Structural and Biochemical Analysis of the Citrate-Responsive Mechanism of the Regulatory Domain of Catabolite Control Protein E from Staphylococcus aureus

Abstract

Catabolite control protein E (CcpE) is a LysR-type transcriptional regulator that positively regulates the transcription of the first two enzymes of the TCA cycle, namely, citZ and citB, by sensing accumulated intracellular citrate. CcpE comprises an N-terminal DNA-binding domain and a C-terminal regulatory domain (RD) and senses citrate with conserved arginine residues in the RD. Although the crystal structure of the apo SaCcpE-RD has been reported, the citrate-responsive and DNA-binding mechanisms by which CcpE regulates TCA activity remain unclear. Here, we report the crystal structure of the apo and citrate-bound SaCcpE-RDs. The SaCcpE-RD exhibits conformational changes between the two subdomains via hinge motion of the central β4 and β10 strands. The citrate molecule is located in a positively charged cavity between the two subdomains and interacts with the highly conserved Ser98, Leu100, Arg145, and Arg256 residues. Compared with that of the apo SaCcpE-RD, the distance between the two subdomains of the citrate-bound SaCcpE-RD is more than ∼3 Å due to the binding of the citrate molecule, and this form exhibits a closed structure. The SaCcpE-RD exhibits various citrate-binding-independent conformational changes at the contacting interface. The SaCcpE-RD prefers the dimeric state in solution, whereas the SaCcpE-FL prefers the tetrameric state. Our results provide insight into the molecular function of SaCcpE.

Functional and structural analysis of catabolite control protein C that responds to citrate

“…4 B). Moreover, the citrate-binding Arg147 and Arg260 residues of BaCcpC-IBD are analogous to the citrate-binding Arg145 and Arg256 residues of CcpE, which are required for CcpE to evoke an appropriate response in the presence of citrate 20 , 30 (Fig. 4 C).…”

Staphylococcal aconitase expression during iron deficiency is controlled by an sRNA-driven feedforward loop and moonlighting activity

“…Staphylococcal citB expression is activated by CcpE a citrate-responsive transcriptional regulator (14,63). Of note, CcpC, the B. subtilis orthologue of CcpE, has been reported to act as a repressor whose activity is relieved by citrate (64).…”

Versatility and Complexity: Common and Uncommon Facets of LysR-Type Transcriptional Regulators

“…Dipoles from four helices of one EBD subunit are organized such that opposing charges in the effector contribute to protein-ligand binding. Some effectors, such as cis,cis-muconate and benzoate in BenM (33), p-toluenesulfate in TsaR (90), salicylate in DntR (29), citrate in CcpE (18), shikimate in QuiR (102), and sulfite in YeiE (51), are strongly polar and form significant hydrogen bond networks with amino acid side chains that determine ligand specificity. In other cases, lipophilic components of the effectors (or designed therapeutics) can reach far into hydrophobic cavities within the EBDs, as in PqsR structures (54,66,137).…”