Binding of the Catabolite Repressor Protein CcpA to Its DNA Target Is Regulated by Phosphorylation of its Corepressor HPr

Abstract

Catabolite repression of a number of catabolic operons in bacilli is mediated by the catabolite control protein CcpA, the phosphocarrier protein HPr from the phosphoenolpyruvate-dependent sugar transport system (PTS), and a cis-acting DNA sequence termed the catabolite-responsive element (cre). We present evidence that CcpA interacts with HPr that is phosphorylated at Ser 46 (Ser(P) HPr) and that these proteins form a specific ternary complex with cre DNA. Titration experiments following the circular dichroism signal of the cre DNA indicate that this complex consists of two molecules of Ser(P) HPr, a CcpA dimer, and the cre sequence. Limited proteolysis experiments indicate that the domain structure of CcpA is similar to other members of the LacI/GalR family of helix-turn-helix proteins, comprised of a helix-turn-helix DNA domain and a C-terminal effector domain. NMR titration of Ser(P) HPr demonstrates that the isolated C-terminal domain of CcpA forms a specific complex with Ser(P) HPr but not with unphosphorylated HPr. Based upon perturbations to the NMR spectrum, we propose that the binding site of the C-terminal domain of CcpA on Ser(P) HPr forms a contiguous surface that encompasses both Ser(P) 46 and His 15 , the site of phosphorylation by enzyme I of the PTS. This allows CcpA to recognize the phosphorylation state of HPr, effectively linking the process of sugar import via the PTS to catabolite repression in bacilli. Catabolite repression (CR)1 in Escherichia coli has provided a general paradigm for understanding the regulation of the synthesis of various catabolic enzymes in response to the availability of rapidly metabolizable carbon sources. The mechanism for CR in bacteria such as E. coli involves the cyclic AMP-dependent action of the catabolite gene activator protein, CAP (1). In Bacillus subtilis, however, the mechanism is completely different, because no cyclic AMP or CAP homologue is present (2). CR in some bacilli and a few other Gram-positive organisms has been shown to be dependent on the catabolite control protein A (CcpA), which is a member of the LacI/GalR family of regulators (3, 4), and a cis-active operator DNA sequence, termed the catabolite-responsive element (cre), which has been identified in the promoter or in the 5Ј region of 29 B. subtilis genes (5).There is growing evidence that CR is mechanistically linked to the phosphoenolpyruvate-dependent sugar transport system (PTS), which is responsible for the import of various sugars (1). Mutation of Ser 46 to Ala in the PTS phosphocarrier protein HPr results in resistance to CR for several catabolic genes in B. subtilis (6). Ser 46 in HPr is known to be phosphorylated by an ATP-dependent kinase that is activated by glycolytic intermediates such as fructose-1,6-diphosphate (7). Confirming the link between CR and the PTS, recent DNase I protection experiments have shown that cre sequences are specifically protected by CcpA only in the presence of HPr phosphorylated at Ser 46 (Ser(P) HPr) (8). Other mechanisms for CR have been established...

X-ray structure of a bifunctional protein kinase in complex with its protein substrate HPr

“…A separate interface involves the C-terminal helix ␣4 of the red subunit, which is in contact with helix ␣B of HPr and also with the first turn of helix ␣A N-capped by His-15. Interestingly, the same region of HPr also interacts with proteins EIIA Glc and EI of the phosphoenolpyruvate:sugar phosphotransferase system (23) and with the catabolite control protein A (24). In the complex with P-Ser-HPr, an additional contact is formed between Arg-245 of the red subunit and the phosphoserine.…”

Carbon Catabolite Control of the Metabolic Network inBacillus subtilis

“…79) In 1995, a complex of CcpA with P-Ser-HPr was verified to recognize the cre of the gnt operon with high affinity in footprinting experiments. 16) This finding led to the present model for the molecular mechanism underlying CCR in B. subtilis ( Fig.…”

“…High affinity binding of the complex of CcpA and P-Ser-HPr to the amyE cre has been confirmed by circular dichroism spectroscopy. 79) Moreover, the structure of the CcpA-(P-Ser-HPr)-cre complex has been determined. 80) CcpA, HPr with Ser-46, and HPr kinase/phosphatase are well conserved among low-GC Gram-positive bacteria, suggesting that this molecular mechanism underlying CCR is operative in these bacteria.…”

Contribution of the phosphoenolpyruvate:mannose phosphotransferase system to carbon catabolite repression in Lactobacillus pentosus

“…HPr can be phosphorylated at a Ser-46 residue by an ATP-dependent HPr kinase activated by fructose 1,6-bisphosphate (Deutscher & Saier, 1983 ;Galinier et al, 1998 ;Reizer et al, 1984Reizer et al, , 1998. HPr(Ser-P) was shown to interact with CcpA, resulting in a protein complex which can bind to cis-acting cataboliteresponsive elements (cre) located in the promoter regions of many catabolic operons (Deutscher et al, 1995 ;Fujita et al, 1995 ;Jones et al, 1997), thereby preventing transcription. The signal for activation of the HPr(Ser-P)\CcpA pathway is generated during glycolysis, especially when the rate of transport and phosphorylation of the repressing sugars is high.…”