Structural Mechanism for the Fine-tuning of CcpA Function by The Small Molecule Effectors Glucose 6-Phosphate and Fructose 1,6-Bisphosphate Maria A. Schumacher 1 ⁎, Gerald Seidel 2 , Wolfgang Hillen 2 and Richard G. Brennan 1 ⁎ 1 Department of Biochemistry and Molecular Biology , Unit 1000, University of Texas, MD Anderson Cancer Center University, 1515 Holcombe Boulevard, Houston, TX 77030, USA 2 Lehrstuhl für Mikrobiologie, Institut für Biologie der Friedrich-Alexander Universität Erlangen-Nürnberg, Staudstrasse 5, 91058, Erlangen, Germany 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, ΔCcpA, 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. © 2007 Elsevier Ltd. All rights reserved. *Corresponding authors Keywords: CcpA; phosphoHPr; CCR; LacI-GalR family; transcription regulation Introduction In order to survive, bacteria must be able to adapt to a variety of environmental conditions, including the availability and supply of different carbon sources. At the heart of this adaptability is the global regulatory mechanism known as carbon catabolite regulation (CCR). In low G+C content Gram-positive bacteria, CCR is effected at the transcriptional level by the carbon catabolite activator protein, CcpA. 1–5 In response to the presence of glucose or other rapidly metabolized carbon sources, the CcpA protein is activated to bind catabolite response elements (cre), DNA operator sites leading to either the transcriptional repression or activation of over 300 genes in Bacillus subtilis that encode proteins involved in carbon metabolism and utilization, including enzymes, transporters and several transcription factors. 6–17 Abbreviations used: CCR, carbon catabolite regulation; CcpA, carbon catabolite activator protein A; HPr, histidine-containing protein; Crh, catabolite repression HPr; PTS, phosphoenolpyruvate:sugar phosphotransferase system; G6P, glucose 6-phosphate; HTH, helix-turn-helix; FBP, fructose 1,6-bisphosphate; MR, molecular replacement; RMSD, root-mean-square deviation. E-mail addresses of the corresponding authors: maschuma@mdanderson.org; rgbrenna@mdanderson.org doi:10.1016/j.jmb.2007.02.054 J. Mol. Biol. (2007) 368, 1042–1050 0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.