Structural analysis of <i>B. subtilis</i> CcpA effector binding site

Chaptal, Vincent; Gueguen-Chaignon, Virginie; Poncet, Sandrine; Lecampion, Cécile; Meyer, Philippe; Deutscher, Josef; Galinier, Anne; Nessler, Sylvie; Moréra, Solange

doi:10.1002/prot.21001

Cited by 9 publications

(9 citation statements)

References 25 publications

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“…The crystal structure of a complex formed between B. subtilis P-Ser-HPr and CcpA missing the first 57 amino acids was very similar to the corresponding structure in the B. megaterium P-Ser-HPr:CcpA complex (106). Interestingly, the B. subtilis protein complex contains two sulfate ions located close to each other.…”

Section: P-ser-hpr Functions As a Catabolite Corepressormentioning

confidence: 56%

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

Deutscher

Francke

Postma

2006

Microbiol Mol Biol Rev

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1,202

769

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SUMMARY The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens.

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Section: P-ser-hpr Functions As a Catabolite Corepressormentioning

confidence: 56%

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

Deutscher

Francke

Postma

2006

Microbiol Mol Biol Rev

Self Cite

1,202

769

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“…DNA binding reactions were performed in 20 µl of binding buffer containing 10 mM Tris-HCl pH 8.5, 50 mM NaCl, 10 mM EDTA, 0.5% Tween-20, 10 mM DTT, and 1 mg of poly(dI-dC)-labelled IRdye-PCR fragments at room temperature for 30 min. For the specific and non-specific competition assays, D(+)-glucose 6-phosphate (30 mM) (Sigma) was added to the binding buffer in the EMSA as CcpA cofactor [35]. Purified rApuR and rCcpA proteins (from 0.5 to 4.0 µM) were incubated, in separate experiments, with the fragment Pr2 and a non-specific competitor fragment that was obtained by PCR amplification of the gene SSU0879 (from 25 to 150 nM).…”

Section: Methodsmentioning

confidence: 99%

Carbohydrate Availability Regulates Virulence Gene Expression in Streptococcus suis

et al. 2014

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Streptococcus suis is a major bacterial pathogen of young pigs causing worldwide economic problems for the pig industry. S. suis is also an emerging pathogen of humans. Colonization of porcine oropharynx by S. suis is considered to be a high risk factor for invasive disease. In the oropharyngeal cavity, where glucose is rapidly absorbed but dietary α-glucans persist, there is a profound effect of carbohydrate availability on the expression of virulence genes. Nineteen predicted or confirmed S. suis virulence genes that promote adhesion to and invasion of epithelial cells were expressed at higher levels when S. suis was supplied with the α-glucan starch/pullulan compared to glucose as the single carbon source. Additionally the production of suilysin, a toxin that damages epithelial cells, was increased more than ten-fold when glucose levels were low and S. suis was growing on pullulan. Based on biochemical, bioinformatics and in vitro and in vivo gene expression studies, we developed a biological model that postulates the effect of carbon catabolite repression on expression of virulence genes in the mucosa, organs and blood. This research increases our understanding of S. suis virulence mechanisms and has important implications for the design of future control strategies including the development of anti-infective strategies by modulating animal feed composition.

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“…All three structures were solved by molecular replacement with MolRep using the same starting model, 2FEP [the B. subtilis CcpA (residues 60–334)–(HPr-Ser46-P) structure] (11,40). After rigid-body refinement in CNS, clear electron density for the DNA-binding region, residues 1–59, and the DNA was revealed.…”

Section: Methodsmentioning

confidence: 99%

Structures of carbon catabolite protein A–(HPr-Ser46-P) bound to diverse catabolite response element sites reveal the basis for high-affinity binding to degenerate DNA operators

Schumacher

Sprehe

Bartholomae

et al. 2010

Nucleic Acids Research

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In Gram-positive bacteria, carbon catabolite protein A (CcpA) is the master regulator of carbon catabolite control, which ensures optimal energy usage under diverse conditions. Unlike other LacI-GalR proteins, CcpA is activated for DNA binding by first forming a complex with the phosphoprotein HPr-Ser46-P. Bacillus subtilis CcpA functions as both a transcription repressor and activator and binds to more than 50 operators called catabolite response elements (cres). These sites are highly degenerate with the consensus, WTGNNARCGNWWWCAW. How CcpA–(HPr-Ser46-P) binds such diverse sequences is unclear. To gain insight into this question, we solved the structures of the CcpA–(HPr-Ser46-P) complex bound to three different operators, the synthetic (syn) cre, ackA2 cre and gntR-down cre. Strikingly, the structures show that the CcpA-bound operators display different bend angles, ranging from 31° to 56°. These differences are accommodated by a flexible linkage between the CcpA helix-turn-helix-loop-helix motif and hinge helices, which allows independent docking of these DNA-binding modules. This flexibility coupled with an abundance of non-polar residues capable of non-specific nucleobase interactions permits CcpA–(HPr-Ser46-P) to bind diverse operators. Indeed, biochemical data show that CcpA–(HPr-Ser46-P) binds the three cre sites with similar affinities. Thus, the data reveal properties that license this protein to function as a global transcription regulator.

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Structural analysis of B. subtilis CcpA effector binding site

Cited by 9 publications

References 25 publications

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

Carbohydrate Availability Regulates Virulence Gene Expression in Streptococcus suis

Structures of carbon catabolite protein A–(HPr-Ser46-P) bound to diverse catabolite response element sites reveal the basis for high-affinity binding to degenerate DNA operators

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