Untitled

Ducat, Thierry; Declerck, Nathalie; Kochoyan, Michel; Déméné, Hélène

doi:10.1023/a:1020264611438

Cited by 7 publications

(6 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…S2). If the mutual orientation of the CAT and PRD1 domains was fixed, both domains would have the same correlation time of 29 ns (as predicted by Hydronmr for a closed structure obtained by combining the CAT C-terminal and PRD1 N-terminal (19). CAT correlation peaks are numbered in red if not shifted and in black (and connected by black lines) if shifted.…”

Section: D99n Mutation Of Prd1 Restores Cat Rna Binding Activity-mentioning

confidence: 85%

“…Fig. 4 shows the chemical shift index plot for backbone and C␤ carbon atoms of native LicT CAT-PRD1 based on the sequential resonance assignment of the triply labeled protein reported previously (19). The predicted secondary structure of CAT-PRD1 in the inactive state deduced from this plot indicates that at least three of the four original ␤-strands of the CAT monomer are present in native CAT-PRD1.…”

Section: D99n Mutation Of Prd1 Restores Cat Rna Binding Activity-mentioning

confidence: 86%

“…Protein Purification-The native (or wild type) CAT-PRD1 fragment (residues 1-167) and the isolated wild type PRD1 fragment (residues 57-169) were produced as 6 histidinetagged proteins in E. coli BL21(DE3) and purified by affinity chromatography and size exclusion chromatography as described previously (19). The activated mutant CAT-PRD1 carrying the D99N substitution was prepared as native CAT-PRD1 except that buffer pH was raised to 7.5 during purification.…”

Section: Methodsmentioning

confidence: 99%

“…15 15 N-1 H HSQC spectra of CAT, CAT-PRD1, and D99N mutant CAT-PRD1 were recorded at 27°C with the protein samples at 0.1 or 1 mM in 200 mM Na 2 SO 4 , pH 6.4, 0.5 mM EDTA, 10 mM sodium phosphate, 1 mM DTT, and 2 mM benzamidine. Titration experiments with the independent CAT and PRD1 domains were performed in the same buffer but higher pH (pH 7) to optimize PRD1 stability and solubility (19). The starting concentration of 15 N-CAT was 0.6 mM, and PRD1 was added up to 1.5 eq of 15 N-CAT (resulting in final concentrations of 0.3 and 0.45 mM, respectively).…”

Section: Methodsmentioning

confidence: 99%

“…For this purpose, we undertook structural studies of a protein fragment containing the RNAbinding domain (CAT, residues 1-52), the first PTS regulation domain (PRD1, residues 68 -167), and the intervening linker region (residues 53-67). We have previously reported a preliminary NMR characterization of the LicT CAT-PRD1, establishing its folding in a symmetrical homodimer (19). In this study, we have further characterized this construct and compared its biochemical and biophysical properties to those of a mutant CAT-PRD1 carrying a D99N substitution which, in vivo, renders LicT activation independent of PTS-mediated phosphorylation (20).…”

mentioning

confidence: 92%

See 4 more Smart Citations

Structural Mechanism of Signal Transduction between the RNA-binding Domain and the Phosphotransferase System Regulation Domain of the LicT Antiterminator

Déméné

Ducat²,

Guillen³

et al. 2008

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

LicT belongs to a family of bacterial transcriptional antiterminators, which control the expression of sugar-metabolizing operons in response to phosphorylations by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Previous studies of LicT have revealed the structural basis of RNA recognition by the dimeric N-terminal co-antiterminator (CAT) domain on the one hand and the conformational changes undergone by the duplicated regulation domain (PRD1 and PRD2) upon activation on the other hand. To investigate the mechanism of signal transduction between the effector and regulation modules, we have undertaken the characterization of a fragment, including the CAT and PRD1 domains and the linker inbetween. Comparative experiments, including RNA binding assays, NMR spectroscopy, limited proteolysis, analytical ultracentrifugation, and circular dichroism, were conducted on native CAT-PRD1 and on a constitutively active CAT-PRD1 mutant carrying a D99N substitution in PRD1. We show that in the native state, CAT-PRD1 behaves as a rather unstable RNAbinding deficient dimer, in which the CAT dimer interface is significantly altered and the linker region is folded as a trypsinresistant helix. In the activated mutant form, the CAT-PRD1 linker becomes protease-sensitive, and the helix content decreases, and the CAT module adopts the same dimeric conformation as in isolated CAT, thereby restoring the affinity for RNA. From these results, we propose that a helix-to-coil transition in the linker acts as the structural relay triggered by the regulatory domain for remodeling the effector dimer interface. In essence, the structural mechanism modulating the LicT RNA antitermination activity is thus similar to that controlling the DNA binding activity of dimeric transcriptional regulators.

