Structural mechanism for the fine-tuning of CcpA function by the small molecule effectors glucose 6-phosphate and fructose 1,6-bisphosphate.
In Gram-positive bacteria, carbon catabolite regulation (CCR) is mediated by the carbon catabolite control protein A (CcpA), a member of the LacI-GalR family of transcription regulators. Unlike other LacI-GalR proteins, CcpA is activated to bind DNA by binding the phosphoproteins HPr-Ser46-P or Crh-Ser46-P. However, fine regulation of CCR is accomplished by the small molecule effectors, glucose 6-phosphate (G6P) and fructose 1,6-bisphosphate (FBP), which somehow enhance CcpA-(HPr-Ser46-P) binding to DNA. Unlike the CcpA-(HPr-Ser46-P) complex, DNA binding by CcpA-(Crh-Ser46-P) is not stimulated by G6P or FBP. To understand the fine-tuning mechanism of these effectors, we solved the structures of the CcpA core, DeltaCcpA, which lacks the N-terminal DNA-binding domain, in complex with HPr-Ser46-P and G6P or FBP. G6P and FBP bind in a deep cleft, between the N and C subdomains of CcpA. Neither interacts with HPr-Ser46-P. This suggests that one role of the adjunct corepressors is to buttress the DNA-binding conformation effected by the binding of HPr-Ser46-P to the CcpA dimer N subdomains. However, the structures reveal that an unexpected function of adjunct corepressor binding is to bolster cross interactions between HPr-Ser46-P residue Arg17 and residues Asp69 and Asp99 of the other CcpA subunit. These cross contacts, which are weak or not present in the CcpA-(Crh-Ser46-P) complex, stimulate the CcpA-(HPr-Ser46-P)-DNA interaction specifically. Thus, stabilization of the closed conformation and bolstering of cross contacts between CcpA and its other corepressor, HPr-Ser46-P, provide a molecular explanation for how adjunct corepressors G6P and FBP enhance the interaction between CcpA-(HPr-Ser46-P) and cognate DNA.
Schumacher
Maria A
MA
Department of Biochemistry and Molecular Biology, Unit 1000, University of Texas, MD Anderson Cancer Center University, 1515 Holcombe Boulevard, Houston, TX 77030, USA. maschuma@mdanderson.org
Seidel
Gerald
G
Hillen
Wolfgang
W
Brennan
Richard G
RG
D001410:Bacillus megaterium / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005635:Fructosediphosphates / Q000737:chemistry* / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry; D010766:Phosphorylation; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D005635:Fructosediphosphates; D012097:Repressor Proteins; C069286:catabolite control proteins, bacteria; D012694:Serine; D019298:Glucose-6-Phosphate; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr; C029063:fructose-1,6-diphosphate