Binding of the catabolite repressor protein CcpA to its DNA target is regulated by phosphorylation of its corepressor HPr.
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 Ser46 (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 His15, 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.
Jones
B E
BE
University of Washington, Department of Biochemistry and Biomolecular Structure Center, Seattle, Washington 98195-7742, USA.
Dossonnet
V
V
Küster
E
E
Hillen
W
W
Deutscher
J
J
Klevit
R E
RE
D001426:Bacterial Proteins; D001665:Binding Sites; D001692:Biological Transport; D050260:Carbohydrate Metabolism; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D019426:Integrases / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D014764:Viral Proteins
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; C069286:catabolite control proteins, bacteria; D004247:DNA; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr; C045073:Cre recombinase; D019426:Integrases