show abstract

Section: D99n Mutation Of Prd1 Restores Cat Rna Binding Activity-mentioning

confidence: 85%

Section: D99n Mutation Of Prd1 Restores Cat Rna Binding Activity-mentioning

confidence: 86%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 92%

See 3 more Smart Citations

Structural Mechanism of Signal Transduction between the RNA-binding Domain and the Phosphotransferase System Regulation Domain of the LicT Antiterminator

Déméné

Ducat²,

Guillen³

et al. 2008

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

Rapid Determination of Protein Solubility and Stability Conditions for NMR Studies Using Incomplete Factorial Design

Ducat

Declerck²,

Gostan³

et al. 2006

J Biomol NMR

View full text Add to dashboard Cite

Sample preparation constitutes a crucial and limiting step in structural studies of proteins by NMR. The determination of the solubility and stability (SAS) conditions of biomolecules at millimolar concentrations stays today empirical and hence time- and material-consuming. Only few studies have been recently done in this field and they have highlighted the interest of using crystallogenesis tools to optimise sample conditions. In this study, we have adapted a method based on incomplete factorial design and making use of crystallisation plates to quantify the influence of physico-chemical parameters such as buffer pH and salts on protein SAS. A description of the experimental set up and an evaluation of the method are given by case studies on two functional domains from the bacterial regulatory protein LicT as well as two other proteins. Using this method, we could rapidly determine optimised conditions for extracting soluble proteins from bacterial cells and for preparing purified protein samples sufficiently concentrated and stable for NMR characterisation. The drastic reduction in the time and number of experiments required for searching protein SAS conditions makes this method particularly well-adapted for a systematic investigation on a large range of physico-chemical parameters.

show abstract

NMR chemical shift assignment of a constitutively active fragment of the antitermination protein LicT

2019

Self Cite

View full text Add to dashboard Cite

LicT belongs to an essential family of bacterial antitermination proteins which bind to nascent mRNAs in order to stimulate transcription of sugar-metabolizing operons. As most of other antitermination proteins involved in carbohydrate metabolism, LicT is composed of a N-terminal RNA-binding module (CAT) and two homologous regulatory modules (PRD1 and PRD2). The activity of the CAT effector module is controlled by antagonist phosphorylations by the phosphotransferase system (PTS). on conserved histidines of the two C-terminal PRDs in response to available carbon sources. Previous studies on truncated and mutant constructs have provided partial structural insight into the mechanism of signal transduction between the N-terminal RNA-binding domain and the two regulation modules. However, no structure at atomic resolution has been ever solved that contain the RNA-binding domain and a regulation module. We report the NMR assignment of a constitutively active fragment of LicT, named D99A-CAT-PRD1 or CAT-PRD1*. This fragment is composed of the RNA-binding module and the first N-terminal regulation module which bears the mutation of Asp99 to an asparagine. Is dimeric as the native protein, with a 40 kD molecular weight. The D99N mutation is sufficient to endow this fragment with a high RNA-binding constitutive activity, in a phosphorylation-free context. The assignment reported here should set the base of future NMR investigation of signal transduction between the regulatory module and the effector module in the active state of the protein, and in the long term enable the structural study of the full length protein structure in interaction with its target RNA.

show abstract

Untitled

Cited by 7 publications

References 9 publications

Structural Mechanism of Signal Transduction between the RNA-binding Domain and the Phosphotransferase System Regulation Domain of the LicT Antiterminator

Structural Mechanism of Signal Transduction between the RNA-binding Domain and the Phosphotransferase System Regulation Domain of the LicT Antiterminator

Rapid Determination of Protein Solubility and Stability Conditions for NMR Studies Using Incomplete Factorial Design

NMR chemical shift assignment of a constitutively active fragment of the antitermination protein LicT

Contact Info

Product

Resources

About