Crystal structures of DntR inducer binding domains in complex with salicylate offer insights into the activation of LysR-type transcriptional regulators.

Activation of LysR-type transcription factors (LTTRs) is thought to result from conformational changes that occur when inducer molecules bind to their Inducer Binding Domains (IBDs). However, the exact nature of these changes remains to be fully elucidated. We present the crystal structures of two truncated constructs of the LTTR DntR in their apo- forms and in complex with its natural inducer molecule, salicylate. These provide a fuller picture of the conformational changes that can occur in LTTR IBDs and offer insights that may be relevant when considering the mechanism of activation of LTTRs. Two of the crystal structures show that DntR IBDs can bind up to two inducer molecules. The full extent of conformational changes observed is achieved only when inducer molecules are bound in both binding sites identified. Point mutations disrupting the putative secondary binding site produce DntR variants with a reduced response to salicylate in a whole cell system, suggesting that this site is functionally relevant.

Factors that influence the response of the LysR type transcriptional regulators to aromatic compounds.

BACKGROUND: The transcriptional regulators DntR, NagR and NtdR have a high sequence identity and belong to the large family of LysR type transcriptional regulators (LTTRs). These three regulators are all involved in regulation of genes identified in pathways for degradation of aromatic compounds. They activate the transcription of these genes in the presence of an inducer, but the inducer specificity profiles are different. RESULTS: The results from this study show that NtdR has the broadest inducer specificity, responding to several nitro-aromatic compounds. Mutational studies of residues that differ between DntR, NagR and NtdR suggest that a number of specific residues are involved in the broader inducer specificity of NtdR when compared to DntR and NagR. The inducer response was also investigated as a function of the experimental conditions and a number of parameters such as the growth media, plasmid arrangement of the LTTR-encoding genes, promoter and gfp reporter gene, and the presence of a His6-tag were shown to affect the inducer response in E.coli DH5α. Furthermore, the response upon addition of both salicylate and 4-nitrobenzoate to the growth media was larger than the sum of responses upon addition of each of the compounds, which suggests the presence of a secondary binding site, as previously reported for other LTTRs. CONCLUSIONS: Optimization of the growth conditions and gene arrangement resulted in improved responses to nitro-aromatic inducers. The data also suggests the presence of a previously unknown secondary binding site in DntR, analogous to that of BenM.

In vivo and in vitro investigation of transcriptional regulation by DntR.

DntR is a bacterial transcription factor that has been isolated from Burkholderia species that are able to degrade the nitro-aromatic compound 2,4-dinitrotoluene. We recently solved the X-ray crystal structure of DntR, which suggested a putative location of an inducer-binding cavity (IBC). In this study, we constructed mutants of DntR in which residues lining the proposed IBC were modified in order to identify the structural elements involved in inducer binding, to modulate the inducer binding specificity, and to investigate the mechanism of transcriptional regulation by DntR. The transcriptional activation of the reporter gene gfp induced by the wild-type and mutant DntRs was monitored by analysing whole-cell fluorescence using flow-cytometry after addition of a number of potential inducer compounds. Three of the mutant proteins (F111L; F111V/H169V and Y110S/F111V) were purified and the binding constants for several of the potential inducers to these mutants were estimated. Furthermore, crystal structures of the F111L and Y110S/F111V mutant proteins were solved and used to explain changes in the inducer binding specificity at an atomic level. A comparison of the inducing capability in the whole-cell system and binding constants for a number of potential inducers suggests a mechanism where binding of an inducer molecule is not the sole requirement for transcriptional activation. In addition, specific interactions between DntR and the inducer molecule resulting in a conformational change of the protein are needed.

The solution configurations of inactive and activated DntR have implications for the sliding dimer mechanism of LysR transcription factors.

