Expression of genes in the gapA operon encoding five enzymes for triose phosphate interconversion in Bacillus subtilis is negatively regulated by the Central glycolytic genes Regulator (CggR). CggR belongs to the large SorC/DeoR family of prokaryotic transcriptional regulators, characterized by an N-terminal DNA-binding domain and a large C-terminal effector-binding domain. When no glucose is present in growth media, CggR binds to its target DNA sequence and blocks the transcription of genes in the gapA operon. In the presence of glucose, binding of the known effector molecule fructose-1,6-bisphosphate abolishes this interaction. We have identified dihydroxyacetone phosphate, glucose-6-phosphate, and fructose-6-phosphate as additional CggR ligands that can bind to the effector-binding site. Crystal structures of C-CggR, the C-terminal effector-binding domain of CggR, both unliganded as well as in complex with the four ligands at resolutions between 1.65 and 1.80 A reveal unique ligand-specific structural changes in the binding site that affect the dimer interface. Binding affinities of these ligands were determined by isothermal titration calorimetry. Chemical cross-linking shows that CggR oligomerization is mediated through its effector-binding domain and that binding of the different ligands differentially affects the distribution of oligomers. Electrophoretic mobility shift assays (EMSAs) confirmed a destabilizing effect of FBP on the CggR/DNA complex and also showed similar effects for dihydroxyacetone phosphate. Our results suggest that CggR stability and function may be modulated by various effectors in a complex fashion.
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Deoxyribonucleoside regulator DeoR from Bacillus subtilis negatively regulates expression of enzymes involved in the catabolism of deoxyribonucleosides and deoxyribose. The DeoR protein is homologous to the sorbitol operon regulator family of metabolic regulators and comprises an N-terminal DNA-binding domain and a C-terminal effector-binding domain. We have determined the crystal structure of the effector-binding domain of DeoR (C-DeoR) in free form and in covalent complex with its effector deoxyribose-5-phosphate (dR5P). This is the first case of a covalently attached effector molecule captured in the structure of a bacterial transcriptional regulator. The dR5P molecule is attached through a Schiff base linkage to residue Lys141. The crucial role of Lys141 in effector binding was confirmed by mutational analysis and mass spectrometry of Schiff base adducts formed in solution. Structural analyses of the free and effector-bound C-DeoR structures provided a structural explanation for mechanism of DeoR function as a molecular switch. This article is protected by copyright. All rights reserved.
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Crystal structures of the effector-binding domain of repressor CggR from Bacillus subtilis reveal ligand-induced structural changes upon binding of several glycolytic intermediates.,Rezacova P, Kozisek M, Moy SF, Sieglova I, Joachimiak A, Machius M, Otwinowski Z Mol Microbiol. 2008 Jun 10;. PMID:18554327<ref>PMID:18554327</ref>
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Structure of the effector-binding domain of deoxyribonucleoside regulator DeoR from Bacillus subtilis.,Skerlova J, Fabry M, Hubalek M, Otwinowski Z, Rezacova P FEBS J. 2014 May 23. doi: 10.1111/febs.12856. PMID:24863636<ref>PMID:24863636</ref>
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br>
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br>
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== References ==
== References ==
Revision as of 05:42, 18 June 2014
Structure of the effector-binding domain of deoxyribonucleoside regulator DeoR from Bacillus subtilis in complex with deoxyribose-5-phosphate
Deoxyribonucleoside regulator DeoR from Bacillus subtilis negatively regulates expression of enzymes involved in the catabolism of deoxyribonucleosides and deoxyribose. The DeoR protein is homologous to the sorbitol operon regulator family of metabolic regulators and comprises an N-terminal DNA-binding domain and a C-terminal effector-binding domain. We have determined the crystal structure of the effector-binding domain of DeoR (C-DeoR) in free form and in covalent complex with its effector deoxyribose-5-phosphate (dR5P). This is the first case of a covalently attached effector molecule captured in the structure of a bacterial transcriptional regulator. The dR5P molecule is attached through a Schiff base linkage to residue Lys141. The crucial role of Lys141 in effector binding was confirmed by mutational analysis and mass spectrometry of Schiff base adducts formed in solution. Structural analyses of the free and effector-bound C-DeoR structures provided a structural explanation for mechanism of DeoR function as a molecular switch. This article is protected by copyright. All rights reserved.
Structure of the effector-binding domain of deoxyribonucleoside regulator DeoR from Bacillus subtilis.,Skerlova J, Fabry M, Hubalek M, Otwinowski Z, Rezacova P FEBS J. 2014 May 23. doi: 10.1111/febs.12856. PMID:24863636[1]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
References
↑ Skerlova J, Fabry M, Hubalek M, Otwinowski Z, Rezacova P. Structure of the effector-binding domain of deoxyribonucleoside regulator DeoR from Bacillus subtilis. FEBS J. 2014 May 23. doi: 10.1111/febs.12856. PMID:24863636 doi:http://dx.doi.org/10.1111/febs.12856
