its reverse phenotype, the test compound proved to be almost devoid of any antibacterial activity; this will be highly beneficial for future applications to control gene expression in bacterial systems.

Diarylpropane-1,3-dione derivatives as TetR-inducing tetracycline mimetics: Synthesis and biological investigations.

Synthesis, biological investigations and molecular docking studies of nonantibiotic and nontetracyclic inducers that feature a minimal key motif of the natural lead tetracycline are presented. The diarylpropane-1,3-dione motif was identified as the minimal substructure responsible for TetR induction by tetracyclines. The first nontetracyclic surrogates of the natural tetracyclines displayed significant inducing effects for TetR(BD)S135L, whereby the chlorohydroxyphenyl-substituted beta-diketone 31 displayed the highest activity. Interestingly, antibiotic activity could not be detected for 31. Homology modeling based on the X-ray structure of 7-chlorotetracycline bound to TetR indicated analogous binding modes for the natural inducer and the synthetic diarylpropane-1,3-dione derivatives.

Structural origins for selectivity and specificity in an engineered bacterial repressor-inducer pair.

The bacterial tetracycline transcription regulation system mediated by the tetracycline repressor (TetR) is widely used to study gene expression in prokaryotes and eukaryotes. To study multiple genes in parallel, a triple mutant TetR(K(64)L(135)I(138)) has been engineered that is selectively induced by the synthetic tetracycline derivative 4-de-dimethylamino-anhydrotetracycline (4-ddma-atc) and no longer by tetracycline, the inducer of wild-type TetR. In the present study, we report the crystal structure of TetR(K(64)L(135)I(138)) in the absence and in complex with 4-ddma-atc at resolutions of 2.1 A. Analysis of the structures in light of the available binding data and previously reported TetR complexes allows for a dissection of the origins of selectivity and specificity. In all crystal structures solved to date, the ligand-binding position, as well as the positioning of the residues lining the binding site, is extremely well conserved, irrespective of the chemical nature of the ligand. Selective recognition of 4-ddma-atc is achieved through fine-tuned hydrogen-bonding constraints introduced by the His64-->Lys substitution, as well as a combination of hydrophobic effect and the removal of unfavorable electrostatic interactions through the introduction of Leu135 and Ile138.

In vivo activation of tetracycline repressor by Cre/lox-mediated gene assembly.

Tetracycline repressor (TetR) bears an unstructured loop region between helices alpha8 and alpha9, which is moderately permissive to amino acid exchanges and length variations. Recognition sites for the site-specific recombinases Flp (FRT) or Cre (lox) were inserted in-frame into tetR, substituting some of this loop's codons. A number of the deduced TetR variants displayed efficient regulation in vivo, thus allowing the establishment of a new mode of TetR activation on the genetic level. Chromosomally encoded tetR in Bacillus subtilis was disrupted and inactivated by insertion of a lox66-aphAIII-lox71 kanamycin resistance cassette. Marker excision by Cre recombinase led to the assembly of a novel tetR allele. The encoded regulator, termed TetR(lox72/1), is distinguished from wt-TetR by a slightly elongated and altered alpha8-alpha9 loop only, harboring an amino acid stretch encoded by lox72. Despite decreased intracellular protein amounts, TetR(lox72/1) displayed efficient in vivo activity in B. subtilis and E. coli, indistinguishable from that of wt-TetR. These results underline the sequence flexibility of TetR in the alpha8-alpha9 loop and demonstrate the possible use of the regulator as a read-out tool for the activity of site-specific recombinases.

An RNA aptamer that induces transcription.

We identified an RNA aptamer that induces TetR-controlled gene expression in Escherichia coli when expressed in the cell. The aptamer was found by a combined approach of in vitro selection for TetR binding and in vivo screening for TetR induction. The smallest active aptamer folds into a stem-loop with an internal loop interrupting the stem. Mutational analysis in vivo and in-line probing in vitro reveal this loop to be the protein binding site. The TetR-inducing activity of the aptamer directly correlates with its stability and the best construct is as efficient as the natural inducer tetracycline. Because of its small size, high induction efficiency, and the stability of the TetR aptamer under in vivo conditions, it is well suited to be an alternative RNA-based inducer of TetR-controlled gene expression.

Functionally important residues of the Tet repressor inducing peptide TIP determined by a complete mutational analysis.

Tet repressor (TetR) is widely used to control gene expression in pro- and eukaryotes. The mechanism of induction by its natural inducer tetracycline is well characterized. A 16-mer oligopeptide, called TIP, fused to thioredoxin A (TrxA) of Escherichia coli is an artificial inducer of TetR. We analyzed the sequence requirements of TIP by directed and random single amino acid substitutions and identified residues important for TetR induction. An alanine scanning analysis of the first twelve residues showed that all except the ones at position eleven and twelve are important for induction. A randomization of residues at positions one to twelve of TIP revealed the properties of each residue necessary for induction. These further insights into the specificity of TIP-TetR interaction are discussed in the light of the X-ray structure of the [TetR-TIP] complex. The last four residues of TIP contribute indirectly to TetR induction by increasing the steady-state level of the fusion protein. TIP mutants fused N-terminally or C-terminally to TrxA in E. coli induce with the same efficiency indicating identical binding and induction mechanisms, and the lack of contribution from TrxA.