Limits...
Structural and functional characterization of the LldR from Corynebacterium glutamicum: a transcriptional repressor involved in L-lactate and sugar utilization.

Gao YG, Suzuki H, Itou H, Zhou Y, Tanaka Y, Wachi M, Watanabe N, Tanaka I, Yao M - Nucleic Acids Res. (2008)

Bottom Line: LldR (CGL2915) from Corynebacterium glutamicum is a transcription factor belonging to the GntR family, which is typically involved in the regulation of oxidized substrates associated with amino acid metabolism.Mutation experiments showed that residues Lys4, Arg32, Arg42 and Gly63 are crucial for DNA binding.The location of the putative ligand binding cavity and the regulatory mechanism of LldR on its affinity for DNA were proposed.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.

ABSTRACT
LldR (CGL2915) from Corynebacterium glutamicum is a transcription factor belonging to the GntR family, which is typically involved in the regulation of oxidized substrates associated with amino acid metabolism. In the present study, the crystal structure of LldR was determined at 2.05-A resolution. The structure consists of N- and C-domains similar to those of FadR, but with distinct domain orientations. LldR and FadR dimers achieve similar structures by domain swapping, which was first observed in dimeric assembly of transcription factors. A structural feature of Zn(2+) binding in the regulatory domain was also observed, as a difference from the FadR subfamily. DNA microarray and DNase I footprint analyses suggested that LldR acts as a repressor regulating cgl2917-lldD and cgl1934-fruK-ptsF operons, which are indispensable for l-lactate and fructose/sucrose utilization, respectively. Furthermore, the stoichiometries and affinities of LldR and DNAs were determined by isothermal titration calorimetry measurements. The transcriptional start site and repression of LldR on the cgl2917-lldD operon were analysed by primer extension assay. Mutation experiments showed that residues Lys4, Arg32, Arg42 and Gly63 are crucial for DNA binding. The location of the putative ligand binding cavity and the regulatory mechanism of LldR on its affinity for DNA were proposed.

Show MeSH

Related in: MedlinePlus

ITC of the interaction of LldR and DNA. Representative plots from ITC experiments are illustrated with raw data in the upper panel and fitting curves (continuous lines) in the lower panel for (A–C). The concentrations of titrated LldR dimer were 200 (A and B) and 100 μM (C). The thermodynamic values calculated by curve fitting were: A (cgl2917-site1 dsDNA), ΔS = –14.1 cal/mol, ΔH = –13.9 ± 0.1 kcal/mol, KB = 1.2 ± 0.2 × 107 M–1 (Kd = 8.1 × 10 nM), N = 1.1 ± 0.01; B (cgl2917-site1–2 dsDNA), ΔS = 0.58, ΔH = –8.0 ± 0.2 kcal/mol, KB = 9.1 ± 1.4 × 105 M–1 (Kd = 1.1 × 103 nM), N = 2.2 ± 0.03; C (cgl1934-site dsDNA), ΔS = –15.1 cal/mol, ΔH = –12.4 ± 1.2 kcal/mol, KB = 5.7 ± 1.6 × 105 M–1 (Kd = 1.7 × 103 nM). N = 0.9 ± 0.05. N is the stoichiometry of bound LldR dimer per dsDNA, and the dissociation constant Kd is the reciprocal of the affinity constant KB (Kd = 1/KB). Note that the initial injection was 3/10 volume of the subsequent injection.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2602784&req=5

Figure 5: ITC of the interaction of LldR and DNA. Representative plots from ITC experiments are illustrated with raw data in the upper panel and fitting curves (continuous lines) in the lower panel for (A–C). The concentrations of titrated LldR dimer were 200 (A and B) and 100 μM (C). The thermodynamic values calculated by curve fitting were: A (cgl2917-site1 dsDNA), ΔS = –14.1 cal/mol, ΔH = –13.9 ± 0.1 kcal/mol, KB = 1.2 ± 0.2 × 107 M–1 (Kd = 8.1 × 10 nM), N = 1.1 ± 0.01; B (cgl2917-site1–2 dsDNA), ΔS = 0.58, ΔH = –8.0 ± 0.2 kcal/mol, KB = 9.1 ± 1.4 × 105 M–1 (Kd = 1.1 × 103 nM), N = 2.2 ± 0.03; C (cgl1934-site dsDNA), ΔS = –15.1 cal/mol, ΔH = –12.4 ± 1.2 kcal/mol, KB = 5.7 ± 1.6 × 105 M–1 (Kd = 1.7 × 103 nM). N = 0.9 ± 0.05. N is the stoichiometry of bound LldR dimer per dsDNA, and the dissociation constant Kd is the reciprocal of the affinity constant KB (Kd = 1/KB). Note that the initial injection was 3/10 volume of the subsequent injection.

