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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.

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The crystal structure of LldR in complex with co-purified Zn2+. (A) Ribbon representation of the LldR-Zn2+ complex in a monomer. The ribbon model is coloured according to the sequence from blue at the N-terminus to red at the C-terminus, and the co-purified zinc ion is shown as a pink ball. (B) Ribbon representation of the LldR-Zn2+ dimer. The two monomers are coloured cyan and blue. (C) Close-up view of the Zn2+ binding site. The amino acid residues and a water molecule binding to Zn2+ are shown as sticks and a cyan ball, respectively. The figures were produced using PyMOL (DeLano Scientific LLC, http://pymol.sourceforge.net).
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Figure 1: The crystal structure of LldR in complex with co-purified Zn2+. (A) Ribbon representation of the LldR-Zn2+ complex in a monomer. The ribbon model is coloured according to the sequence from blue at the N-terminus to red at the C-terminus, and the co-purified zinc ion is shown as a pink ball. (B) Ribbon representation of the LldR-Zn2+ dimer. The two monomers are coloured cyan and blue. (C) Close-up view of the Zn2+ binding site. The amino acid residues and a water molecule binding to Zn2+ are shown as sticks and a cyan ball, respectively. The figures were produced using PyMOL (DeLano Scientific LLC, http://pymol.sourceforge.net).

Mentions: The crystal structure of LldR was determined using the Se-MAD method, and the structure (Figure 1) was refined to 2.05-Å resolution using native data. The final model contains two monomers (monomer A, Ser2–Leu232; monomer B, Ser2–Ala231) (Figure 1B), two zinc ions and 400 water molecules in an asymmetric unit, and has been deposited in the Protein Data Bank (code 2DI3). The two copies were well superimposed with a root mean square deviation (r.m.s.d.) of 1.3 Å on Cα atoms except for residues 58–72, which form an anti-parallel β-sheet and have distinct conformations (the maximum and average distances between Cα atoms are 13.1 and 6.3 Å, respectively). The two monomers form a dimer, which is consistent with the results of gel filtration experiments (data not shown).Figure 1.


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)

The crystal structure of LldR in complex with co-purified Zn2+. (A) Ribbon representation of the LldR-Zn2+ complex in a monomer. The ribbon model is coloured according to the sequence from blue at the N-terminus to red at the C-terminus, and the co-purified zinc ion is shown as a pink ball. (B) Ribbon representation of the LldR-Zn2+ dimer. The two monomers are coloured cyan and blue. (C) Close-up view of the Zn2+ binding site. The amino acid residues and a water molecule binding to Zn2+ are shown as sticks and a cyan ball, respectively. The figures were produced using PyMOL (DeLano Scientific LLC, http://pymol.sourceforge.net).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: The crystal structure of LldR in complex with co-purified Zn2+. (A) Ribbon representation of the LldR-Zn2+ complex in a monomer. The ribbon model is coloured according to the sequence from blue at the N-terminus to red at the C-terminus, and the co-purified zinc ion is shown as a pink ball. (B) Ribbon representation of the LldR-Zn2+ dimer. The two monomers are coloured cyan and blue. (C) Close-up view of the Zn2+ binding site. The amino acid residues and a water molecule binding to Zn2+ are shown as sticks and a cyan ball, respectively. The figures were produced using PyMOL (DeLano Scientific LLC, http://pymol.sourceforge.net).
Mentions: The crystal structure of LldR was determined using the Se-MAD method, and the structure (Figure 1) was refined to 2.05-Å resolution using native data. The final model contains two monomers (monomer A, Ser2–Leu232; monomer B, Ser2–Ala231) (Figure 1B), two zinc ions and 400 water molecules in an asymmetric unit, and has been deposited in the Protein Data Bank (code 2DI3). The two copies were well superimposed with a root mean square deviation (r.m.s.d.) of 1.3 Å on Cα atoms except for residues 58–72, which form an anti-parallel β-sheet and have distinct conformations (the maximum and average distances between Cα atoms are 13.1 and 6.3 Å, respectively). The two monomers form a dimer, which is consistent with the results of gel filtration experiments (data not shown).Figure 1.

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