LysR Type Transcriptional Regulators (LTTRs) regulate basic metabolic pathways or virulence gene expression in prokaryotes. Evidence suggests that the activation of LTTRs involves a conformational change from an inactive compact apo- configuration that represses transcription to an active, expanded holo- form that promotes it. However, no LTTR has yet been observed to adopt both configurations. Here, we report the results of structural studies of various forms of the LTTR DntR. Crystal structures of apo-DntR and of a partially autoinducing mutant H169T-DntR suggest that active and inactive DntR maintain a compact homotetrameric configuration. However, Small Angle X-ray Scattering (SAXS) studies on solutions of apo-, H169T- and inducer-bound holo-DntR indicate a different behaviour, suggesting that while apo-DntR maintains a compact configuration in solution both H169T- and holo-DntR adopt an expanded conformation. Models of the SAXS-obtained solution conformations of apo- and holo-DntR homotetramers in complex with promoter-operator region DNA are consistent with previous observations of a shifting of LTTR DNA binding sites upon activation and a consequent relaxation in the bend of the promoter-operator region DNA. Our results thus provide clear evidence at the molecular level which strongly supports the 'sliding dimer' hypothesis concerning LTTR activation mechanisms.

A novel flow cytometry-based method for analysis of expression levels in Escherichia coli, giving information about precipitated and soluble protein.

A high throughput method for screening of protein expression is described. By using a flow cytometer, levels of both soluble and precipitated protein can simultaneously be assessed in vivo. Protein fragments were fused to the N-terminus of enhanced GFP and the cell samples were analysed using a flow cytometer. Data concerning whole cell fluorescence and light scattering was collected. The whole cell fluorescence is probing intracellular concentrations of soluble fusion proteins. Concurrently, forward scattered light gives data about inclusion body formation, valuable information in process optimisation. To evaluate the method, the cells were disrupted, separated into soluble and non-soluble fractions and analysed by gel electrophoresis. A clear correlation between fluorescence and soluble target protein was shown. Interestingly, the distribution of the cells regarding forward scatter (standard deviation) correlates with the amount of inclusion bodies formed. Finally, the newly developed method was used to evaluate two different purification tags, His(6) and Z(basic), and their effect on the expression pattern.

Directed evolution of the transcriptional regulator DntR: isolation of mutants with improved DNT-response.

The transcriptional regulator DntR, which previously has been isolated from bacterial strains capable of degrading 2,4-dinitrotoluene (DNT), was engineered in order to improve the ability to detect DNT. A directed evolution strategy was employed, where sequence diversity first was created by random mutagenesis in three subsequent rounds, followed by recombination of previously selected mutants. A gfp gene was used as a reporter for transcriptional activity mediated by DntR and cells with higher GFP expression after addition of DNT were sorted out using fluorescence-activated cell sorting (FACS). A DntR mutant, which displayed 10 times higher induction levels than wild-type DntR in response to DNT was isolated. This mutant still maintained low levels of gfp expression in the absence of DNT. The detection limit was ∼10 µM, a 25-fold improvement compared to wild-type DntR. The functional role of some substitutions found in this clone is discussed in the framework of the structural changes observed when comparing the recently determined structures of DntR with and without bound inducer ligand.

Formation of supported lipid bilayers on silica particles studied using flow cytometry.

Silica colloidal particles with functionalized surfaces are used, for example, in studies of membrane proteins or for drug delivery, where novel applications are based on the use of particles covered by lipid membrane bilayers. The mechanism by which such supported lipid bilayers are formed on spherical support is not fully understood. Here, we present results from studies of this process using a new method based on flow cytometry. The approach enabled us to detect particle populations coated and uncoated with lipids in the same sample according to the vesicle:particle surface area ratio. The data suggest that DOPC lipid vesicles efficiently break upon interaction with the silica colloidal particle surface; only a small fraction of the adsorbed vesicles remain unbroken. Furthermore, the data support earlier observations showing that formation of the lipid bilayer at the surface is a cooperative process, where bilayer formation is catalyzed by previously bound membrane fragments.