Mentions: The lengths of the two LldR-binding sequences are quite different as determined by footprint analyses. Taking the size of the LldR protein into account, it seems that two LldR dimers bind to the long sequence of cgl2917 (cgl2917-site1–2, 34 bp), and one dimer to the sequence of cgl1934. Four types of dsDNA representing the full-length (cgl2917-site1–2) or the two halves (cgl2917-site1, cgl2917-site2) of cgl2917, or the cgl1934-site (Figure 4A and B), were thus prepared to perform ITC experiments. The results of ITC showed that the stoichiometry of protein and DNA was clearly one LldR dimer to cgl2917-site1 and cgl1934-site, and two LldR dimers to cgl2917-site1–2, corresponding to Kd values of 8.1 × 10 nM, 1.1 × 103 nM and 1.7 × 103 nM, respectively (Figure 5). The Kd values indicated that LldR has the highest affinity for cgl2917-site1, which contains an inverted repeat sequence with more precise symmetry (5′-−4TtgTGGTCTGACCAtgA+13-3′, the centre of symmetry and the half-site of the inverted repeat are underlined and shown in italic type, respectively) (Figure 4A and D). The typical fitting curve was not observed for the titration of LldR to cgl2917-site2-dsDNA, implying a weak LldR–DNA interaction, which was consistent with the results of in vitro binding assay by EMSA (data not shown). These results, taken together, suggest that binding of one LldR dimer to cgl2917-site1 enhances binding of the second dimer to cgl2917-site2, but the close proximity of the two dimers binding decreases the affinity for cgl2917-site1, with Kd from 8.1 × 10 nM to 1.1 × 103 nM.Figure 5.


Structural and functional characterization of the LldR from Corynebacterium glutamicum: a transcriptional repressor involved in L-lactate and sugar utilization.

Gao YG, Suzuki H, Itou H, Zhou Y, Tanaka Y, Wachi M, Watanabe N, Tanaka I, Yao M - Nucleic Acids Res. (2008)

ITC of the interaction of LldR and DNA. Representative plots from ITC experiments are illustrated with raw data in the upper panel and fitting curves (continuous lines) in the lower panel for (A–C). The concentrations of titrated LldR dimer were 200 (A and B) and 100 μM (C). The thermodynamic values calculated by curve fitting were: A (cgl2917-site1 dsDNA), ΔS = –14.1 cal/mol, ΔH = –13.9 ± 0.1 kcal/mol, KB = 1.2 ± 0.2 × 107 M–1 (Kd = 8.1 × 10 nM), N = 1.1 ± 0.01; B (cgl2917-site1–2 dsDNA), ΔS = 0.58, ΔH = –8.0 ± 0.2 kcal/mol, KB = 9.1 ± 1.4 × 105 M–1 (Kd = 1.1 × 103 nM), N = 2.2 ± 0.03; C (cgl1934-site dsDNA), ΔS = –15.1 cal/mol, ΔH = –12.4 ± 1.2 kcal/mol, KB = 5.7 ± 1.6 × 105 M–1 (Kd = 1.7 × 103 nM). N = 0.9 ± 0.05. N is the stoichiometry of bound LldR dimer per dsDNA, and the dissociation constant Kd is the reciprocal of the affinity constant KB (Kd = 1/KB). Note that the initial injection was 3/10 volume of the subsequent injection.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2602784&req=5

