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 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 Sep(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.