Structural analysis of <i>B. subtilis</i> CcpA effector binding site

摘要

In Gram-positive bacteria, the catabolite control protein A (CcpA) is the central regulator of a fundamental signal transduction pathway called carbon catabolite repression.1 In response to the availability of glucose or other rapidly metabolizable carbohydrates in the growth medium, CcpA recognizes and binds cis-acting palindromic DNA sequences called catabolite response elements (cre)2 and regulates the transcription of numerous carbon catabolic operons. CcpA belongs to the LacI/GalR family of bacterial regulatory proteins characterized by an N-terminal helix–turn–helix type DNA-binding domain and a large C-terminal effector-binding domain.3 Most of the family members bind to their cognate DNA depending upon the presence or absence of low-molecular-weight effectors. The effector-binding site is formed by a cleft between the two subdomains of the core protein that resembles periplasmic sugar-binding proteins.4 Unlike other members of the family, CcpA is activated by a protein that acts as co-repressor, PserHPr, the Ser46-phosphorylated form of HPr, a phosphocarrier protein of the bacterial phosphotransferase system.5 In bacilli, PserCrh, the Ser46-phosphorylated form of Crh, a protein homologous to HPr, is an alternative coregulator of CcpA.6 Small-molecule effectors have also been shown to regulate CcpA activity. Fructose-1, 6-bisphosphate (FBP) enhances the affinity of CcpA for PserHPr.5 The ternary complex CcpA/PserHPr/FBP shows the highest affinity for DNA.7 Glucose 6-phosphate (G6P) triggers cooperative binding of CcpA to cre sites at low pH.8 Interestingly, the effects of FBP and G6P on the CcpA–PserHPr complex are not observed when PserCrh is used as co-repressor of CcpA instead of PserHPr.9 It has also been proposed that NADP/NADPH could enhance interaction of CcpA with the transcription machinery.7 Recent results suggesting direct interaction of CcpA with the RNA polymerase confirm this hypothesis.10 The X-ray structures of Bacillus megaterium apo CcpA and of ternary complex with a cre site and PserHPr or PserCrh have been reported.11, 12 The co-repressor molecule binds to the N-subdomain of CcpA's core, inducing a conformation change and suggesting an allosteric regulatory mechanism. The molecular mechanism of CcpA fine regulation by the small molecule effectors remains nevertheless unclear. We therefore decided to crystallize CcpA in presence of FBP. Here, we report the 2.45-Å resolution X-ray structure of the binary complex between B. subtilis PserHPr and ΔCcpA, a truncated form of CcpA lacking the N-terminal DNA-binding domain. We focus on the effector-binding mode. The B. subtilis CcpA gene deleted of the first 57 codons encoding the DNA binding domain was cloned into a pQE30 plasmid (Qiagen). The resulting ΔCcpA (residues Ala57 to Ser334) carrying a N-terminal histidine tag was expressed in the Escherichia coli strain M15 and purified by IMAC chromatography followed by gel filtration. B. subtilis HPr was produced in E. coli with a N-terminal histidine tag as described.13 ATP-dependent seryl-phosphorylation of HPr was carried out as described using the B. subtilis HPr kinase/phosphorylase.14 Crystals were grown in sitting drops containing 70 μM ΔCcpA, 138.5 μM PserHPr, 4 mM FBP, 0.5% (v/v) MPD, 50 mM Mes (pH 6.5), 1.75 M ammonium sulfate over pits containing 1% (v/v) MPD, 100 mM Mes (pH 6.5), and 3.5 M ammonium sulfate. The asymmetric unit contains one ΔCcpA monomer bound to one PserHPr molecule. Diffraction data were collected at 100 K on an ADSC Quantum Q4 detector at the European Synchrotron Radiation Facility (Grenoble) on beamline ID14-H1 with a single crystal directly flash frozen in liquid nitrogen. Diffracted intensities, evaluated using MOSFLM,15 and further processed with the CCP4 program suite,16 were 99.9% complete at 2.45 Å resolution (Table I). The structure of the ΔCcpA–PserHPr complex was solved by molecular replacement with Phaser17, 18 using coordinates from the CcpA-PserHPr-DNA complex (PDB co