Figure 5: ITC of the interaction of LldR and DNA. Representative plots from ITC experiments are illustrated with raw data in the upper panel and fitting curves (continuous lines) in the lower panel for (A–C). The concentrations of titrated LldR dimer were 200 (A and B) and 100 μM (C). The thermodynamic values calculated by curve fitting were: A (cgl2917-site1 dsDNA), ΔS = –14.1 cal/mol, ΔH = –13.9 ± 0.1 kcal/mol, KB = 1.2 ± 0.2 × 107 M–1 (Kd = 8.1 × 10 nM), N = 1.1 ± 0.01; B (cgl2917-site1–2 dsDNA), ΔS = 0.58, ΔH = –8.0 ± 0.2 kcal/mol, KB = 9.1 ± 1.4 × 105 M–1 (Kd = 1.1 × 103 nM), N = 2.2 ± 0.03; C (cgl1934-site dsDNA), ΔS = –15.1 cal/mol, ΔH = –12.4 ± 1.2 kcal/mol, KB = 5.7 ± 1.6 × 105 M–1 (Kd = 1.7 × 103 nM). N = 0.9 ± 0.05. N is the stoichiometry of bound LldR dimer per dsDNA, and the dissociation constant Kd is the reciprocal of the affinity constant KB (Kd = 1/KB). Note that the initial injection was 3/10 volume of the subsequent injection.
Mentions: The lengths of the two LldR-binding sequences are quite different as determined by footprint analyses. Taking the size of the LldR protein into account, it seems that two LldR dimers bind to the long sequence of cgl2917 (cgl2917-site1–2, 34 bp), and one dimer to the sequence of cgl1934. Four types of dsDNA representing the full-length (cgl2917-site1–2) or the two halves (cgl2917-site1, cgl2917-site2) of cgl2917, or the cgl1934-site (Figure 4A and B), were thus prepared to perform ITC experiments. The results of ITC showed that the stoichiometry of protein and DNA was clearly one LldR dimer to cgl2917-site1 and cgl1934-site, and two LldR dimers to cgl2917-site1–2, corresponding to Kd values of 8.1 × 10 nM, 1.1 × 103 nM and 1.7 × 103 nM, respectively (Figure 5). The Kd values indicated that LldR has the highest affinity for cgl2917-site1, which contains an inverted repeat sequence with more precise symmetry (5′-−4TtgTGGTCTGACCAtgA+13-3′, the centre of symmetry and the half-site of the inverted repeat are underlined and shown in italic type, respectively) (Figure 4A and D). The typical fitting curve was not observed for the titration of LldR to cgl2917-site2-dsDNA, implying a weak LldR–DNA interaction, which was consistent with the results of in vitro binding assay by EMSA (data not shown). These results, taken together, suggest that binding of one LldR dimer to cgl2917-site1 enhances binding of the second dimer to cgl2917-site2, but the close proximity of the two dimers binding decreases the affinity for cgl2917-site1, with Kd from 8.1 × 10 nM to 1.1 × 103 nM.Figure 5.

Bottom Line: LldR (CGL2915) from Corynebacterium glutamicum is a transcription factor belonging to the GntR family, which is typically involved in the regulation of oxidized substrates associated with amino acid metabolism.Mutation experiments showed that residues Lys4, Arg32, Arg42 and Gly63 are crucial for DNA binding.The location of the putative ligand binding cavity and the regulatory mechanism of LldR on its affinity for DNA were proposed.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.

ABSTRACT
LldR (CGL2915) from Corynebacterium glutamicum is a transcription factor belonging to the GntR family, which is typically involved in the regulation of oxidized substrates associated with amino acid metabolism. In the present study, the crystal structure of LldR was determined at 2.05-A resolution. The structure consists of N- and C-domains similar to those of FadR, but with distinct domain orientations. LldR and FadR dimers achieve similar structures by domain swapping, which was first observed in dimeric assembly of transcription factors. A structural feature of Zn(2+) binding in the regulatory domain was also observed, as a difference from the FadR subfamily. DNA microarray and DNase I footprint analyses suggested that LldR acts as a repressor regulating cgl2917-lldD and cgl1934-fruK-ptsF operons, which are indispensable for l-lactate and fructose/sucrose utilization, respectively. Furthermore, the stoichiometries and affinities of LldR and DNAs were determined by isothermal titration calorimetry measurements. The transcriptional start site and repression of LldR on the cgl2917-lldD operon were analysed by primer extension assay. Mutation experiments showed that residues Lys4, Arg32, Arg42 and Gly63 are crucial for DNA binding. The location of the putative ligand binding cavity and the regulatory mechanism of LldR on its affinity for DNA were proposed.

Show MeSH
Related in: MedlinePlus