Solution structure and DNA binding of the effector domain from the global regulator PrrA (RegA) from Rhodobacter sphaeroides: insights into DNA binding specificity

Abstract

Prr/RegA response regulator is a global transcription regulator in purple bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus, and is essential in controlling the metabolic changes between aerobic and anaerobic environments. We report here the structure determination by NMR of the C-terminal effector domain of PrrA, PrrAC. It forms a three-helix bundle containing a helix-turn-helix DNA binding motif. The fold is similar to FIS protein, but the domain architecture is different from previously characterised response regulator effector domains, as it is shorter than any characterised so far. Alignment of Prr/RegA DNA targets permitted a refinement of the consensus sequence, which contains two GCGNC inverted repeats with variable half-site spacings. NMR titrations of PrrAC with specific and non-specific DNA show which surfaces are involved in DNA binding and suggest residues important for binding specificity. A model of the PrrAC/DNA complex was constructed in which two PrrAC molecules are bound to DNA in a symmetrical manner.

Figure 1

Stereo view of 20 overlaid PrrAC structures. The 20 best energy, water refined, structures of PrrAC calculated from NMR restraints, with the residues in helices coloured in red. The residues N-terminal to the area represented (137–184) are disordered. PrrAC amino acid sequence and secondary structure are indicated.

Figure 2

Alignment of DNA sequences recognised by PrrA/RegA. DNA sequences are identified by DNase I protection with R.capsulatus RegA* (,,,–27) and by gel retardation assays with B.japonicum RegR (7). The consensus sequence found for RegR, the RegR box, is indicated. A consensus sequence determined from this alignment is also indicated and the identities with this sequence outlined. N stands for any nucleotide, Y for pyrimidine, R for purine and M for A or T. The sequences have been sorted by increasing distance (in bp) between the GCG and CGC repeats, ranging from 3 (top) to 9 (bottom).

Figure 3

¹⁵N-HSQC titration of PrrAC with puc DNA fragment: side chain region of the spectrum. Increasing quantities of the 25 bp DNA fragment were added to PrrAC; in black before addition, and in red, green and blue for 0.5, 1 and 1.5 DNA/protein molar ratios, respectively. The residue numbers of the side chains involved in puc binding are represented in red. Residues involved in DNA binding have peaks exhibiting chemical shift variations and/or exchange broadening, which alters the peak shapes. The intensity of the peaks are corrected for dilution.

Figure 4

Representation of chemical shift variations of PrrAC (residues 137–184) upon binding to R.capsulatus puc DNA sequence. The variations were comparable for puc and cycAP2 promoter sequences. Large and medium backbone HN chemical shift variations are, respectively, in red and orange. Side chains experiencing chemical shift variations are shown, in green for the ones affected only by puc binding and in blue the ones affected by both specific and non-specific DNA binding. The protein is shown with the same orientation in (A) and surface representation (B). A continuous surface is affected by DNA binding: the recognition helix α8 and the α6–α7 loop, plus the beginning of α6. A perpendicular view (C) close to the orientation that PrrAC would have when bound in the major groove of DNA (see Fig. 6) shows that most of the contact made by PrrAC would mainly involve the floor and one side of the major groove. The main difference between puc and cycAP2 titrations is the Q175 Hε side chain, which is affected only by binding to puc.

Figure 5

Proposed contacts between PrrAC and the GCG conserved motif. Pattern of contacts suggested between the residues proposed to be in contact with bases and the GCG motif according to the contact code (35). The residues on the recognition helix are represented between the two strands of DNA. The lines represent observed contacts in HTH/DNA complexes (35). The continuous lines represent the contacts with the best chemical and stereochemical scores and the dashed lines the possible contacts, considering the length of the amino acid side chains. The best scores contacts were used to make the model in Figure 6. Arginines bind almost exclusively guanine bases, so make highly specific protein/DNA contacts, whereas glutamines are observed to be able to make contacts with any base.

Figure 6

Model of two PrrAC monomers bound to a 20 bp DNA fragment. (Top) A view of both monomers. (Bottom) A detail of PrrAC/DNA interactions. PrrAC monomers were fitted to respect the protein/DNA contacts predicted in Figure 5, the DNA titrations and the DNA and protein surfaces. R171, R172, Q175 and R176 side chain positions are at a distance where the predicted contacts would be possible. The recognised GCG inverted repeats have been coloured by DNA strand (brown and olive). The PrrAC colour code is as in Figure 4. R166 and R181 have been omitted for clarity.